Recent Advances in Transthyretin Evolution, Structure and Biological Functions
Samantha J. Richardson
l
Vivian Cody
Recent Advances in Transthyretin Evolution, Structure and Biological Functions
Dr. Samantha J. Richardson RMIT University School of Medical Sciences Bundoora VIC 3083 Bundoora West Campus Australia
[email protected]
Dr. Vivian Cody Hauptman-Woodward Medical Research Institute 700 Elliott Street Buffalo NY 14203 USA
[email protected]
ISBN 978-3-642-00645-6 e-ISBN 978-3-642-00646-3 DOI: 10.1007/978-3-642-00646-3 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009926025 # Springer-Verlag Berlin Heidelberg 2009 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)
Preface
Since its first description in 1942 in both serum and cerebrospinal fluid, transthyretin (TTR) has had an eventful history, including changes in name from “prealbumin” to “thyroxine-binding prealbumin” to “transthyretin” as knowledge increased about its functions. TTR is synthesised in a wide range of tissues in humans and other eutherian mammals: the liver, choroid plexus (blood- cerebrospinal fluid barrier), retinal pigment epithelium of the eye, pancreas, intestine and meninges. However, its sites of synthesis are more restricted in other vertebrates. This implies that the number of tissues synthesising TTR during vertebrate evolution has increased, and raises questions about the selection pressures governing TTR synthesis. TTR is most widely known as a distributor of thyroid hormones. In addition, TTR binds retinol-binding protein, which binds retinol. In this way, TTR is also involved with retinoid distribution. More recently, TTR has been demonstrated to bind a wide variety of endocrine disruptors including drugs, pollutants, industrial compounds, heavy metals, and some naturally occurring plant flavonoids. These not only interfere with thyroid hormone delivery in the body, but also transport such endocrine disruptors into the brain, where they have the potential to accumulate. The X-ray crystal structure of TTR from vertebrates (fish, chicken, rat, mouse and human) has not changed in its overall structure. Despite this, TTRs in fish, amphibians, reptiles and birds bind T3 with higher affinity than T4, whereas in mammals, while maintaining the same three-dimensional structure in the binding site, TTR binds T4 with higher affinity than T3. The high conservation of quaternary, tertiary, secondary and primary structure, in combination with the sequencing of many non-vertebrate genomes, has allowed the identification of genes coding for TTR-Like Proteins (TLPs) in microbes, plants and non-vertebrate animals. TLPs studied to date do not bind thyroid hormones, but have 5-hydroxyisourate hydrolase activity. The change in function from 5-HIUase in bacteria to distributor of T3 in fish, amphibians, reptiles and birds, to distributor of T4 in mammals, yet maintaining the same overall three-dimensional structure, renders TLP/TTR an excellent model for the study of protein evolution. The function of TTR is still considered controversial by some researchers, as TTR null mice were originally reported to be without an overt phenotype. There exists the possibility that the lack of phenotype is due to redundancies in thyroid v
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Preface
hormone and retinoid metabolism, and also that mice living in animal houses in laboratories are not subjected to the same stresses as those in the wild; thus the phenotype is not seen under laboratory conditions. Intriguingly, there are no reports of humans lacking TTR. However, more recent studies have revealed a variety of phenotypes in TTR null mice, shedding light on previously unknown roles of TTR in development and neurobiology. TTR is also widely implicated in human health and disease. TTR is used as a nutritional marker, and also as an indicator of recovery following some diseases and surgery. Perhaps the most prolific area of TTR research concerns human TTR amyloid formation. There are two main types of TTR amyloid: familial amyloidotic polyneuropathy (FAP) is an autosomal dominant inherited mutation in the TTR gene causing polyneuropathy, whereas senile systemic amyloidosis (SSA; also known as senile cardiac amyloidosis, SCA) is age-dependent and the amyloid fibrils contain wild type TTR. Of the 127 amino acids in the polypeptide, there are at least 100 point mutations that result in FAP. Therefore, there is an urgent need for continued research into the mechanisms of TTR amyloid formation, and for the development of therapeutics including drugs, gene therapy and organ transplants. Thus, progress in medical research into TTR is fundamental to human health. TTR is a fascinating protein from the stand point of protein evolution, and also in medicine. Thus, this is an exciting time for experts in TTR research to come together to write this monograph that covers both the basic and the clinical research in TTR. This monograph describes each of the above-mentioned aspects of TTR and brings the reader up to date on the latest developments and discoveries.
March 2009
Samantha J. Richardson Vivian Cody
Contents
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Mechanisms of Molecular Recognition: Structural Characteristics of Transthyretin Ligand Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vivian Cody and Andrzej Wojtczak
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Transthyretin Synthesis During Development and Evolution: What the Marsupials Revealed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Samantha J. Richardson
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Evolution of Transthyretin Gene Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . Porntip Prapunpoj
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Evolutionary Insights from Fish Transthyretin . . . . . . . . . . . . . . . . . . . . . . . Deborah M. Power, Isabel Morgado, and Joa˜o C.R. Cardoso
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5
The Salmonella sp. TLP: A Periplasmic 5-Hydroxyisourate Hydrolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sarah Hennebry
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Vertebrate 5-Hydroxyisourate Hydrolase Identification, Function, Structure, and Evolutionary Relationship with Transthyretin . . . . . . Giuseppe Zanotti, Ileana Ramazzina, Laura Cendron, Claudia Folli, Riccardo Percudani, and Rodolfo Berni
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Transthyretin-Related and Transthyretin-like Proteins . . . . . . . . . . . . . . 109 A. Elisabeth Sauer-Eriksson, Anna Linusson, and Erik Lundberg
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The Transthyretin–Retinol-Binding Protein Complex . . . . . . . . . . . . . . . 123 Hugo L. Monaco
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Transthyretin and Retinol-Binding Protein: Implications in Fish Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Sancia Gaetani and Diana Bellovino
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Contents
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Transthyretin and Endocrine Disruptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Kiyoshi Yamauchi and Akinori Ishihara
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Genetics: Clinical Implications of Transthyretin Amyloidosis . . . . . . . 173 Merrill D. Benson
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Molecular Pathogenesis Associated with Familial Amyloidotic Polyneuropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Maria Joa˜o Saraiva
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Histidine 31: The Achilles’ Heel of Human Transthyretin. Microheterogeneity is Not Enough to Understand the Molecular Causes of Amyloidogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Klaus Altland and Samantha J. Richardson
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New Therapeutic Approaches for Familial Amyloidotic Polyneuropathy (FAP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Yukio Ando, Masaaki Nakamura, Mistuharu Ueda, and Hirofumi Jono
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Liver Transplantation for Transthyretin Amyloidosis . . . . . . . . . . . . . . . 239 Bo-Goran Ericzon, Erik Lundgren, and Ole B. Suhr
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Mouse Models of Transthyretin Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . 261 Sadahiro Itoand Shuichiro Maeda
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What Have We Learned from Transthyretin-Null Mice: Novel Functions for Transthyretin? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Joa˜o Carlos Sousa and Joana Almeida Palha
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Transthyretin Null Mice: Developmental Phenotypes . . . . . . . . . . . . . . . . 297 Julie A. Monk and Samantha J. Richardson
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Transthyretin Null Mice as a Model to Study the Involvement of Transthyretin in Neurobiology: From Neuropeptide Processing to Nerve Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 Carolina Estima Fleming, Ana Filipa Nunes, Ma´rcia Almeida Liz, and Mo´nica Mendes Sousa
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Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Yves Ingenbleek
Index
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Contributors
Klaus Altland Justus-Liebig-University, Institute of Human Genetics Schlangenzahl 14, 35392 Giessen, Germany
[email protected] Yukio Ando Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan
[email protected] Diana Bellovino National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy
[email protected] Merrill D. Benson Indiana University School of Medicine, 635 Barnhill Drive, MS-128, Indianapolis, IN 46202-5126, USA
[email protected] Rodolfo Berni Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy
[email protected] Joa˜o C.R. Cardoso Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005139 Faro, Portugal
[email protected] Laura Cendron Department of Chemistry, University of Padua, and ICB-CNR, Section of Padua, Via Marzolo 1, 35131 Padova, Italy and Venetian Institute of Molecular Medicine, Via Orus 2, 35127 Padua, Italy
[email protected] Vivian Cody Structural Biology Department, Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203, USA
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Bo-Goran Ericzon Head Division of Transplantation Surgery, CLINTEC, Karolinska Institutet, Karolinska University Hospital Huddinge, F82, 141 86 Stockholm, Sweden
[email protected] Carolina Estima Fleming Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal
[email protected] Claudia Folli Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy
[email protected] Sancia Gaetani National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy
[email protected] Sarah Hennebry Human Neurotransmitters Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia
[email protected] Yves Ingenbleek Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur, Strasbourg 1, France
[email protected] and 15 bis, rue de la Vise, 34540 Balaruc-le-Vieux, France
[email protected] Akinori Ishihara Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka, Japan
[email protected] Sadahiro Ito Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan and Center for Life Science Research, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan
[email protected] Hirofumi Jono Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan
[email protected]
Contributors
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Anna Linusson Department of Chemistry, Umea˚ University, 90187 Umea˚, Sweden Ma´rcia Almeida Liz Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal
[email protected] Erik Lundgren Department of Molecular Biology, Umea˚ University, 901 87 Umea˚, Sweden
[email protected] and Department of Chemistry, Umea˚ University, 90187 Umea˚, Sweden Shuichiro Maeda Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan
[email protected] Hugo L. Monaco Biocrystallography Laboratory, Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy
[email protected] Julie A. Monk ProScribe Medical Communications, 481 Gilbert Rd., Preston, VIC 3072, Australia
[email protected], url: www.proscribe.com.au Isabel Morgado Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005139 Faro, Portugal and Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany
[email protected] Masaaki Nakamura Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan
[email protected] Ana Filipa Nunes Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal
[email protected]
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Contributors
Joana Almeida Palha Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
[email protected] Riccardo Percudani Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy
[email protected] Deborah M. Power Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005-139 Faro, Portugal
[email protected] Porntip Prapunpoj Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
[email protected] Ileana Ramazzina Department of Biochemistry and Molecular Biology, University of Parma, Viale delle Scienze 23/A, 43100 Parma, Italy
[email protected] Samantha J. Richardson School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora, 3083 VIC, Australia
[email protected] Maria Joa˜o Saraiva Molecular Neurobiology Group, IBMC – Instituto de Biologia Molecular e Celular, R Campo Alegre 823, 4150 Porto, Portugal
[email protected] and ICBAS – Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Porto, Portugal A. Elisabeth Sauer-Eriksson Department of Chemistry, Umea˚ University, 90187 Umea˚, Sweden
[email protected] Joa˜o Carlos Sousa Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
[email protected] Mo´nica Mendes Sousa Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, R Campo Alegre 823, 4150 Porto, Portugal
[email protected]
Contributors
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Ole B. Suhr Department of Medicine, Section for Gastroenterology and Hepatology, Umea˚ University Hospital, 901 85 Umea˚, Sweden
[email protected] Mistuharu Ueda Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan
[email protected] Andrzej Wojtczak Chemistry Department, N. Copernicus University, Torun, Poland
[email protected] Kiyoshi Yamauchi Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka, Japan
[email protected] Giuseppe Zanotti Department of Chemistry, University of Padua, and ICB-CNR, Section of Padua, Via Marzolo 1, 35131 Padova, Italy and Venetian Institute of Molecular Medicine, Via Orus 2, 35127 Padua, Italy
[email protected]
Chapter 1
Mechanisms of Molecular Recognition: Structural Characteristics of Transthyretin Ligand Interactions Vivian Cody and Andrzej Wojtczak
Abstract Transthyretin (TTR) is a homotetrameric serum protein responsible for the transport through the general circulation of thyroid hormones and their metabolites, and also binds retinol-binding protein (RBP) that transports retinol. Structure-activity data show that many pharmacological agents can compete for the binding of thyroid hormone to TTR and that this competition can interfere with their normal physiological functions. This review surveys more than 100 TTR crystal structures currently (October 2008) reported in the Protein Data Bank, representing only five species of vertebrates. Despite the highly conserved nature of protein sequences, TTR can bind a wide range of compounds with a high degree of flexibility. TTR is composed of four identical monomeric subunits that assemble around a central channel such that the tetramer possesses molecular 222-symmetry with two hormone-binding sites per tetramer. At physiological conditions, only one domain is occupied; however, negative cooperativity in binding to the second domain causes the second hormone to bind with a lower affinity. To date, more than 100 variants at 67 amino acid sites of the 127 residues of the human TTR monomer have been implicated in amyloid fibril formation that gives rise to familial amyloid polyneuropathy (FAP). Although the mechanism underlying TTR tetramer stability is not well understood, it has been shown that tight binding ligands can reduce the propensity for amyloid fibril formation. Structural data of TTR ligand complexes provide new insight into the design of novel inhibitors that can stabilize specific mutants and thereby delay the onset of fibril formation. Keywords Transthyretin, Conformational flexibility, Active site topology, Proteinprotein interactions
V. Cody (*) Structural Biology Department, Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, New York 14203, USA
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_1, # Springer‐Verlag Berlin Heidelberg 2009
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V. Cody and A. Wojtczak
1.1
Introduction
Transthyretin (TTR) is one of the three thyroid hormone-binding proteins found in the blood of vertebrates (Robbins 1996; Cody 2005). Produced in the liver, it binds retinol-binding protein that carries retinol (vitamin A) and thyroxine (3,5,30 ,50 tetraiodo-L-thyronine, T4) and differs from thyroxine-binding globulin (TBG) and serum albumin (SA) that has several nonspecific hormone-binding sites. Human TTR is a 55 kDa homotetramer composed of four identical 127 residue subunits that form an extensive b-sheet structure. The homotetramer assembles to form an internal channel in which two T4 molecules are bound per tetramer (Fig. 1.1). Biochemical data revealed that in the general circulation only one site is filled and that there is negative cooperativity in the binding of the second T4 (Cheng et al. 1977). Structure activity data revealed that the thyroid hormones and their metabolites have different binding affinities for TTR, depending on their substituent patterns, that is, the number of iodine atoms present or the type of side chain (propionic or acetic acid) (Table 1.1) (Cody 1980). In addition, many pharmacologic agents and natural products, such as plant flavonoids, nonsteroidal analgesic drugs, and inotropic bipyridines, are strong competitors for T4 binding to TTR and have binding affinities greater than T4 (Robbins 1996; Cody 2002). Recent studies have shown that TTR is one of about 20 human proteins that form fibrils in vivo, and to date more than 100 point mutations in TTR have been associated with the formation of fibrils, highly organized filamentous structures that form by self assembly of aggregated polypeptides. The propensity of TTR variants to form fibrils is the basis of familial amyloidotic polyneuropathy (FAP), a fatal genetic disease that is characterized by specific organ damage that targets the
a
b
Fig. 1.1 (a) TTR assembles as a tetramer with four identical subunits that form a channel with two hormone binding sites. The tetramer is generated by a twofold symmetry axis along the channel with monomers A-A0 (green, violet) and B-B0 (Cyan, gold). (b) Independent monomers of TTR illustrating forward and reverse binding of T4 acetic acid (T4A, Fig. 1.2)
1 Mechanisms of Molecular Recognition
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Table 1.1 Relative binding affinities and biologic potencies of selected thyroid hormone analogues (Cody 1980) Compound TGB (%) TTR (%) Membrane (%) Nuclear (%) Potencya (%) L-Thyroxine
100.0
D-Thyroxine
54.0
39.3 0.95
96.6
12.5
63.0
–
18.1 3.0
3.6 76.4 23.0 – 3.0 T4-propionic acid 1.7 100.0 30.3 – 0.25 T4-acetic acid L-T3 (3,5,30 -triiodo) 9.0 1.4 100.0 100.0 100.0 38.0 3.1 67.5 0.1 <0.1 rT3 (3,5,30 -triiodo) a Potency is percentage of L-T3 (triiodothyronine; 30 ,3,5-triiodothyronine) rT3 reverse triiodothyronine, T3 triiodothyronine, T4 thyroxine, TGB thyroxine-binding globulin, TTR transthyretin
Fig. 1.2 TTR bound ligand classes reported in the PDB (October 2008)
heart, liver, brain, and peripheral nerves (Benson and Kincaid 2007; Westermark et al. 2007). The main focus of this review is to analyze TTR structures to determine preferences in ligand-bound conformation and geometry. In this review, a survey of all TTR protein complexes reported in the Protein Data Bank (PDB) (Berman et al. 2000) will be made, along with a comparison of the variants studied in order to understand the criteria for the formation of amyloid fibrils. Interactions between TTR and retinol-binding protein will not be discussed in this chapter as they are detailed in another chapter. Currently (October 2008), there are over 100 reported TTR crystal structures and their variants listed in the PDB (Berman et al. 2000), representing five species of protein (human, rat, mouse, chicken, and fish) with 37 different ligands in nine structure classifications (Fig. 1.2). Sequence analysis shows that TTRs from various species are more than 85% identical to human (Table 1.2). Of the structural data reported for TTR, the human protein has been the most studied.
hTTR rTTR mTTR cTTR sbTTR
EEEFVEGIYK DEKFTEGVYR DEKFVEGVYR EEQFVEGVYR EQQFPAGVYR
61
APL APTPT
VEIDTKSYWK VELDTKSYWK VELDTKSYWK VEFDTSSYWK VEFDTKAYWT
71 ALGISPFHEH ALGISPFHEY TLGISPFHEF GLGLSPFHEY NQSTPFHEVA
81 AEVVFTANDS AEVVFTANDS ADVVFTANDS ADVVFTANDS EVVFDAHPEG
91
LSPYSYSTTA LSPYSYSTTA LSPYSYSTTA LSPFSYSTTA LSPFSYTTTA
111
WEPFASGKTS WEPFASGKTA WEPFASGKTA WQDFATGKTT WTQIATGVTD
GPRRYTIAAL GHRHYTIAAL GHRHYTIAAL GHRHYTIAAL HRHYTILALL
101
HVFRKAADDT KVFKKTADGS KVFKKTSEGS KVFKKAADGT KVSQKTADGG
VVTNPKE VVSNPQN VVSNPQN VVSDPQE VVSSVHE
121
51
RGSPAINVAV RDSPAVDVAV RGSPAVDVAV RGSPAANVAV KGTPAGSVAL
ESGELHGLTT ESGELHGLTT ESGELHGLTT EFGEIHELTT ATGEIHNLIT
PLMVKVLDAV PLMVKVLDAV PLMVKVLDAV PLMVKVLDAV PLMVKILDAV
hTTR rTTR mTTR cTTR sbTTR
GPTGTGESKC GPGGAGESKC GPAGAGESKC VSHGSVDSKC DKHGGSDTRC
Table 1.2 Sequence comparison of human, rat, mouse, chicken, and sea bream TTR for which crystal structures have been determined 1 11 21 31 41
4 V. Cody and A. Wojtczak
1 Mechanisms of Molecular Recognition
1.2
5
TTR Monomer Assembly and Stability
Each monomer of the human TTR homotetramer is defined as a b-barrel built from a single polypeptide chain of 127 amino acids forming eight strands of A-H organized into two anti-parallel b-sheets (Fig. 1.1b). Each barrel contains a short a-helix between strands E and F (Blake et al. 1978). Among the TTR structures in the PDB, majority of the human (h) TTR structures (73/83) crystallize in the orthorhombic space group P21212 with two independent monomers in the asymmetric unit, designated A and B, that form a dimer that assembles into a tetramer (A-A0 , B-B0 ), forming a dimer of dimers by applying the twofold axis along the thyroid hormone-binding channel (Fig. 1.1a) (Blake and Oately 1977). The resulting 222 symmetry in the tetramer results in twofold symmetry for each of the ligandbinding sites formed by pairs of monomers positioned across the channel axis. As TTR ligands reported to date (Fig. 1.2) do not have twofold symmetry, the electron density for the ligand appears as an average of two symmetry-related positions. Thus, for many hTTR complexes, the specific details of ligand binding interactions are obscured by the twofold disorder observed for the ligand orientation. As observed in some TTR ligand complexes, interpretation of the density is further complicated by the presence of alternate binding modes that simultaneously occupy the same binding pocket (Cody et al. 1991; Ghosh et al. 2000; Muziol et al. 2001a, b). A scan of TTR structures deposited in the PDB revealed that other packing arrangements have been observed for human, as well as other species of TTR, which result in an independent tetramer in the asymmetric unit that defines the unique environment for ligand binding. Each of these packing environments results in the assembly of alternating columns of TTR tetramers that may reflect patterns for fibril formation (Fig. 1.3). Interestingly, crystal packing in other space groups has revealed asymmetric units that contain unique dimers, tetramers, and tetramer– dimer combinations. It is of interest to note that the majority of structures from other species (e.g., rat, mouse, fish) tend to crystallize in space groups that have a unique tetramer in the asymmetric unit of the crystal lattice. This variety of packing interactions provides insight into the role of surface residues in the dynamics of monomer assembly. For example, all the structures of rat TTR crystallized in the tetragonal space group P43212 and provided details of the first unique binding environment for T4 in TTR (Wojtczak et al. 2001a; Fig. 1.3b). Similarly, all the structures reported for fish (sea bream) TTR crystallize with a tetramer in the asymmetric unit in the space groups P41 or C2 (Folli et al. 2003; Eneqvist et al. 2004; Fig. 1.3c). Mouse TTR crystallizes with one tetramer and two unique dimers in the asymmetric unit in the space group P3121 (Reixach et al. 2008). On the other hand, chicken TTR crystallizes in the space group P6522 with a unique dimer in the asymmetric unit (Sunde et al. 1996). One example of unusual monomer assembly and TTR binding was observed for the hTTR T4 complex in space group P21. These data showed two independent tetramers in the asymmetric unit. What makes this structure unusual is that the hormone is bound in only one tetramer (Wojtczak et al. 2001b) (Fig. 1.3d).
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Fig. 1.3 Representative packing diagrams for TTR assemblies: (a) two views of hTTR-T4 P21212 (2rox) packing of the unique homodimers (red) showing lattice packing (green, cyan), (b) two views of rTTR-T4 P43212 (1ie4) packing of the unique tetramer (green, cyan, violet, yellow) showing the lattice packing (green), (c) two views of piscine TTR-T4 P41 (1sn0) packing of the unique tetramer (green, cyan, violet, yellow) showing lattice packing (green), (d) two views of hTTR-T4 P21 (1ict) packing of two unique tetramers (green, cyan, violet, yellow: pink, grey, purple, gold) showing the lattice packing (green). One tetramer has T4 bound in the two binding domains and the other tetramer is empty
The crystal packing is such that there are intermolecular interactions involving the tips of the alpha helices and loops near Arg21, Glu61, and Ser100 of all monomers. The binding of T4 in this structure differs between the two binding domains. In one, T4 is bound in the orientation observed in the orthorhombic P21212 lattice, while in the other, the hormone is bound deeper in the channel, similar to that observed for 30 ,50 -dinitro-N-acetyl-L-thyronine (Wojtczak et al. 1996). The size of the binding channel in the apo tetramer is smaller than that for the T4 bound tetramer. Another unusual monomer assembly was observed for the hTTR diethylstilbestrol (DES) complex (Fig. 1.2) in space group C2, in which there are two sets of unique dimers that form two tetrameric structures (Morais-de-Sa et al. 2004). In this
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structure, there are two independent conformers of DES bound in the first tetramer and only one DES bound in the second tetramer. This arrangement differs from that observed in the C2 lattice of the Leu55Pro mutant that formed a tetramer and two independent dimers (Sebastiao et al. 1998). In this case, the structure shows that the L55P mutation disrupts the hydrogen bonds between the D and A strands, resulting in different packing contacts. Tetramers of human wild-type TTR or its variants can dissociate at low pH and assemble into amyloid fibrils. Some models for such transformations include the conformational changes of TTR monomers followed by aggregation of such misfolded monomers to build the amyloid fibril (Jiang et al. 2001; Liu et al. 2000; Lai et al. 1996; Lashuel et al. 1998; Palaninathan et al. 2008; Hurshman-Babbes et al. 2008). Recent studies on the denaturation of TTR have addressed the relationship between protein stability and amyloidosis. Characterization of the thermodynamics of dissociation of wild-type human TTR and the disease-associated variants V122I, L55P, V30M, and A25T have revealed a correlation between the quaternary and tertiary structural stability (Hurshman-Babbes et al. 2008). These data showed that the two-step dissociation of tetramers to folded monomers and then to unfolded monomers was more complicated for the more unstable monomers. These stability measures also correlated with the aggressiveness of the clinical manifestations of the amyloid disease. More recent data indicate that long-lived monomers of the non-amyloidogenic T119M variant can be successfully incorporated into heterotetramers that are less amyloidogenic and that these monomers can function as inhibitors of TTR aggregation (Palhano et al. 2009). Other data suggest that TTR amyloid fibrils may assemble from dimers or tetramers of the protein rather than from altered monomers (Ferrao-Gonzales et al. 2000; Eneqvist et al. 2000; Serag et al. 2001, 2002; Olofsson et al. 2001, 2004; Kelly 1998; Hammarstro¨m et al. 2003). On the other hand, the use of small molecule inhibitors has proven to be an efficient way of preventing the formation of TTR-related amyloid fibrils by tetramer stabilization or prevention of conformational changes independent of the particular mechanism of transformation into amyloid filbrils (Johnson et al. 2005, 2008; Miroy et al. 1996; Peterson et al. 1998; Klabunde et al. 2000; Green et al. 2003). Kelly and coworkers observed that TTR can assemble around bivalent inhibitors that span both T4 binding sites of the tetramer simultaneously. The crystal structures of these complexes are isomorphous with the native TTR, although these compounds do not bind to the native TTR, but rather intercept TTR during folding and assembly (Green et al. 2003). Miroy et al. have shown that binding of T4 stabilizes the TTR tetramer and consequently inhibits amyloid formation (Miroy et al. 1996). White and Kelly investigated the amyloid formation from TTR in the presence of holo RBP, T4, and small molecular inhibitors (White and Kelly 2001) and found that T4 significantly inhibited TTR fibril formation in both TTR and its complex with holo RBP. They observed synergism in the RBP and in the small molecule inhibitor actions against the formation of fibrils of the most amyloidogenic L55P TTR variant.
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Negative Cooperativity in TTR Binding
Thyroxine (T4), its analogues, and derivatives can be bound to both binding sites inside the central channel of the TTR tetramer. However, binding of the second ligand is much weaker than that of the first molecule. Equilibrium dialysis experiments had shown that TTR binding affinity for T4, measured by the K1 and K2 association constants for the first and the second T4 molecule bound to TTR, differed by a factor of 100 (K1 = 1.0 108 M1 and K2 = 9.5 105 M1). This difference is explained by the negative cooperativity (NC) effect and has been described for several iodinated T4 analogues with similar binding constants (Cheng et al. 1977; Ferguson et al. 1975). However, the fluorescent probe 8-anilino-1-naphtalenesulfonic acid (ANS) revealed a much smaller difference between the first and the second binding constants, with the K1/K2 ratio of 4.5–3.0 (Cheng et al. 1977; Ferguson et al. 1975). An increase in the difference between the two binding constants with the decreasing pH was observed for 3-(4-hydroxy-3,5-diiodophenyl)-propionic acid (DIPA) (Cheng et al. 1977). This change was caused by a decrease of K2, while K1 remained almost unchanged. This observation was explained by the pH dependence of ionization of the DIPA phenolic hydroxyl group. These data suggest that electrostatic interactions may contribute to the NC effect. A general mechanism can be introduced that involves conformational changes upon binding of a ligand to one subunit, which alter the interactions between the TTR subunits and consequently affects the binding affinity of the second ligand molecule. Studies by ultraviolet difference spectroscopy and ultraviolet fluorescence methods (Peterson et al. 1998; Gonzalez and Tapia 1992; Gonzalez 1989; Irace and Edelhoch 1978) have suggested minor conformational changes of the protein or alteration of the H-bond network linking the two sites upon binding of T4 in the first site. The changes are coupled with the reported quenching of the Trp-79 signal (Irace and Edelhoch 1978). Such changes should also induce conformational restrictions on the protein, making the second site more rigid, so that transport of a second hormone molecule into the channel might be obstructed. Analysis of the crystal structure of monoclinic hTTR provided a unique opportunity to compare the structure of apo hTTR and of its T4 complex at the same level of accuracy as the two forms co-exist in one crystal (Wojtczak et al. 2001b) and permitted examination of the conformational changes caused by T4 binding in the hTTR tetramers. The detailed comparison of several structures of native TTR and TTR complexes with small molecule ligands provided insights into the mechanism of ligand-induced conformational changes on ligand binding (Neumann et al. 2001). The comparison was performed for the TTR channel diameter for the apo protein structures and for different complexes. The channel diameter was calculated in each binding site as the Ca–Ca distances between equivalent amino acids from ˚ and larger than the two monomers. Many of these differences were larger than 0.5 A three times the Luzzati error. In all cases, similar trends were found, although the magnitude of the changes varied.
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A model of the ligand-induced changes in the TTR binding channel was based on the apo TTR and complex structures (Muziol et al. 2001a; Wojtczak et al., 1992 1993, 1996, 2001a, b; Sunde et al. 1996; Kelly and Lansbury 1994; Ciszak et al. 1992; Hamilton et al. 1993; Wojtczak 1997; Neumann et al. 2005). Comparison of the diameter of the TTR channel revealed that the two hormone-binding sites are not identical. The primary site has a slightly larger channel diameter when measured between equivalent amino acids belonging to b-strands D, A, and H, while the diameter for residue pairs 105–109 of strands G is smaller. The secondary binding site has the opposite characteristics and is narrower at both the outer and the inner part of the channel, while the middle of this binding site is wider. The first hormone molecule binds to the site with a larger diameter near the channel entrance (strands D and A). Binding interactions at the primary site of the first hormone molecule cause an increase in the diameter of the primary site measured at strand G (residues 105–109) and slight collapse of the channel entrance (strands D and A) and the inner part of the binding site (strand H) (Fig. 1.1b). The conformational changes caused by the binding of the first ligand molecule in the primary site trigger the changes in the secondary site, transmitted by the relatively rigid antiparallel b-sheet structure of the channel. The second binding site becomes wider in both the outer and the inner parts of the channel (strands D, A, and H), while the middle part (strand G) collapses. The resulting characteristics of the second binding site are similar to that of an empty primary binding site. Binding of the second ligand reverses the alterations described in both the sites, but the amplitude of the changes is relatively small because of the binding interactions that are already formed by the first ligand. Therefore, the collapse of the second site is not as significant as that of the first site, and the second hormone molecule is bound less tightly. The mechanism of negative cooperativity in TTR based on structural data is consistent with fluorescence spectroscopy studies (Irace and Edelhoch 1978), showing that quenching of tryptophanyl emission is caused mostly by the first ligand molecule bound to the hTTR tetramer. Similar results have been obtained with molecular dynamics simulations by Wang et al. (Wang et al. 2007). They investigated the influence of different ligands on the conformational flexibility of the TTR channel by exploring their interactions with human TTR complexes with flufenamic acid (FLU) and 10-(m-trifluoromethylphenyl)-phenoxazine-4,6-dicarboxylate (BPD). The FLU complex revealed a pattern change similar to that reported for the NC effect (Neumann et al. 2001). However, BPD exhibited a different pattern of channel alterations, with both binding sites slightly collapsed. This was attributed to the independent (positive) cooperativity of that ligand. These results were also supported by an all-atom molecular dynamics simulation study associated with tetramer dissociation, which showed that only the dimeric form of TTR can undergo initial fibril formation (Sorensen et al. 2007). More recently, molecular dynamics simulations carried out on wild-type monomer and amyloidogenic variants at neutral and low pH revealed that the D strand dissociated from the b-sheet to expose the A strand, in agreement with the structural data (Steward et al. 2008). These calculations further showed that the T119M protective variant suppressed a-sheet formation by changing
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the local conformation at this site, in contrast to the other variants that had a negative cooperative conformational change that was influenced by specific variants.
1.4
Ligand Binding Architecture
Biochemical studies revealed that the two thyroid hormone-binding domains of the TTR tetramer, although chemically equivalent, are biochemically different as indicated by the negative cooperativity observed for binding the second site (Cheng et al. 1977). Also, analysis of structural data for TTR complexes with T4 and its analogues showed that there are three pairs of halogen binding pockets (P1-P10 , P2-P20 , and P3-P30 ) in each binding domain (Fig. 1.4a) (Blake and Oately 1977; Cody et al. 1991). The pair of pockets positioned near the center of the TTR tetramer (P3 and its twofold-related P30 ) is defined by the hydroxyl groups of the side chains of Ser112, Ser115, Ser117, and Thr119. The central P2 and P20 pockets are hydrophobic and are formed between the side chains of Leu17, Thr106, Ala108, Leu110, as well as the methylene carbons of the Lys15 side chain. The outermost P1 and P10 pockets near the channel entrance are defined by Glu54, His56, Lys15 and the hydrophobic groups of the Lys15, Ala108, and Thr106 side chains. Structural data for thyroid hormone-binding complexes with hTTR revealed that T4 binds in a ‘‘forward’’ mode, with its 40 -hydroxyl bound deep within the channel
Fig. 1.4 (a) Superposition of 3,30 -diiodothyronine (red) on that of T4 (green) in hTTR dimer A-A0 (green and violet). The halogen binding pockets P1, P2, P3, and their symmetry-related pairs are highlighted. (b) Superposition of T4 in h (green), h in P21 lattice (cyan), piscine (violet), and rat (yellow) TTR complexes. (c) Superposition of T4A in the forward and reverse orientation for h (green) and rat (cyan) TTR. (d) Flavone EMD21388 in forward and reverse binding mode in domain A for hTTR. (e) Two alternative forward binding modes for EMD21388 in domain A for rTTR. (f) Superposition of EMD21388 (green and cyan) with T4A (violet) in hTTR
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near the tetramer interface and the phenolic ring iodine atoms positioned in the P2 and P30 pockets of the two monomers forming the binding site. The tyrosyl ring iodine atoms occupy the P1 and P10 pockets and the amino side chain that binds near the channel entrance interacts with Lys15 and Glu54. The presence of a twofold symmetry axis along the dimer interface of the P21212 space group found for most hTTR crystal structures requires the ligand to either have twofold symmetry or have a 50% statistical disorder in its binding orientation (Blake and Oately 1977). The orientation of T4 in the second binding site is different, as the phenolic ring iodine atoms occupy both the P3 and P30 pockets, the tyrosyl ring iodine atoms occupy the P1 and P10 pockets, and the two P2 pockets contain water molecules. The observed position of T4 deep in the channel results in the involvement of Ser117 hydroxyl in the binding interactions with the hormone’s 40 -OH group (Fig. 1.4a). The structural data suggest that changes in thyroxine’s position in the binding channel of TTR are coupled with the replacement of water molecules in the binding pockets. This picture is consistent with the presence of strongly bound water molecules stabilizing the TTR tetramer as was shown by the molecular dynamics investigation of TTR binding (Wang et al. 2007; Sorensen et al. 2007). ˚ between phenolic Analysis also revealed a short contact of approximately 3.0 A ring iodines and the Ala109 carbonyl. Similar interactions have been found in the small molecular crystal structures deposited in the Cambridge Structure Database (Cody and Murray-Rust 1984). A survey of TTR structural data revealed significant flexibility in the orientation of many ligands. Comparison of the seven TTR–T4 complexes from human, rat, mouse, and fish TTR revealed that there is variability in the orientation of hormonebinding that depends on the pH of the crystallization conditions and the species of TTR. As illustrated in Fig. 1.4b, the superposition of T4 complexes indicates that the T4 binding mode observed in the fish TTR complex (Eneqvist et al. 2004) differs from that reported for rat TTR and monoclinic human TTR (Wojtczak et al. ˚ closer to the channel entrance 2001a, b). The hormone molecule is bound about 1 A 0 0 and iodine atoms occupy the P2/P2 and P1/P1 pockets. Because of the differences in the orientation of the hormone, no significant interaction between the iodine atoms of T4 and the nucleophilic groups of the protein main chain was observed. The T3 complex of the sea bream TTR (Eneqvist et al. 2004) revealed another binding mode of the hormone. The steric restrictions are smaller than those for T4 and permit the ligand to adopt a slightly different conformation of the T3 ether bridge. The single phenolic iodine atom of T3 is positioned near the channel axis between the pair of P3 pockets, while the hydroxyl group penetrates deep into the P3 pocket and interacts with the main chain polar groups of Leu109 and Leu110. The two tyrosyl iodine atoms are positioned in the P1 and P10 binding pockets. Data for T4 metabolites revealed novel binding orientations. The structure analysis of 3,30 -T2 (3,30 -diiodo-L-thyronine) (Fig. 1.4a) placed the weak binding metabolite deeper in the channel with a shift in orientation that showed the iodine atoms bound in the P3 and P10 pockets (Wojtczak et al. 1992). In the structure of the 30 ,50 -dinitro-N-acetyl-L-thyronine complex, the nitro groups bind in the P3 and P30 pockets, while the 40 -OH forms almost identical H-bonds to Ser117 of two
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monomers constituting each binding site of TTR (Wojtczak et al. 2001a). Comparison of the H-bond network in the P3 pockets of both binding sites as well as those found at the interface between the two sites revealed that ligand binding disrupts the H-bond link between these sites across the tetramer interface (Wojtczak et al. 2001b). This appears to be an element of the mechanism of signal transmission between the two binding sites of TTR and could be coupled with both the molecular mechanism of the tetramer stabilization and negative cooperativity observed in the binding of the second domain. In both cases, T4 or other tightly binding analogues should prevent the destabilization of the TTR tetramer and its conversion into amyloid fibrils. The tightly binding metabolite, tetraiodothyroacetic acid (T4A) (Fig. 1.2), turned out to be unique in its binding. The structures of its complexes with both rat and human TTRs (Muzioł et al. 2001a; Neumann et al. 2005) revealed that it can bind in both the forward and the reverse orientations (Figs. 1.1b, 1.4c). In the forward mode, the position of the ring system with iodine atoms is similar to that observed for T4. It was found that in the forward mode the iodine atoms are positioned in the P3, P20 , P1, and P10 pockets, while the carboxylic group interacts with Lys15 ammonium group in P1 pocket. In the reverse mode, the iodine atoms are positioned in both the P2/P20 and the P1/P10 pockets and form several polar interactions with the main-chain nucleophilic groups of Ala109 and Leu110. In each binding site, the carboxylic group of T4A bound in the reverse mode is positioned deep in the channel in the P3 pocket. It forms polar interactions with the Ser117 residues of two monomers forming the binding site and with Ser115 of the second binding site across the dimer–dimer interface. Such penetration of the interface between two binding sites is unique for T4A and has not been found for any other T4 derivative in TTR. The reverse mode of T4A binding is exceptional, as it is the first report on the ligand bridging two halves of its binding site and simultaneously penetrating the interface between the two sites (Neumann et al. 2005). This ability seems to be coupled with the replacement of the nonplanar alanyl moiety of T4 or its analogues with relatively smaller and flat carboxylic group capable of forming numerous polar interactions with the hydroxyl groups of amino acids present at the interface between the two thyroid hormone-binding sites of TTR. The lack of bulky groups in TTR ligand inhibitors allows penetration of the narrow space between two binding sites at the tetramer center, as found for the first time for T4A (Wojtczak et al. 2001a; Neumann et al. 2005). The network of hydrophobic interactions is formed between each ligand and the surrounding side chains in the central P2 pockets. All these interactions bridge either two monomeric barrels forming each binding site of TTR or even link two dimers to stabilize the tetramer, as found for T4A. Also, the significance of ligand interactions with Glu54 and Lys15 at the channel entrance cannot be overestimated, as there are reports of the D strand destabilization leading to the TTR transformation into amyloid (Peterson et al. 1998; Armen et al. 2004; Laidman et al. 2006). These conclusions constituted the basis for the design of different groups of ligands that should prevent the TTR transformation into amyloid and might be efficient drugs in TTR-related amyloidosis.
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Biochemical data also showed that other agents can be competitive inhibitors of hormone binding and showed that the inotropic bipyridine milrinone has an affinity of 59% that of T4 (Davis et al. 1987). Structural data revealed that milrinone was bound with the cyano substituent positioned deep in the P3 pocket, and the carbonyl group formed the H-bond to the Ser117 side chain at the center of the TTR tetramer (Wojtczak et al. 1993). Another cardiac sparing analogue, SKF94901, was designed with liver-sensitive, cardiac-sparing properties, with the potential to reduce cholesterol. Structural data showed that the 3,5-bromine atoms occupy the same positions as the tyrosyl ring iodines of T4, and the 30 -methyl-pyridazinone group interacts in a hydrophobic pocket with contacts to Thr118 and Thr119 (Cody 2005). Plant flavonoids are another class of molecules that was shown to competitively bind to TTR (Cody 2005). Biochemical data revealed that dibromoaurones and bromoflavones are the strongest competitors for T4 binding to TTR. Structural data revealed that the dibromoaurone binds in the forward mode (Ciszak et al. 1992) while the dibromoflavone EMD21388 binds in both the forward and the reverse modes to both human and rat TTR (Muzioł et al. 2001a, b). As illustrated in Figs. 1.4d–f, in the human structure, EMD21388 binds simultaneously in a forward and reverse mode in domain A, while it was observed only in the forward mode in domain B (Cody 2005). This is in contrast to the observation of two alternative binding positions in the forward mode of the bromoflavone in domain A of rat TTR (Muzioł et al. 2001a). The increasing exposure of the halogenated organic compounds, among them polychlorinated phenols, might affect the TTR transport of T4. The structures of 2,4,6-tribromophenol and pentabromophenol complexes of human TTR revealed that these relatively small ligands bind along the channel axis and not as deep as T4 (Ghosh et al. 2000). Both bromophenol ligands were found to bind exclusively in the reverse mode, similar to that reported for T4A, which means the hydroxyl group oriented towards the channel entrance and interacted with Lys15 ammonium group. The core of both ligands participated in the hydrophobic interactions with Lys15, Leu17, Ala108, and Leu110. The results of the TTR-tribromophenol complex structure determination suggest that the outer P1 pockets are preferred for halogen binding (at least for bromine substituents). Their binding interactions are also assisted by the H-bonds formed between the phenolic OH and Lys15 in the reverse mode of binding. It is well known that the halides bind nonspecifically to proteins, interacting with the positively charged ammonium groups or hydroxyl groups. The binding pockets in the TTR channel have been tested for their ability to bind not only halogenated organic compounds but also iodide and chloride ions that have been found to stabilize the tetramers (Hornberg et al. 2005). Such effects were much less pronounced for chloride binding, despite similarity of binding interactions for both ions. These results had shown that the P1 and P3 pockets of each monomeric barrel bind I or Cl ions. One nonspecific site of halogen binding was also found for each monomer between Lys35 and His90 on the surface of the cylindrical cavity of the TTR tetramer.
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Fig. 1.5 (a) Monomer of TTR highlighting locations of variants (violet) that give rise to amyloid fibril formation. (b) Superposition of the binding orientation of T4A (green), Phenox (gold) of hTTR. (c) Superposition of T4A (yellow), DES (cyan), and DNNAT (gold) in hTTR
1.5
TTR Variants and Amyloid Disease
There are two types of inherited variations in human TTR: those associated with amyloidosis and those associated with hyperthyroxinemia (Cody 1980). TTR amyloidosis, known more commonly as familial amyloidotic polyneuropathy, is an autosomal dominant disorder that results from a mutation in the gene encoding plasma TTR, which results in weaker hormone binding and destabilization of the tetramers. Distinct clinical presentations of the disease have been related to different point mutations (Benson and Kincaid 2007; Westermark et al. 2007). In hyperthyroxinemia, the mutations result in tighter binding of the hormone. To date, more than 100 point mutations at 67 sites within the 127 residue monomer of human TTR have been implicated in FAP disease (Kelly 1998; Hammarstro¨m et al. 2003). As illustrated in Fig. 1.5a, these variants are located throughout the length of the monomer. The crystal structures of 29 variants have been reported for 21 single mutants, six double mutants, and two structures of a triple mutant. Of these, only the T119M and A109T variants are protective, in that they enhance T4 affinity and counter the dissociative effects of V30M, the most prevalent variant found in patients. Structural data for the T4 complex with these variants revealed enhanced monomer–monomer and dimer–dimer contacts and a shift in the water hydrogen bonding network in the binding channel that increases T4 binding (Sebastiao et al. 2001; Steinrauf et al. 1993; Schormann et al. 1996). Data for the V30M variant also revealed a distortion of the b-sheets that causes a distortion of the T4 binding channel that weakens the hormone interactions (Hamilton et al. 1993). The structure of the most amyloidogenic mutant Leu55Pro reveals a disruption of the hydrogen bonds between strands D and A that produces different interface contacts and, as mentioned previously, results in eight monomers that have a different packing arrangement than described for the wild-type human TTR (Fig. 1.3d; Sebastiao et al. 1998; Morais-de-Sa et al. 2006). Structural data for the amyloidogenic variants I84S and I84A were determined at low and neutral pH and revealed significant differences in conformation; at low pH, there was an unwinding
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of the helix and a change in the loop connecting the helix to strand F that was not observed at neutral pH (Pasquato et al. 2007). The highly amyloidogenic triple mutant G53S/E54D/L55S revealed a new conformation with a novel b-slip conformation, which results in a three-residue shift in strand D that places Leu58 at the position occupied by Leu55. This new conformation impacts the binding interactions with retinol-binding protein. This unique interaction shows the TTR helices to pack as a double helix (Eneqvist et al. 2000). Several hypotheses have been proposed based on monomeric or dimeric amyloidogenic intermediates to explain fibril formation from TTR monomers. One model proposes head-to-tail polymerization of monomeric intermediates (Quintas et al. 1997). Another model is based on the formation of linear aggregates of TTR molecules, each linked by a pair of disulfide bonds involving Cys-10 (Blake and Serpell 1996; Gales et al. 2007). Data for the Y114C variant revealed three forms for the mutant, which suggests that TTR aggregation follows two pathways: amyloid fibril formation or disulfide-bonded polymerization that prevents fibril formation (Eneqvist et al. 2002; Karlsson et al. 2005). Another model is based on the data from two engineered amyloidogenic mutants and requires dimers that are associated by antiparallel organization of the F and H strands of the native protein. This model requires the destabilization of the tetramer prior to fibril formation (Klabunde et al. 2000; Adamski-Werner et al. 2004). Yet another model invokes proteolytic cleavage as the initiation step in fibril formation (Pasquato et al. 2007). Although the mechanism of tetramer stabilization is still unclear, it has been shown that ligand binding stabilizes tetramer formation in all variant human TTRs. Therefore, one means of intervention for disease treatment could involve binding of non-thyromimetic analogues that can stabilize the TTR tetramer and possibly delay the onset of fibril formation. To this end, numerous compounds have been screened (Klabunde et al. 2000; Adamski-Werner et al. 2004; Oza et al. 2002; Petrassi et al. 2000, 2005). Structural data for the TTR complexes with flufenamic acid and diflunisal show that they mediate inter-subunit hydrophobic interactions and inter-subunit hydrogen bonds that stabilize the normal tetrameric fold (Klabunde et al. 2000; Adamski-Werner et al. 2004). Data for the TTR complex with another analgesic analogue, VCP-6, that has 628% the affinity of T4, show that the molecule forms strong hydrogen bond interactions of its 2-carboxylate with Lys-15 and with the 3,5-dichloro atoms in symmetric hydrophobic pockets near the tetramer interface (Cody 2005). These results suggest that the strategy of stabilization by strong competitors may prove fruitful. Optimization of the inhibitor molecules was performed with the ring system and the bridging linker (Johnson et al. 2008). One aryl ring usually has a polar substituent, capable of interacting with Ser117 or Thr119 at the tetramer center or with Lys15 and Glu54 in P1 pockets. Several groups of ligands were design to investigate the components of the molecule responsible for efficient inhibition. The important group among those investigated was the nonsteroidal anti-inflammatory drugs (NSAIDs) and their derivatives (Klabunde et al. 2000), which increased the kinetic barrier of TTR tetramer dissociation (Oza et al. 2002). These competitors possessed polar interactions with Lys15 in the P1 pockets, while the hydrophobic core was
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involved in the interactions with amino acids constituting the central P2 pockets. In particular, the commonly used drugs diclofenac and flurbiprofen were competitive based on their ability to form the polar interactions at the channel entrance and fill the P3 pockets with their hydrophobic ring moieties. The carboxylate of diclofenac that binds in the P3 pockets corresponds to the reverse mode of binding found for T4A (Muziol et al. 2001b; Neumann et al. 2005). Diclofenac and diflunisal were selected for optimization as the promising inhibitors of amyloid fibril formation (Adamski-Werner et al. 2004; Oza et al. 2002) against wild-type TTR (senile systemic amyloidoses; SSA) and V30M and L55P (familial amyloid polyneuropathy and familial amyloidotic cardiomyopathy, respectively). Compounds derived from diclofenac showed binding in the reverse mode analogous to that of diclofenac (Oza et al. 2002). The molecular architecture of these compounds is analogous to the structure of T4A. Diflunisal analogs are linear molecules derived from biphenyl. Investigation of these compounds suggested that the para-carboxylic group is required for high affinity, because of its interactions with Lys15 or Ser117 in the forward and reverse binding, respectively, while the hydroxyl is not crucial for inhibition (Oza et al. 2002). The presence of halogen substituents is required, but their exact positions are not vital for the efficiency of inhibition, probably due to the alternative positions able to be adopted in the halogen binding pockets. The series of N-phenyl phenoxazine derivatives has been designed to bind simultaneously to the pockets across the TTR channel axis exploiting its twofold symmetry (Petrassi et al. 2000). These compounds proved to be good FAP inhibitors. The phenoxazine moiety was complementary to the space available at the entrance to the TTR channel (P1 pockets) (Fig. 1.5b). Ligands substituted with two carboxylic groups in positions 4 and 6 of the ring system increased the binding affinity by interacting with the symmetry-related pair of Lys15 residues. The metasubstituted phenyl moieties were positioned in the inner part of the binding site. The m-CF3-substituted phenyl filled the innermost P3 pocket, similar to what was found for flufenamic acid (Klabunde et al. 2000). Promising dibenzofuran inhibitors of TTR amyloidogenesis were also investigated for a kinetic stabilization of the TTR native state (Petrassi et al. 2000; 2005). These compounds presumably bind with the ring system positioned in two P1 pockets analogous to their parent compound investigated earlier (Klabunde et al. 2000). The C1-substituted compounds are excellent inhibitors of wild-type TTR acid-mediated fibril formation and revealed high binding selectivity. Their molecular architecture also includes the two polar groups interacting with the pair of Lys15 at the P1 pockets and one or two fluorine/chlorine substituents of C1-phenyl to be bound in the inner pockets.
1.6
Summary
This review of TTR structures was based on the three-dimensional coordinates reported in the PDB (October 2008) for more than 100 listings representing X-ray crystallography from five species and one solution NMR data from a TTR fragment.
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Structures from human TTR have been studied the most, as they represent direct concern for treatment of genetic disorders resulting in amyloid fibril formation. Despite the highly conserved nature of the ligand binding sites, TTR can bind highly flexible ligands that can adopt alternative conformations as shown by the positional disorder reflecting different occupancies. The observation of ligandinduced conformational changes between the two domains has provided insight into the mechanism of negative cooperativity in the binding affinity of the second ligand. In addition, the fact that a ligand can bind in multiple orientations, for example, forward or reverse, as well as at different depths within the channel, has provided insight into the design of new ligands that target interactions with specific residues, in particular those variants resulting in FAP that could stabilize TTR to prevent amyloid fibril formation.
Acknowledgement
We thank Melda Tugac for her help with the schematic figures.
References Adamski-Werner SL, Palaninathan SK, Sacchettini JC, Kelly JW (2004) Diflunisal analogues stabilize the native state of transthyretin. Potent inhibition of amyloidogenesis. J. Med. Chem. 47:355–374 Armen RS, DeMarco ML, Alonso DOV, Daggett V (2004) Pauling and Cory’s alpha-pleated sheet structure may define the prefibrillar amyloidogenic intermediate in amyloid disease. Proc. Natl. Acad. Sci. USA 101:11622–11627 Benson MD, Kincaid JC (2007) The molecular biology and clinical features of amylobid neuropathy. Muscle Nerve 36:411–423 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res. 28:235–242 Blake CCF, Geisow MJ, Oatley SJ, Rerat C, Rerat B (1978) Structure of prealbumin: secondary, ´˚ tertiary and quaternary interactions determined by fourier refinement at 1.8A . J. Mol. Biol. 121:339–356 Blake CCF, Oately SJ (1977) Protein-DNA and protein-hormone interactions in prealbumin: a model of the thyroid hormone nuclear receptor? Nature 268:115–129 Blake CCF, Serpell L (1996) Synchrotron X-ray studies suggest that the core of the transthyretin fibril is a continuous b-sheet helix. Structure 4:989–998 Cheng S-Y, Pages RA, Saroff HA, Edelhock H, Robbins J (1977) Analysis of thyroid hormone binding to human serum prealbumin by 8-anilinonapthalene-1-sulfonate fluorescence. Biochemistry 16:3707–3713 ˚ resolution of human Ciszak E, Luft J, Cody V (1992) Crystal structure determination at 2.3-A transthyretin-30 ,50 -dibromo-20 ,4,40 ,6- tetrahydroxyaurone complex. Proc. Natl. Acad. Sci. USA. 89:6644–6648 Cody V (1980) Thyroid–hormone interactions: molecular conformation, protein binding, and hormone action. Endocr. Rev. 1:140–166 Cody V (2002) Mechanisms of molecular recognition: crystal structure analysis of human and rat transthyretin inhibitor complexes. Clin. Chem. Lab. Med. 40:1237–1243
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Cody V (2005) Thyroid hormone structure-function relationships. In: Braverman LE and Utiger RD (eds) Werner & Ingbar’s The Thyroid: a fundamental and clinical text, 9th edn. Lippincott Williams & Wilkins, Philadelphia, pp 151–157 Cody V, Murray-Rust P (1984) Iodine . . . X(O,N,S) intermolecular contacts: models of thyroid hormone-protein binding interactions using information from the Cambridge Crystallographic Data Files. J. Mol. Struct. 112:189–199 Cody V, Wojtczak A, Ciszak E, Luft JR (1991) Differences in inhibitor and stubstrate binding in transthyretin crystal complexes. In: Gordon A, Gross J, Hennemann G (eds) Progress in thyroid research, Balema, Rotterdam, pp. 793–796 Davis PJ, Cody V, Davis FB, Warnick PR, Schoenl M, Edwards L (1987) Milrinone, a noniodinated bipyridine, competes with thyroid hormone for binding sites on human serum prealbumin (TBPA). Biochem. Pharmacol. 36:3635–3640 Eneqvist T, Andersson K, Olofsson A, Lundgren E, Sauer-Eriksson AE (2000) The b-slip: a novel concept in transthyretin amyloidosis. Mol. Cell 6:1207–1218 Eneqvist T, Lundberg E, Karlsson A, Huang S, Santos CRA, Power DM, Sauer-Eriksson AE (2004) High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J. Biol. Chem. 279:26411–26416 Eneqvist T, Olofsson A, Ando Y, Miyakawa T, Katsuragi S, Jass J, Lundgren E, Sauer-Eriksson AE (2002) Disulfide-bond formation in the transthyretin mutant Y114C prevents amyloid fibril formation in vivo and in vitro. Biochemistry 41:13143–13151 Ferguson RN, Edeldoch H, Saroff HA, Robbins J (1975) Negative cooperativity in the binding of thyroxine to human serum transthyretin. Biochemistry 14:282–289 Ferrao-Gonzales AD, Souto SO, Silva JL, Foguel D (2000) The preaggregated state of an amyloidogenic protein: hydrostatic pressure converts native transthyretin into the amyloidogenic state. Proc. Natl. Acad. Sci. USA. 97:6445–6450 Folli C, Pasquato N, Ramazzina I, Battistutta R, Zanotti G, Berni R (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett. 555:279–284 Gales L, Saraiva MJ, Damas AM (2007) Structural basis for the protective effect of sulfite against transthyretin amyloid formation. Biochim. Biophys. Acta 1774:59–61 Ghosh M, Meerts IATM, Cook A, Bergman A, Brouwer A, Johnson LN (2000) Structure of human transthyretin complexed with bromophenols: a new mode of binding. Acta Cryst. D56:1085–1095 Gonzalez G (1989) Fluorescent derivative of cysteine-10 reveals thyroxine-dependent conformational modifications in human serum prealbumin. Arch. Biochem. Biophys. 271:200–205 Gonzalez G, Tapia G (1992) Fluorescence study of the thyroxine-dependent conformational changes in human serum transthyretin. FEBS Lett. 297:253–256 Green NS, Palaninathan SK, Sacchettini JC, Kelly JW (2003) Synthesis and characterization of potent bivalent amyloidosis inhibitors that bind prior to transthyretin tetramerization. J. Am. Chem. Soc. 125:13404–13414 Hamilton JA, Steinrauf LK, Braden BC, Liepniks J, Benson MD, Holmgren G, Sandgren O, Steen L (1993) The X-ray crystal structure refinements of normal human transthyretin and the amyloi˚ resolution. J. Biol. Chem. 268:2416–2424 dogenic Val30Met variant to 1.7 A Hammarstro¨m P, Wiseman RL, Powers ET, Kelly JW (2003) Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science 299:713–716 Ho¨rnberg A, Hultdin UW, Olofsson A, Sauer-Eriksson AE (2005) The effect of iodide and chloride on transthyretin structure and stability. Biochemistry 44:9290–9299 Hurshman-Babbes AR, Powers ET, Kelly JW (2008) Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: the relationship between stability and amyloidosis. Biochemistry 47:6969–6984. Irace G, Edelhoch H (1978) Thyroxine-induced conformational changes in prealbumin. Biochemistry 17:5729–5733 Jiang X, Smith CS, Petrassi HM, Hammarstrom P, White JT, Sacchettini JC, Kelly JW (2001) An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured. Biochemistry 40:11442–11452
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Johnson SM, Connelly S, Wilson IA, Kelly JW (2008) Toward optimization of the linker substructure common to transthyretin amyloidogenesis inhibitors using biochemical and structural studies. J. Med. Chem. 51:6348–6358 Johnson SM, Wiseman RL, Sekijima Y, Green NS, Adamski-Werner SL, Kelly JW (2005) Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Acc. Chem. Res. 38:911–921 Karlsson A, Oloffson A, Eneqvist T, Sauer-Eriksson AE (2005) Cys-114-linked dimers of transthyretin are compatible with amyloid formation. Biochemistry 44:13063–13070 Kelly JW (1998) The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr. Opin. Struc. Biol. 8:101–106 Kelly JW, Lansbury PTJ (1994) A chemical approach to elucidate the mechanism of transthyretin and beta-protein amyloid fibril formation. Amyloid 1:186–205 Klabunde T, Petrassi HM, Oza VB, Raman P, Kelly JW, Sacchettini JC (2000) Rational design of potent human transthyretin amyloid disease inhibitors. Nat. Struct. Biol. 7:312–321 Lai Z, Colon W, Kelly JW (1996) The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. Biochemistry 35:6470–6482 Laidman J, Forse GJ, Todd O, Yeates TO (2006) Conformational change and assembly through edge beta strands in transthyretin and other amyloid proteins. Acc. Chem. Res. 39:576–583 Lashuel HA, Lai Z, Kelly JW (1998) Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation. Biochemistry 37:17851–17864 Liu K, Cho HS, Lashuel HA, Kelly JW, Wemmer DE (2000) A glimpse of a possible amyloidogenic intermediate of transthyretin. Nat. Struct. Biol. 7:754–757 Miroy GJ, Lai Z, Lashuel HA, Peterson SA, Strang C, Kelly JW (1996) Inhibiting transthyretin amyloid fibril formation via protein stabilization. Proc. Natl. Acad. Sci. USA. 93:15051–15056 Morais-de-Sa E, Neto-Silva RM, Bereira PJB, Saraiva MJ, Damas AM (2006) The binding of 2,4dinitrophenol to wild-type and amyloidogenic transthyretin. Acta Cryst. D62:512–519 Morais-de-Sa E, Pereira PJB, Saraiva MJ, Damas AM (2004) The crystal structure of transthyretin in complex with diethylstilbestrol. J. Biol. Chem. 279:53483–53490 Muzioł T, Cody V, Luft JR, Pangborn W, Wojtczak A (2001a) Complex of rat transthyretin with ´˚ tetraiodothyroacetic acid refined at 2.1 and 1.8A resolution. Acta Biochim. Polonica 48:877–884 Muzioł T, Cody V, Wojtczak A (2001b) Comparison of binding interactions of dibromoflavonoids with transthyretin. Acta Biochim. Polonica 48:885–892 Neumann P, Cody V, Wojtczak A (2001) Structural basis of negative cooperativity in transthyretin. Acta Biochim. Polonica 48:867–875 Neumann P, Cody V, Wojtczak A (2005) Ligand binding at the transthyretin dimer-dimer ˚ resolution. Acta interface: crystal structure of the transthyretin-T4Ac complex at 2.2 A Cryst. D61:1313–1319 Olofsson A, Ippel HJ, Baranov V, Horstedt P, Wijmenga S, Lundgren E (2001) Capture of a dimeric intermediate during transthyretin amyloid formation. J. Biol. Chem. 276:39592–39599 Olofsson A, Ippel JH, Wijmenga SS, Lundgren E, Ohman A (2004) Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy. J. Biol. Chem. 279:5699–5707 Oza VB, Smith C, Raman P, Koepf EK, Lashuel HA, Petrassi HM, Chiang KP, Powers ET, Sachettinni J, Kelly JW (2002) Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors. J. Med. Chem. 45:321–332 Palaninathan SK, Mohamedmohaideen NN, Snee QC, Kelly JW, Sacchettini JC (2008) Structural insight into pH-induced conformational changes within the native human transthyretin tetramer. J. Mol. Biol. 382:1157–1167 Palhano FL, Leme LP, Gusnardo RG, Foguel D (2009) Trapping the monomer of a non-amyloidogenic variant of transthyretin: exploring its possible use as a therapeutic strategy against transthyretin amyloidogenic diseases. J. Biol. Chem. 284:1443–1453 Pasquato N, Berni R, Folli C, Alfieri B, Cendron L, Zanotti G (2007) Acidic pH-induced conformational changes in amyloidogenic mutant transthyretin. J. Mol. Biol. 366:711–719
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Peterson SA, Klabunde T, Lashuel HA, Purkey H, Sacchettini JC, Kelly JW (1998) Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc. Natl. Acad. Sci. USA. 95:12956–12960 Petrassi HM, Johnson SM, Purkey H, Chiang KP, Walkup T, Jiang X, Powers ET, Kelly JW (2005) Potent and selective structure-based dibenzofuran inhibitors of transthyretin amyloidogenesis: kinetic stabilization of the native state. J. Am. Chem. Soc. 127:6662–6671 Petrassi HM, Klabunde T, Sacchettini JC, Kelly JW (2000) Structure-based design of N-phenyl phenoxazine transthyretin amyloid fibril inhibitors. J. Am. Chem. Soc. 122:2178–2192 Quintas A, Saraiva MJM, Brito RMM (1997) The amyloidogenic potential of transthyretin variants correlates with their tendency to aggregate in solution. FEBS Lett. 418:297–300 Reixach N, Foss TR, Santelli E, Pascual J, Kelly JW, Buxbaum JN (2008) Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic. J. Biol. Chem. 283:2098–2107 Robbins J (1996) Thyroid hormone transport proteins and the physiology of hormone binding. In: Braverman LE, Utidger RD (eds) Werner & Ingbar’s The Thyroid: a fundamental and clinical text, 7th edn. Lippincott Williams & Wilkins, Philadelphia, pp. 96–110 Schormann N, Murrell JR, Benson MD (1996) Tertiary structures of amyloidogenic and non-amyloidogenic transthyretin variants: new model for amyloid fibril formation. Amyloid 5:175–187 Sebastiao MP, Lamzin V, Saraiva MJ, Damas AM (2001) Transthyretin stability as a key factor in amyloidogenesis: X-ray analysis at atomic resolution. J. Mol. Biol. 306:733–744 Sebastiao MP, Saraiva MJ, Damas AM (1998) The crystal structure of amyloidogenic Leu55Pro transthyretin variant reveals a possible pathway for transthyretin polymerization into amyloid fibrils. J. Biol. Chem. 273:24715–24722 Serag AA, Altenbach C, Gingery M, Hubbell WL, Yeates TO (2001) Identification of a subunit interface in transthyretin amyloid fibrils: evidence for self-assembly from oligomeric building blocks. Biochemistry 40:9089–9096 Serag AA, Altenbach C, Gingery M, Hubbell WL, Yeates TO (2002) Arrangement of subunits and ordering of beta-strands in an amyloid sheet. Nat. Struct. Biol. 9:734–739 Steinrauf LK, Hamilton JA, Braden BC, Murrell JR, Bensen MD (1993) X-ray crystal structure of the Ala-109-Thr variant of human transthyretin which produces euthyroid hyperthyroxinemia. J. Biol. Chem. 268:2425–2430 Steward RE, Armen RS, Daggett V (2008) Different disease-causing mutations in transthyretin trigger the same conformational conversion. Prot. Eng. Design Selec. 21:187–195 Sorensen J, Hamelberg D, Schiott B, McCammon JA (2007) Comparative MD analysis of the stability of transthyretin providing insight into the fibrillation mechanism. Biopolymers 86:73–82 Sunde M, Richardson SJ, Chang L, Pettersson TM, Schreiber G, Blake CCF (1996) The Crystal structure of transthyretin from chicken. Eur. J. Biochem. 236:491–499 Wang H, Tang Y, Ming Lei M (2007) Models for binding cooperativities of inhibitors with transthyretin. Arch. Biochem. Biophys. 466:85–97 Westermark P, Benson MD, Buxbaum JN, Cohen AS, Brangione B, Ikeda S-I, Masters CL, Merlini G, Saraiva MJ, Sipe JD (2007) A primer of amyloid nonmenclature. Amyloid 14:179–1834 White JT, Kelly JW (2001) Support for the multigenic hypothesis of amyloidosis: the binding stoichiometry of retinol-binding protein, vitamin A, and thyroid hormone influences transthyretin amyloidogenicity in vitro. Proc. Natl. Acad. Sci. USA 98:13019–13024 ˚ resolution: First report on a Wojtczak A (1997) Crystal structure of rat transthyretin at 2.5 A unique tetrameric structure. Acta Biochim. Polon. 44:505–518 Wojtczak A, Luft JR, Cody V (1992) Mechanism of molecular recognition: Structural aspects of 3, 30 -diiodo-L-thyronine binding to human serum transthyretin. J. Biol. Chem. 267:353–357
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Wojtczak A, Luft JR, Cody V (1993) Structural aspects of inotropic bipyridine binding: crystal ˚ of the human serum transthyretin–milrinone complex. J. Biol. structure determination to 1.9 A Chem. 268:6202–6206 Wojtczak A, Cody V, Luft JR, Pangborn W (1996) Structures of human transthyretin complexed ˚´ resolution. ˚´ resolution and 30 ,50 -dinitro-N-acetyl-L-thyronine at 2.2A with thyroxine at 2.0A Acta Cryst. D52:758–765 Wojtczak A, Cody V, Luft J, Pangborn W (2001a) Structure of rat transthyretin (rTTR) complex ´˚ with thyroxine at 2.5A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Cryst. D57:1061–1070 Wojtczak A, Neumann P, Cody V (2001b) Structure of a new polymorphic monoclinic form ˚´ resolution reveals a mixed complex between unliganded and of human transthyretin at 3A T4-bound tetramers of TTR. Acta Crsyt. B57:957–967
Chapter 2
TTR Synthesis During Development and Evolution: What the Marsupials Revealed Samantha J. Richardson
Abstract Many of our current concepts about biological systems are based on the data from eutherian mammals. Taking the comparative approach gives a broader evolutionary perspective. By analysing the situation also in fish, amphibians, reptiles, birds, monotremes and marsupials, we are forced to consider the differences in physiology (e.g. poikilothermic vs. endothermic, aquatic vs. terrestrial, carnivore vs. herbivore vs. omnivore, modes of reproduction and development, etc.) and we are forced to consider the selection pressures involved. This results in a greater understanding of the biological system in question. In the case of transthyretin (TTR), investigation into tissue specificity of synthesis during both development and evolution revealed that not only are the mammalian TTRs the exception (all other TTRs preferentially bind T3, not T4), but that TTR synthesis appears spatially and temporally where greater thyroid hormone (TH) distribution is required. In the case of TTR evolution, the study of marsupials provided several ‘missing links’, even regarding the structure–function relationships. Thus, we can now see a sequential change in the structure of the N-terminal regions of the TTR subunits, sequential change in affinity of TTRs for THs, and a sequential increase in affinity of TTR for THs from amphibians to mammals. Studying marsupials also allowed us to understand the evolution of TTR synthesis during ontogeny. By elucidating the evolutionary history of a protein in terms of its structure, function and sites of synthesis and timing of gene expression, this allowed us to obtain a comprehensive picture of its biological roles. Keywords Evolution, Eutherians, Marsupials, Monotremes, Birds, Reptiles, Amphibians, Fish, Thyroid hormones, Development, Blood, Cerebrospinal fluid, Liver, Choroid plexus, Gene expression, Albumin, Thyroxine-binding globulin, Amyloid
S.J. Richardson School of Medical Sciences, RMIT University, PO box 71, Bundoora, 3083, Victoria, Australia e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_2, # Springer‐Verlag Berlin Heidelberg 2009
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2.1 2.1.1
S.J. Richardson
Marsupials and Other Mammals Eutherians, Marsupials and Monotremes
Mammals (Class Mammalia) can be divided into three subclasses: Eutheria (often erroneously referred to as ‘placental mammals’), Metatheria (marsupials) and Prototheria (monotremes). In brief, all mammals suckle their young. Eutherian mammals (e.g. humans, mice, bats, whales) are the predominant mammalian subclass and are found on all continents. Marsupials (e.g. kangaroos, koalas, opossums) are born at a very early stage in development compared with other mammals; some females have pouches where the young live for most of their development, and there is no corpus callosum in the brain (only the anterior commissure joins the two hemispheres). Marsupials live in the United States and in Australia. Monotremes (echidnas, platypus and zaglossus) are mammals that lay eggs, and live in Australia.
2.1.2
Evolutionary History of the Marsupials
The mammalian lineage probably diverged from the avian/reptilian lineage around 300 million years ago (MYA). The monotremes probably diverged from the marsupial/eutherian lineage about 200 MYA (van Rheede et al. 2006), and the marsupials probably diverged from the eutherian lineage about 90 MYA (Meredith et al. 2007) (Fig. 2.1). 400
300
200
100
0 Eutherians
+
Diprotodont Marsupials Polyprotodont Marsupials
+ Stem Reptiles LD
++
Monotremes
?LD
Reptiles
LD
Birds
+
400
300
200
100
LD
Amphibians
LD
Fish 0
MYA
Fig. 2.1 Evolutionary/developmental tree for hepatic and choroid plexus synthesis of TTR. Double plus, onset of TTR synthesis in the choroid plexus; LD, hepatic TTR synthesis during development only; Plus, hepatic TTR synthesis throughout life; MYA, million years ago (from Richardson et al. 2005)
2 TTR Synthesis During Development and Evolution: What the Marsupials Revealed
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Today, marsupials are found in North America, South America and Australia. There are many classification systems for marsupials (see Meredith et al. 2007 and references therein). One of the simplest (but not necessarily most accurate) classifications of marsupials is based on dentition. This classification divides marsupials into two Orders: Polyprotodonta and Diprotodonta. Diprotodont marsupials (e.g. kangaroos and wombats) have two procumbent incisors in the lower jaw. The upper jaw has the normal number of incisors facing in the usual direction. Coupled with diastema and well-developed premolars and molars, this tooth arrangement makes them very effective herbivores. Polyprotodont marsupials (e.g. Tasmanian devils and dunnarts) have many teeth on their upper and lower jaws and are carnivores or omnivores. The polyprotodont/diprotodont classification is used in this review because it fits exactly with patterns of hepatic transthyretin (TTR) synthesis in marsupials. According to the fossil record, marsupials originated in Laurasia (now North America) and were polyprotodont (Tyndale-Biscoe 1973). They then migrated to what is now South America and those in the north died out. From South America, some marsupials migrated back to (what is now) North America and others migrated across Gondwanaland. About 45 million years ago, Gondwanaland separated into South America, Antarctica and Australia (Talent 1984). Fossils of marsupials have been found in Antarctica, e.g. on Seymour Island (Woodburne and Zinsmeister 1982), and some marsupials were isolated on the Australian continent after the land masses drifted too far apart for terrestrial animals to traverse. Soon after Gondwanaland broke up, there was a radiation of marsupials in Australia, which included the divergence of diprotodont marsupials from polyprotodont marsupials (Tyndale-Biscoe 1973; Fig. 2.2).
N.A. 1
Fig. 2.2 Migration of marsupials during evolution. Landmasses currently known as North America (N.A.), South America (S. A.), Africa (Afr), India (Ind), Antarctica (Ant) and Australia (Aus) are shown as they were in relation to each other about 150 million years ago. Major marsupial migration patterns are indicated by arrows: (1) North America to South America; (2) South America to North America and South America via Antarctica to Australia; (3) marsupial radiation throughout Australia (from Richardson 2007)
2
Afr
S.A.
Ind Aus Ant 3
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2.1.3
S.J. Richardson
Development of Marsupials
Marsupials are born at a much earlier developmental stage than eutherian mammals. For example, the South American grey short-tailed opossum Monodelphis domestica is born 14 days after conception. At birth, it is comparable to an E14 rat embryo or 6 week human embryo with respect to brain development (Saunders et al. 1989). Many marsupials have a pouch in which the new born live during the time they are attached to the teat. The majority of differentiation and organ development occurs during pouch life (Tyndale-Biscoe and Janssens 1988). At the time of pouch exit, many marsupials are at a similar developmental stage to that of many eutherian species at the time of birth (Setchell 1974; TyndaleBiscoe and Janssens 1988). Thus, much of the development that occurs in utero for eutherians occurs postnatally in marsupials. Therefore, marsupials are an ideal model for developmental studies as the young are easily accessible and the mothers do not need to be sacrificed.
2.2
TTR as a Thyroid Hormone Distributor Protein (THDP)
The major thyroid hormone distributor proteins (THDPs) in human blood are albumin, TTR and thyroxine-binding globulin (TBG). THs are extremely lipophilic, having a partition coefficient of 20,000:1 between lipid and aqueous environments (Dickson et al. 1987). In human blood, 99.97% of T4 and 99.70% of T3 are bound to the THDPs (Mendel et al. 1989). Of these, TBG has the highest affinity for T4 and T3 (1.0 1010 M1 and 4.6 108 M1, respectively), TTR has intermediate affinity (7.0 107 M1 and 1.4 107 M1, respectively) and albumin has the lowest affinity (7.0 105 M1 and 1.0 105 M1, respectively). Together, these three THDPs form a buffering network for free T4 in blood, which could help to protect against hypothyroidism (abnormally low levels of free TH in blood) or hyperthyroidism (abnormally high levels of free TH in blood) (Schreiber and Richardson 1997). The older literature and some modern text books (e.g. Alberts et al. 2002) state that the role of THDPs in blood is to overcome low TH solubility. However, the concentration of free T4 in human blood is about 25 pM and the maximum solubility of T4 in physiological saline is 2.3 mM, that is about 100,000 times the concentration of free T4 (Schreiber and Richardson 1997). Therefore, THDPs must play a role other than to aid the solubility of THs in the blood and cerebrospinal fluid (CSF).
2.2.1
The Role of THDPs in Blood
The function of THDPs in the blood was demonstrated using perfused rat livers. When the livers were perfused with medium containing 125I-T4 in the absence of THDPs, all the 125I-T4 partitioned into the first cells it came into contact with.
2 TTR Synthesis During Development and Evolution: What the Marsupials Revealed
*** Mean of Tunel positive cells per section
untreated PTU treated
15
Fig. 2.3 Absence of TTR in the CSF of adult mice results in a hypothyroid phenotype in the subventricular zone of the brain. Wildtype mice (left bars) and TTR null (TTR KO; right bars) mice were either untreated (open bars) or treated with propylthiouracil (PTU) to induce hypothyroidism (filled bars). Untreated TTR null mice had the same level of apoptosis in neural stem cells and progenitor cells as the hypothyroid wildtype mice. Means S.E.M. ***p < 0.001 (from Richardson et al. 2007)
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10
5
0 Wild–type
TTR KO
When the livers were perfused with medium containing 125I-T4 and THDPs, the 125 I-T4 was evenly distributed throughout the liver lobule and was present in the perfusate (Mendel et al. 1987).
2.2.2
TTR as a THDP in the CSF
The role of TTR as a THDP in the CSF was demonstrated by the analysis of adult TTR null mice. Compared to wild-type mice, TTR null mice were found to have reduced delivery of TH to the stem cells and progenitor cells in the subventricular zone (SVZ) of their brains. The main fate of stem and progenitor cells in the SVZ of adult mammals is apoptosis. The cell cycle of these cells is regulated by TH and apoptosis is reduced during hypothyroidism (Lemkine et al. 2005). The level of apoptosis in the SVZ of TTR null mice was equivalent to that in the SVZ of hypothyroid wild-type mice (Richardson et al. 2007; Fig. 2.3). Thus, the function of THDPs is to ensure an even distribution of TH throughout tissues and to maintain a circulating TH pool of sufficient size in the blood and CSF.
2.2.3
THDPs Differ Between Species: Mammalian TTR is the Exception, not the Rule
In mammals, TTR, albumin and TBG have higher affinity for T4 than T3, and as the concentration of T4 is higher than that of T3 in mammalian blood, T4 is often referred to as the ‘transport form’ of TH. As T3 has higher affinity for the TH
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S.J. Richardson
nuclear receptors than T4 (Sandler et al. 2004), T3 is often referred to as the ‘active form’ of TH. However, fish, amphibians, reptiles and birds do not have TBG in their blood (see below) and TTR is the major THDP in their blood. Moreover, TTR in fish, amphibians, reptiles and birds has higher affinity for T3 than for T4 (see below). Thus, mammalian TTRs are the exception in binding T4 rather than T3! The function of TTR has changed from binding T3 to binding T4. This is one example of the importance of studying species other than mammals in order to understand the function of a protein. This raises the question as to why TTR changed from distributing T3 to distributing T4.
2.3 2.3.1
Sites of TTR Synthesis in Eutherian Mammals Liver
As with all plasma proteins, TTR is synthesised by the liver and secreted into the blood (see Schreiber 1987). In the blood, TTR binds TH and up to two molecules of retinol-binding protein/retinol complex (for more details see chapters ‘The Transthyretin–Retinol-Binding Protein Complex’ and ‘TTR and RBP: Implications in Fish Physiology’). Thus, hepatic TTR is involved in the distribution of THs and retinol throughout the body via the blood.
2.3.2
Choroid Plexus
TTR is synthesised by the choroid plexus (Dickson et al. 1987) epithelial cells (Stauder et al. 1986) and secreted into the CSF (Schreiber et al. 1990). In rats, this TTR is involved in the movement of T4 (but not T3) from the blood into and within the brain (Chanoine et al. 1992; Dickson et al. 1987; Southwell et al. 1993); for review see Richardson (2005). TTR in the CSF and interstitial fluid is involved in the delivery of TH to stem cells and progenitor cells within the brain (Richardson et al. 2007), which require TH for regulation of cell cycle (Lemkine et al. 2005).
2.3.3
Visceral Yolk Sac and Placenta
Synthesis of TTR and retinol-binding protein (RBP) in the visceral yolk sac (VYS) of rats and placenta of humans is involved in the transport of TH and retinol from the maternal circulation to the developing fetus (McKinnon et al. 2005; Sklan and Ross 1987; Soprano et al. 1986). In rats, TTR and RBP are secreted across the basolateral membrane towards the fetal circulation and could be the source of plasma proteins for the fetus prior to functioning of the fetal liver (Thomas et al. 1990).
2 TTR Synthesis During Development and Evolution: What the Marsupials Revealed
2.3.4
29
Retinal and Ciliary Pigment Epithelia
TTR synthesised by the retinal pigment epithelium (RPE) (Cavallaro et al. 1990) is secreted across the apical membrane into the extracellular matrix together with RBP (Ong et al. 1994). This TTR and RBP is involved in the delivery of retinol to Mu¨ller and amacrine cells (Ong et al. 1994), where it is converted to retinal, which is required for photoreceptor function (Bridges et al. 1984). More recently, TTR synthesis by the ciliary pigment epithelium (CPE) was identified as a result of amyloid deposits (Kawaji et al. 2005), but no function for this TTR has been ascribed. Although TTR synthesis has been described in the RPE of several mammalian species, it has been described only in the CPE of rabbits.
2.3.5
Intestine, Pancreas and Meninges
TTR synthesis has been described in human intestines during fetal development (Loughna et al. 1995), but not in the intestine of adult rats (Dickson et al. 1985). TTR synthesis in the islets of Langerhans in the pancreas has been described in several studies (e.g. Kato et al. 1985). More recently, Refai et al. (2005) described a role for TTR in promoting glucose-induced increases in cytoplasmic calcium ion concentration and insulin release in pancreatic beta cells. A role for TTR in protection against beta cell apoptosis was also proposed, having implications for type 1 diabetes. Blay et al. (1993) described extremely low levels of TTR synthesis by the meninges. However, a function for TTR synthesised by the intestine, pancreas and meninges has not yet been proposed.
2.4 2.4.1
Sites of TTR Synthesis During Development in Vertebrates Liver
Fish, amphibians, reptiles and polyprotodont marsupials do not have TTR in their blood as adults (see below). However, during specific stages of development, i.e. when there is a rise in TH levels in blood, the TTR gene is turned on in the liver of these animals. In fish, this is during early development while they are still absorbing the yolk (Funkenstein et al. 1999; Santos and Power 1999) and during smoltification in salmonids (Richardson et al. 2005; Yamauchi et al. 1999). In amphibians, TTR is synthesised by the liver just prior to or at the climax of metamorphosis (Prapunpoj et al. 2000a; Yamauchi et al. 1998), which involves a sharp rise in TH levels in blood. In the only species of reptiles and polyprotodont marsupials investigated, hepatic TTR synthesis was detected during early development at the time of organ
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Table 2.1 Vertebrate species with an additional THDP with higher affinity for TH in blood during development Development Adult Reference (for each species) S. aurata
Fish
O. masou
Amphibians
Reptile
Mammals Marsupials –Polyprotodonta –Diprotodonta
Eutherians –Rodentia
S. salar
TTR
O. tshawytscha
Albumin
X. laevis
TTR
R. catesbeiana
Albumin
C. porosus
TTR
S. crassicaudata M. eugenii
R. norvegicus M. musculus
Albumin
Albumin
Prapunpoj et al. (2000) Yamauchi et al. (1993) Richardson et al. (2005)
Albumin
Albumin
TTR Albumin
Albumin
TBG TTR Albumin
TTR Albumin
TBG
TTR Albumin Modified from Richardson et al. 2005. From Richardson 2007
Funkenstein et al. (1999); Santos et al. (1999) Yamauchi et al. (1999) Richardson et al. (2005) Richardson et al. (2005)
TTR Albumin
Richardson et al. (2005) Richardson et al. (2002); Richardson et al. (2005) Vranckx et al. (1990a) Vrancks et al. (1990b)
maturation (Richardson et al. 2005). TH levels in blood during development have not yet been characterised in these animals. TTR is synthesised in the livers of adult eutherians, diprotodont marsupials and birds (see below). The ontogeny of TTR gene expression in the liver has been studied in eutherians (rat, mouse), birds (chicken, pigeon, quail) and a diprotodont marsupial (tammar wallaby). Hepatic TTR synthesis was identified in all species from the earliest time points investigated (Fung et al. 1988; Richardson et al. 2002; Southwell et al. 1991). Thus, in these animals, hepatic TTR synthesis appears not to be regulated by fluctuations in TH distribution requirements. By contrast, some eutherians and diprotodont marsupials synthesise TBG in times of increased TH distribution requirements (Richardson et al. 2005 and references therein). (Table 2.1).
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2.4.2
31
Onset of Hepatic TTR Synthesis in Juveniles Only Could be Related to the Developmental Surge of Thyroid Hormones in Blood at this Time
In all vertebrates studied to date, there is a characteristic rise in TH levels in blood during critical periods of TH-regulated development. As THs are lipophilic and >99% is bound to THDPs, this increase in TH concentration would require additional TH distribution capacity. This could be the selection pressure for the developmentally regulated hepatic synthesis of TTR in species where TTR is not synthesised by adult liver. In these species, albumin is the only THDP in adult blood. TTR has higher affinity for THs than for albumin, and would contribute to the distribution of elevated TH levels in blood during these crucial stages of TH-regulated development. Investigations into marsupials were fundamental in developing the hypothesis that an augmented THDP network is required at the time of elevated TH levels in blood during development: (1) Polyprotodont marsupials only synthesise TTR in the liver during development, probably during the elevated TH levels in blood (Richardson et al. 2005). (2) Diprotodont marsupials only synthesise TBG in liver when there are elevated levels of TH in the blood, which boosts their capacity for TH distribution (above that of albumin and TTR, which are synthesised in the liver throughout life) (Richardson et al. 2002, 2005).
2.4.3
Choroid Plexus
The choroid plexus has the highest concentration of TTR mRNA per gram tissue weight in the body. The adult rat choroid plexus has 4.4 mg TTR mRNA per gram wet weight tissue compared to only 0.39 mg TTR mRNA per gram wet weight liver (Schreiber et al. 1990). In chickens, the adult choroid plexus has 7.2 mg TTR mRNA per gram wet weight, while the liver has only 0.33 mg TTR mRNA per gram wet weight (Duan et al. 1991). The pattern of TTR synthesis by the choroid plexus during development differs between precocial (relatively independent soon after birth) and altricial (very dependent on their mothers after birth) animals. At the time of birth, the brain of precocial animals (e.g. sheep) is significantly further developed compared with the brains of altricial animals (e.g. rats). Therefore, it was of interest to compare TTR gene expression in the choroid plexus of altricial with precocial animals. In rats (altricial), TTR mRNA was detected in the choroid plexus primordia from E12.5 (i.e. 12.5 days post conception). The proportion of TTR mRNA in total RNA increased 40-fold from E12.5 until birth. The maximum of TTR mRNA as a proportion of total brain mRNA (about 140% level in adults) occurred 2 days before birth, just prior to the period of fastest brain growth. By 8 days after birth, TTR mRNA levels had decreased to adult levels (Fung et al. 1988).
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In sheep (precocial), TTR mRNA was detected in choroid plexus from embryos just a couple of days after gestation. At E40 (40 days post conception), the proportion of TTR mRNA in total mRNA was 34% of that for adult choroid plexus. By E90, this had increased to 70% of the adult value and remained constant throughout the rest of gestation (Tu et al. 1990). In sheep (precocial), the maximal increase in brain weight occurred 50 days before birth (Tu et al. 1990), while in rats (altricial), the maximum increase in brain weight occurred about 9 days after birth (Fung et al. 1988). For both species, the choroid plexus had its maximal growth rate prior to that of the rest of the brain. The critical period of brain development that is dependent on the THs is earlier for precocial animals than for altricial animals (Fisher and Polk 1989). The maximum TTR mRNA level in the choroid plexus of sheep occurs during the first half of gestation, while in rats, it occurs 2 days prior to birth. If TTR is involved in transporting THs across the blood–CSF barrier into the brain and acts as a THDP in the CSF, in each of these species, the timing of the maximal expression of the TTR gene is highly appropriate. Given that (1) THs have a profound effects on brain development; (2) the blood– brain barrier starts to develop as soon as the first blood vessel grows into the brain (Saunders et al. 1999); (3) the choroid plexus develops more rapidly than other parts of the brain; (4) the choroid plexus has an important role in regulating the composition of the CSF; (5) the timing of maximal TTR mRNA in the choroid plexus of altricial and precocial animals is just prior to the maximal growth rate of the brain and (6) TTR null mice have reduced TH levels in the subventricular zone (Richardson et al. 2007), it would follow that the synthesis of TTR from the stage of the choroid plexus primordia would appear to have an important regulatory role in determining the delivery of THs to the brain.
2.4.4
Visceral Yolk Sac and Placenta
TTR synthesis has been documented in both the placenta and VYS of the rat. Levels of TTR mRNA in the VYS were higher than those in the fetal liver throughout gestation. As stated previously, TTR synthesised by the VYS is involved in the transfer of THs from the maternal circulation to fetus, and also provides TTR to the fetal circulation before the fetal liver has reached full TTR synthesis rates. TTRRBP secreted by the VYS facilitates retinol transport to the fetus. More recent papers have confirmed these findings, added quantitative data and implicated specific transcription factors in the regulation of TTR gene expression in rodents (see Richardson 2007). A recent study reported the synthesis of TTR by trophoblasts in 38–40 weeks gestation in human placentas (McKinnon et al. 2005). However, earlier time points, including those where sufficient TH levels are crucial for normal fetal development, particularly during first trimester, have not been reported.
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2.4.5
33
Other Tissues
Scant data are available on TTR or its mRNA levels in other tissues during development. TTR mRNA in the RPE of rats was first detected at E16 and increased gradually until E21. There was a surge in TTR mRNA levels in the RPE at P0 followed by an increase to adult levels by P7. This surge was suggested to be a consequence of an external stimulus response, such as incident light (Mizuno et al. 1992). TTR mRNA has been reported in human fetuses at 12 weeks gestation in the islets of Langerhans (Gray et al. 1985) and in mid-term fetuses in the pancreatic endocrine A-cells (Jacobsson 1989). At 20–23 weeks gestation, over-expression of the TTR gene in the intestine was reported in human trisomy 18 (Edward’s Syndrome) (Loughna et al. 1995). TTR mRNA was also detected at E9 in the rat foregut endoderm (Makover et al. 1989).
2.5
TTR Synthesis During Evolution (Adult Animals)
2.5.1
Liver
2.5.1.1
Albumin is the Oldest THDP in Vertebrates
For a comprehensive analysis, serum from adult individuals from about 150 species was analysed for the presence of THDPs. All species studied were found to have albumin, and in some species (e.g. fish, amphibians, reptiles and some mammals) albumin was the only THDP (e.g. Richardson et al. 1994; Schreiber and Richardson 1997). Therefore, it was concluded that albumin is the phylogenetically oldest THDP in adult vertebrates. 2.5.1.2
No Clear Phylogeny for TBG in Vertebrates
By contrast, TBG-like proteins were detected in serum from several eutherian, marsupial and monotreme species, but no clear phylogeny was apparent (Richardson et al. 1994). Furthermore, many of these TBG-like proteins were not unambiguously identified, and could feasibly include TBG, lipoproteins that bind 125I-T4, or other 125I-T4 binding proteins that migrate as globulins. 2.5.1.3
Hepatic TTR Synthesis in Adult Birds, Diprotodont Marsupials and Eutherians
Birds and eutherians had TTR in addition to albumin. An intriguing situation became apparent among the Australian marsupial species: some had albumin as their only THDP, while others had TTR in addition to albumin. Those that had TTR
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in their serum belonged to the Order Diprotodonta, while those without TTR in their serum belonged to the Order Polyprotodonta (Richardson et al. 1993, 1994; Fig. 2.1). As stated previously, this classification is not currently favoured by taxonomists, but we have used this classification because it fits exactly with the serum TTR data for the 67 species of adult Australian marsupials studied. Thus, it appears that hepatic TTR synthesis remains ‘turned on’ in the adult in the lineages leading to eutherians, birds and diprotodont marsupials, and is ‘turned off’ after development in the lineages leading to polyprotodont marsupials, reptiles, amphibians, fish and possibly monotremes. Although the hypothesis for hepatic TTR synthesis during development is related to the transient increased levels of THs in blood, there are two current hypotheses concerning possible selection pressures for maintaining hepatic TTR synthesis during adult life. 2.5.1.4
Hepatic TTR Synthesis and the Increase in Lipid Pool to Body Mass Ratio: Consider Marsupials
A study by Hulbert and Else (1989) compared many physiological parameters of an adult reptile and an adult eutherian of similar body mass. Amongst other data, they showed that the eutherian had larger sizes of internal organs (including liver, brain, kidney and heart, which are among the metabolically most active), and that these tissues contained about 50% more phospholipid than the corresponding tissues in the reptile. As THs are lipophilic and preferentially partition into the lipid phase rather than the aqueous phase (Dickson et al. 1987; Hillier 1970), the increase in the relative size of the lipid pool could have been a selection pressure for maintaining hepatic TTR synthesis during adult life of eutherians compared with reptiles (Richardson et al. 1994). As TTR has higher affinity for THs than albumin has, the presence of TTR in the blood would augment the pool of circulating THs, thereby counteracting the increased lipid pool (sink) into which THs could partition. Another example that supports this hypothesis is that of the Australian marsupials. The diprotodont marsupials have hepatic TTR synthesis as adults, but the polyprotodont marsupials do not. The digestive tracts of herbivorous (diprotodont) marsupials are longer than those of carnivorous (polyprotodont) marsupials (Hume 1982). The intestines have a substantial lipid content and are the extra-thyroidal tissue with the highest TH content (Nguyen et al. 1993), and it has been suggested that the THDPs may be responsible for the regulation of delivery of THs to the intestines (Distefano et al. 1993). Thus, the diprotodont marsupials with longer digestive tracts have TTR in their blood, while the polyprotodont marsupials with shorter intestines do not. 2.5.1.5
Hepatic TTR Synthesis and Homeothermy: Marsupials Fall between the Cracks
Most adult animals that synthesise TTR in their livers throughout life are homeothermic (maintain their body temperature at or near 37 C by metabolic means).
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TTR was found in serum from all studied species of birds and eutherians, which are known homeotherms. However, TTR was not detected in the serum from adult fish, amphibians, reptiles, which are ectotherms (body temperature is determined by behaviour and the environment) (see Richardson 2007; Fig. 2.1). As THs are involved in the regulation of oxygen consumption, basal metabolic rates and homeothermy, it is conceivable that there is a link between the increase in the size of the most metabolically active organs and increase in the TH distribution capacity in blood (TTR) and homeothermy. Thus, a selection pressure for maintaining hepatic TTR synthesis throughout life could have been to enable appropriate distribution of THs throughout the body to maintain homeothermy. Marsupials are ‘poor homeotherms’ and there has been no evidence that the presence of TTR in marsupial blood is correlated with better control of body temperature.
2.5.1.6
The TTR–RBP Complex: Marsupials Raise a Question
TTR binds many ligands, including naturally occurring compounds, pollutants and non-steroidal anti-inflammatory drugs (see chapter ‘TTR and Endocrine Disruptors’). The only protein ligand known to be bound by TTR is RBP, which itself binds retinol (Kanai et al. 1968). It was suggested that the TTR–RBP/retinol complex (80 kDa) or the retinol/RBP–TTR–RBP/retinol complex (100 kDa) prevented loss of RBP/retinol (21 kDa) through glomerular filtration of the kidneys (Raz and Goodman 1969). This hypothesis may hold true for eutherians, but it is not immediately convincing when considering adult marsupials. Diprotodonta have TTR in their blood while Polyprotodonta do not (Richardson et al. 1994). This raises the questions as to whether there is a difference in glomerular filtration size cut-off in diprotodont marsupials compared with that of polyprotodont marsupials, or if glomerular filtration has a lower molecular weight cut-off in marsupials than in eutherians, or if a plasma protein other than TTR binds to RBP in all marsupials or in Polyprotodonta.
2.5.2
Choroid Plexus
TTR synthesis was not detected in the choroid plexus from fish or amphibians, but was the major protein synthesised and secreted by the choroid plexus from reptiles, birds, monotremes, marsupials and eutherians (see Richardson 2005). It appears that the TTR gene in the choroid plexus was turned on once at the stage of the stemreptiles, the closest common ancestor to reptiles, birds and mammals, but not of amphibians and fish (see Fig. 2.1). The stem-reptiles were the first to develop traces of a cerebral neocortex (Kent 1987), resulting in an increase in brain volume. As THs are lipophilic and readily partition into cell membranes, the increase in brain volume may have been a selection pressure for ‘turning on’ the TTR gene in the choroid plexus. This resulted in TTR assisting movement of THs from the blood
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across the blood–CSF barrier into the brain, and also acting as a THDP in the CSF (Schreiber and Richardson 1997). It is most probable that the stem-reptiles had the TTR gene in their genomes, possibly expressed in the liver during development. A change in the specificity of transcription factors could have been all that was required to activate expression of the TTR gene in the choroid plexus. The major protein synthesised and secreted by the choroid plexus of juvenile and adult amphibians is the lipocalin prostaglandin D synthetase (PGDS) (Achen et al. 1992), also known as beta-trace (Beuckmann et al. 1999) and Cpl1 (Lepperdinger 2000). PGDS is a monomeric 20 kDa protein that belongs to the lipocalin superfamily. Lipocalins have a calyx (cup) structure and are specialized in binding small molecules (Godovac-Zimmermann 1988). This raises the question as to whether PGDS was the functional evolutionary precursor to TTR in the choroid plexus.
2.5.3
Other Tissues
The majority of data in this section is derived from the studies of eutherian mammals. Therefore, to obtain a more global picture, investigations in species from other classes of vertebrates in both juveniles and adults are required. In adult eutherian mammals, TTR synthesis has been reported in the placenta, RPE, CPE, intestine, pancreas and meninges (in addition to the liver and choroid plexus). In adult reptiles, TTR mRNA has been identified in the choroid plexus from adult animals of four species: stumpy-tailed lizards (Tiliqua rugosa) (Achen et al. 1993), red-eared slider turtle (Trachemys scripta), the common snapping turtle (Chelydra serpentine) (Richardson et al. 1997) and in whole eyes and choroid plexus of the salt-water crocodile (Crocodylus porosus) (Prapunpoj et al. 2002). One study of an adult fish species (Sparus aurata, sea bream) detected TTR mRNA by RT-PCR (i.e. higher levels of sensitivity compared to the above mentioned studies) in the liver, intestine, brain, skin, heart, skeletal muscle, kidney, testis, gills and pituitary (Santos and Power 1999). Thus, given the paucity of data from noneutherian species, a generalized picture of TTR synthesis in vertebrate’s tissues other than the liver and choroid plexus is not yet possible.
2.6
Evolution of TTR Structure and Function: Marsupials are a ‘Missing Link’
In earlier studies of 125I-T4 binding to TTRs from various species, it had been noted that chicken TTR appeared to have a lower affinity for T4 than human TTR did (S. Richardson, unpublished observations). However, the amino acids in the TH binding sites are identical in human and chicken TTRs (Duan et al. 1991). As the
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37
N-termini had been shown to be located in the vicinity of the opening to the THbinding channel of TTR (Hamilton et al. 1993), this raised the question as to whether the N-terminal regions of TTR might be affecting the entry of THs into the binding sites. The X-ray crystal structure of chicken TTR was determined (Sunde et al. 1996). It did not show diffraction for the N-terminal regions, implying that the four N-terminal regions were moving freely in solution. However, it did confirm that the TH binding sites were identical in chicken and human TTRs (Sunde et al. 1996). This was surprising, as a difference in binding affinity was expected to be due to a difference in ligand binding site. To further investigate the possibility that the N-terminal regions influenced TH affinity, TTRs were purified from several species of eutherians (short hydrophilic N-terminal regions), birds (long hydrophobic N-terminal regions) and marsupials (intermediate N-terminal regions). The literature revealed that, depending on the method used, Kd values for human TTR and T4 ranged from 0.3 to 128 nM (see Chang et al. 1999). Therefore, a precise, accurate and reproducible method for assaying Kd values of TTRs with THs was developed (Chang et al. 1999). As expected, eutherian TTRs bound T4 with higher affinity than T3. However, avian TTRs bound T3 with higher affinity than T4. Marsupial TTRs had intermediate affinities, but bound T4 with higher affinity than T3 (Chang et al. 1999). However, similar to avian TTRs, reptilian and amphibian TTRs were also found to bind T3 with higher affinity than T4 (Prapunpoj et al. 2002, 2000b; Yamauchi et al. 1998; Table 2.2). Piscine TTRs bind T3 with higher affinity than T4 (Santos and Power 1999; Yamauchi et al. 1999), but specific Kd values are yet to be determined. Thus, shorter hydrophilic N-terminal regions are correlated with higher
Table 2.2 Dissociation constants of T3 and T4 from TTRs from a variety of species Kd T3 (nM) Kd T3/Kd T4 Source of TTR Kd T4 (nM) Eutherians Human 13.6 56.6 4.2 Sheep 11.3 63.5 3.2 Rat 8.0 67.2 8.4 Marsupials Wombat 21.8 97.8 4.5 Possum 15.9 206.1 12.9 Wallaby 13.8 65.3 4.7 Birds Emu 37.4 18.9 0.51 Chicken 28.8 12.3 0.43 Pigeon 25.3 16.1 0.64 Reptile Crocodile 36.7 7.56 0.21 Amphibian Toad 508.0 248.0 0.49 Data are from Chang et al. 1999; Prapunpoj et al. 2000; Prapunpoj et al. 2002. From Richardson 2007
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affinity for T4 binding, and longer more hydrophobic N-terminal regions are correlated with higher affinity for T3 binding. Chimeric TTRs were synthesised, where N-terminal regions from one species were replaced with those from another species (e.g. human/crocodile; Xenopus/ crocodile). Kd values for T3 and T4 were determined (Prapunpoj et al. 2002, 2006). As expected, the N-termini altered the affinity and specificity of T3 and T4 binding to TTRs. More specifically, it appeared that the core of TTR had a major influence on determining the affinity of T3 and the N-terminal regions mainly affected the affinity of T4. (For further details, see chapter ‘Evolution of Transthyretin Gene Structure’.) These data supported the hypothesis that the character of the N-terminal regions of TTR affects the affinities for THs, and revealed that mammalian TTRs are the exception rather than the rule, in that they preferentially bind T4, while TTRs from fish, amphibians, reptiles and birds preferentially bind T3. Marsupial TTRs can be considered as a ‘missing link’ between avian/reptilian TTRs and eutherian TTRs in terms of structure and function. Marsupial TTR N-terminal regions are intermediate in structure (length and hydrophobicity) between those of avian/reptilian TTRs and eutherian TTRs, and provided one of the ‘steps’ in the step-wise change in the position of the intron 1/exon 2 splice site that caused the change in structure from longer and more hydrophobic to shorter and more hydrophilic (see chapter ‘Evolution of Transthyretin Gene Structure’). While marsupial TTRs bind T4 with higher affinity than T3 (similarly to other mammalian TTRs), they have lower affinity for T3 than eutherian, avian or reptilian TTRs and intermediate affinities for T4 between those of eutherian TTRs and avian and reptilian TTRs. This is consistent with the structure of the N-terminal regions of marsupial TTRs being intermediate between those of eutherian and avian/ reptilian TTRs, and the hypothesis that the N-terminal region affects affinity for T4.
2.7
Marsupial Models for Studying TTR Amyloid Formation
The evolutionary changes in TTR are clustered around the N-terminal regions, while the point mutations in human TTR that result in amyloidosis are evenly spread throughout the molecule (see Richardson 2007). In general, the amino acid residues that occur as point mutations in human TTR and result in amyloid formation are not found in other species. One notable exception is amino acid 68. In wild-type human TTR residue 68 is Ile, but if it is mutated to Leu, TTR amyloidosis results. However, Leu is the normal amino acid in this position in several marsupial TTRs (Schreiber and Richardson 1997). These marsupials are apparently without symptoms of amyloidosis. Elucidation of the 3D structure of a marsupial TTR with Leu68 could give us valuable insights into how Leu is accommodated at position 68 in a marsupial TTR, but not in a human TTR. A second example of a marsupial TTR that is of interest for the study of TTR amyloid formation is wallaby TTR. Human TTR is believed to dissociate from
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tetramers into dimers, and from dimers into monomers before forming amyloid fibrils. All amyloidogenic mutations in human TTR destabilise the tetramer (Altland et al. 2007). Under specific conditions that cause the human TTR dimers to dissociate into monomers (Altland et al. 1999), wallaby TTR dimers remain intact (see chapter ‘Histidine 31: The Achilles’ Heel of Human Transthyretin. Microheterogeneity is Not Enough to Understand the Molecular Causes of Amyloidogenicity’). Again, structural data on key residues in wallaby TTR (e.g. His31) could give insight into the mechanism of TTR amyloid formation.
2.8
Conclusions
1. Taking together the data for TTR synthesis in the liver and in the choroid plexus, during both development and evolution, we arrive at the following profile (Fig. 2.1): (a) TTR synthesis only in the liver, only during development: fish, amphibians Hypothesis: hepatic TTR synthesis is required during the developmental surge of THs in blood. (b)TTR synthesis in the liver during development and in the choroid plexus throughout life: reptiles, polyprotodont marsupials and probably monotremes Hypothesis: hepatic TTR synthesis is required during the developmental surge of THs in blood; and the increase in brain volume required TTRmediated transport of T4 across the choroid plexus and TTR as a THDP in the CSF. (c) TTR synthesis in the liver and in the choroid plexus throughout life: birds, diprotodont marsupials, eutherians Hypothesis: hepatic TTR synthesis is required during the developmental surge of THs in blood; increased lipid volume to body mass ratio and/or homeothermy required an additional THDP in blood; and the increase in brain volume required TTR-mediated transport of T4 across the choroid plexus and TTR as a THDP in the CSF. The complex patterns of tissue-specific TTR synthesis imply that TTR synthesis is tightly regulated during development and during evolution. It also implies a multi-factorial set of selection pressures governing its synthesis. 2. Marsupial TTRs are intermediate in structure and in function, between those of reptilian/avian TTRs and eutherian TTRs. They provide evidence of a step-wise shift in the evolution of TTR structure and function. TTR changed from distributing T3 in fish, amphibians, reptiles and birds to distributing T4 in mammals. A possibility for the selection pressure leading to
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this could be an additional level of regulation of TH gene expression by TH. Instead of the ‘active’ form of the hormone being distributed around the body and brain (i.e. T3), in mammals, TTR distributes the precursor form (i.e. T4). This could require tissue-specific or cell-specific regulation of activation by deiodinases from T3 to T4. This could give tighter control over regulation of TH-responsive gene expression. 3. Marsupials should be considered as models for studying TTR amyloid formation. Why Leu68 can be tolerated in marsupial TTRs but causes amyloid formation in human TTR is highly intriguing and should be investigated. Furthermore, features of wallaby TTR that render it more stable than human TTR under specific conditions should be elucidated.
References Achen, M. G., et al. (1993) Transthyretin gene expression in choroid plexus first evolved in reptiles. American Journal of Physiology 265:R982–R989 Achen, M. G., et al. (1992) Protein synthesis at the blood-brain barrier. The major protein secreted by amphibian choroid plexus is a lipocalin. Journal of Biological Chemistry 267:23170–23174 Alberts, B., et al. (2002) Molecular Biology of the Cell. Garland Science, New York Altland, K., et al. (2007) Genetic microheterogeneity of human transthyretin detected by IEF. Electrophoresis 28:2053–2064 Altland, K., et al. (1999) Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients. Electrophoresis 20:1349–1364 Beuckmann, C. T., et al. (1999) Binding of biliverdin, bilirubin, and thyroid hormones to lipocalintype prostaglandin D synthase. Biochemistry 38:8006–8013 Blay, P., et al. (1993) Transthyretin expression in the rat brain: effect of thyroid functional state and role in thyroxine transport. Brain Research 632:114–120 Bridges, C. D., et al. (1984) Visual cycle in the mammalian eye. Retinoid-binding proteins and the distribution of 11-cis retinoids. Vision Research 24:1581–1594 Cavallaro, T., et al. (1990) The retinal pigment epithelium is the unique site of transthyretin synthesis in the rat eye. Investigative Ophthalmology and Visual Science 31:497–501 Chang, L., et al. (1999) Evolution of thyroid hormone binding by transthyretins in birds and mammals. European Journal of Biochemistry 259:534–542 Chanoine, J. P., et al. (1992) Role of transthyretin in the transport of thyroxine from the blood to the choroid plexus, the cerebrospinal fluid, and the brain. Endocrinology 130:933–938 Dickson, P. W., et al., (1985) High prealbumin and transferrin messenger RNA levels in the choroid plexus of rat brain. Biochemical and Biophysical Research Communications 127:890–895 Dickson, P. W., et al. (1987) Thyroxine transport in choroid plexus. Journal of Biological Chemistry 262:13907–13915 Distefano, J. J., et al., (1993) Transfer kinetics of 3,5,30 -triiodothyronine and thyroxine from rat blood to large and small intestines, liver, and kidneys in vivo. Endocrinology 132:1735–1744 Duan, W., et al., (1991) Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin. Implications for the evolution of structure and function of transthyretin in vertebrates. European Journal of Biochemistry 200:679–687 Fisher, D. A., Polk, D. H. (1989) Development of the thyroid. Bailliere’s Clinical Endocrinology and Metabolism 3:627–657
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Fung, W. P., et al., (1988) Structure and expression of the rat transthyretin (prealbumin) gene. Journal of Biological Chemistry 263:480–488 Funkenstein, B., et al., (1999) Cloning of putative piscine (Sparus aurata) transthyretin: developmental expression and tissue distribution. Molecular and Cellular Endocrinology 157:67–73 Godovac-Zimmermann, J. (1988) The structural motif of beta-lactoglobulin and retinol-binding protein: a basic framework for binding and transport of small hydrophobic molecules? Trends in Biochemical Sciences 13:64–66 Gray, H. D., et al., (1985) Sites of prealbumin production in the human fetus using the indirect immunoperoxidase technique. Virchows Archive A: Pathological Anatomy and Histopathology 406:463–473 Hamilton, J. A., et al., (1993) The x-ray crystal structure refinements of normal human transthyretin and the amyloidogenic Val-30 ! Met variant to 1.7-A resolution. Journal of Biological Chemistry 268:2416–2424 Hillier, A. P. (1970). The binding of thyroid hormones to phospholipid membranes. Journal of Physiology 211:585–597 Hulbert, A. J., Else, P. L. (1989) Evolution of mammalian endothermic metabolism – mitochondrial activity and cell composition. American Journal of Physiology 256:R63–R69 Hume, I. D. (1982) Digestive Physiology and Nutrition of Marsupials. Cambridge University Press, Cambridge Jacobsson, B. (1989) In situ localization of transthyretin-mRNA in the adult human liver, choroid plexus and pancreatic islets and in endocrine tumours of the pancreas and gut. Histochemistry 91:299–304 Kanai, M., et al., (1968) Retinol-binding protein: the transport protein for vitamin A in human plasma. Journal of Clinical Investigation 47:2025–2044 Kato, M., et al., (1985) Plasma and cellular retinoid-binding proteins and transthyretin (prealbumin) are all localized in the islets of Langerhans in the rat. Proceedings of the National Academy of Sciences of the United States of America 82:2488–2492 Kawaji, T., et al., (2005) Transthyretin synthesis in rabbit ciliary pigment epithelium. Experimental Eye Research 81:306–312 Kent, G. C. (1987) Comparative anatomy of the vertebrates. Time Mirror/Mosby College, St Louis. Lemkine, G. F., et al. (2005) Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor. The FASEB Journal 19:863–865 Lepperdinger, G. (2000) Amphibian choroid plexus lipocalin, Cpl1. Biochimica et Biophysica Acta 1482:119–126 Loughna, S., et al., (1995) Molecular analysis of the expression of transthyretin in intestine and liver from trisomy 18 fetuses. Human Genetics 95:89–95 Makover, A., et al., (1989) An in situ-hybridization study of the localization of retinol-binding protein and transthyretin messenger RNAs during fetal development in the rat. Differentiation 40:17–25 McKinnon, B., et al., (2005) Synthesis of thyroid hormone binding proteins transthyretin and albumin by human trophoblast. Journal of Clinical Endocrinology and Metabolism 90:6714–6720 Mendel, C. M., et al., (1989) Thyroxine transport and distribution in Nagase Analbuminemic rats. Journal of Clinical Investigation 83:143–148 Mendel, C. M., et al., (1987) Thyroid hormone binding proteins in plasma facilitate uniform distribution of thyroxine within tissues – A perfused rat liver study. Endocrinology 120:1742– 1749 Meredith, R. W., et al., (2007) A phylogeny and time scale for marsupial evolution based on sequences for five nuclear genes. Journal of Mammalian Evolution 15:1–36 Mizuno, R., et al., (1992) Temporal expression of the transthyretin gene in the developing rat eye. Investigative Ophthalmology and Visual Science 33:341–349
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Nguyen, T. T., et al., (1993) Steady-state organ distribution and metabolism of thyroxine and 3,5,30 -triiodothyronine in intestines, liver, kidneys, blood, and residual carcass of the rat invivo. Endocrinology 133:2973–2983 Ong, D. E., et al., (1994) Synthesis and secretion of retinol-binding protein and transthyretin by cultured retinal pigment epithelium. Biochemistry 33:1835–1842 Prapunpoj, P., et al., (2006) Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3. FEBS Journal 273:4013–4023 Prapunpoj, P., et al., (2000a) The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia. Molecular Biology and Evolution 17:1199–1209 Prapunpoj, P., et al., (2002) Crocodile transthyretin: structure, function, and evolution. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 283:R885–R896 Prapunpoj, P., et al., (2000b) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 279:R2026–R2041 Raz, A., Goodman, D. S. (1969) The interaction of thyroxine with human plasma prealbumin and with the prealbumin-retinol-binding protein complex. Journal of Biological Chemistry 244:3230–3237 Refai, E., et al., (2005) Transthyretin constitutes a functional component in pancreatic beta-cell stimulus-secretion coupling. Proceedings of the National Academy of Sciences of the United States of America 102:17020–17025 Richardson, S. J. (2005) Expression of transthyretin in the choroid plexus: Relationship to brain homeostasis of thyroid hormones. In: W. Zheng, A. Chodobski (eds.), The Blood-Cerebrospinal Fluid Barrier. CRC Press, Boca Raton, pp. 275–304 Richardson, S. J. (2007) Cell and molecular biology of transthyretin and thyroid hormones. International Review of Cytology 258:137–193 Richardson, S. J., et al., (2002) Developmental profile of thyroid hormone distributor proteins in a marsupial, the tammar wallaby Macropus eugenii. General and Comparative Endocrinology 125:92–103 Richardson, S. J., et al., (1993) The expression of the transthyretin gene in liver evolved during the radiation of diprotodont marsupials in Australia. General and Comparative Endocrinology 90:177–182 Richardson, S. J., et al., (1994) Evolution of marsupial and other vertebrate thyroxine-binding plasma proteins. American Journal of Physiology 266:R1359–R1370 Richardson, S. J., et al., (1997) Abundant synthesis of transthyretin in the brain, but not in the liver, of turtles. Comparative Biochemistry and Physiology B – Biochemistry and Molecular Biology 117:421–429 Richardson, S. J., et al., (2007) Cell division and apoptosis in the adult neural stem cell niche are differentially affected in transthyretin null mice. Neuroscience Letters 421:234–238 Richardson, S. J., et al., (2005) Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish. American Journal of Physiology – Regulatory Integrative and Comparative Physiology 288:R1264–R1272 Sandler, B., et al., (2004) Thyroxine-thyroid hormone receptor interactions. Journal of Biological Chemistry 279:55801–55808 Santos, C. R. A., Power, D. M. (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinology 140:2430–2433 Saunders, N. R., et al., (1989) Monodelphis domestica (grey short-tailed opossum): an accessible model for studies of early neocortical development. Anatomy and Embryology (Berlin) 180:227–236 Saunders, N. R., et al., (1999) Barrier mechanisms in the brain, II. Immature brain. Clinical and Experimental Pharmacology and Physiology 26:85–91 Schreiber, G. (1987) Synthesis, processing, and secretion of plasma proteins by the liver and other organs and their regulation. In: F. W. Putnam (ed.), The Plasma Proteins. Academic Press, New York, pp. 293–363
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Schreiber, G., et al., (1990) Thyroxine transport from blood to brain via transthyretin synthesis in choroid plexus. American Journal of Physiology 258:R338–R345 Schreiber, G., Richardson, S. J. (1997) The evolution of gene expression, structure and function of transthyretin. Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology 116:137–160 Setchell, P. J. (1974) Development of thermoregulation and thyroid function in marsupial Macropus eugenii (Desmarest). Comparative Biochemistry and Physiology 47:1115–1121 Sklan, D., Ross, A. C. (1987) Synthesis of retinol-binding protein and transthyretin in yolk sac and fetus in the rat. Journal of Nutrition 117:436–442 Soprano, D. R., et al., (1986) Retinol-binding protein and transthyretin mRNA levels in visceral yolk sac and liver during fetal development in the rat. Proceedings of the National Academy of Sciences of the United States of America 83:7330–7334 Southwell, B. R., et al., (1993) Thyroxine transport to the brain: role of protein synthesis by the choroid plexus. Endocrinology 133:2116–2126 Southwell, B. R., et al., (1991) Ontogenesis of transthyretin gene expression in chicken choroid plexus and liver. Comparative Biochemistry and Physiology B: Comparative Biochemistry 100:329–338 Stauder, A. J., et al., (1986) Synthesis of transthyretin (pre-albumin) mRNA in choroid plexus epithelial cells, localized by in situ hybridization in rat brain. Journal of Histochemistry and Cytochemistry 34:949–952 Sunde, M., et al., (1996) The crystal structure of transthyretin from chicken. European Journal of Biochemistry 236:491–499 Talent, J. (1984) Australian biogeography past and present: determinants and implications. In: J. J. Veevers (ed.), Phanerozoic Earth History of Australia. Oxford University Press (Clarendon), London, pp. 57–93 Thomas, T., et al., (1990) Plasma protein synthesis and secretion in the visceral yolk sac of the fetal rat: gene expression, protein synthesis and secretion. Placenta 11:413–430 Tu, G. F., et al., (1990) Expression of the genes for transthyretin, cystatin C and beta A4 amyloid precursor protein in sheep choroid plexus during development. Brain Research Developmental Brain Research 55:203–208 Tyndale-Biscoe, H. (1973). Life of Marsupials. Arnold, London. Tyndale-Biscoe, H. C., Janssens, P. A. (1988) The Delevoping Marsupial: Models for Biomedical Research. Springer-Verlag, Berlin van Rheede, T., et al., (2006) The platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and Therians. Molecular Biology and Evolution 23:587–597 Woodburne, M. O., Zinsmeister, W. J. (1982) Fossil Land Mammal from Antarctica. Science 218:284–286 Yamauchi, K., et al., (1999). Purification and characterization of thyroid-hormone-binding protein from masu salmon serum – A homolog of higher-vertebrate transthyretin. European Journal of Biochemistry 265:944–949 Yamauchi, K., et al., (1998) Structural characteristics of bullfrog (Rana catesbeiana) transthyretin and its cDNA – comparison of its pattern of expression during metamorphosis with that of lipocalin. European Journal of Biochemistry 256:287–296 Yamauchi, K., et al., (1993) Purification and characterization of a 3,5,30 -L-triiodothyroninespecific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinology 132, 2254–2261.
Chapter 3
Evolution of Transthyretin Gene Structure Porntip Prapunpoj
Abstract Transthyretin (TTR) is one of the three major thyroid hormone (TH)binding proteins in plasma and cerebrospinal fluid (CSF) of vertebrates. Binding of TTR to THs is necessary to counteract the loss of these hormones from the vascular and interstitial compartments, resulting from high lipid solubility and from the tendency to partition into and accumulate in the cell membranes. TTR and the other two components of the TH-binding protein system (albumin and thyroxine-binding globulin) function as a network. The loss of one of the proteins could be compensated by increased binding to the remaining plasma proteins. The gene structure of TTR at genomic and mRNA levels from many animals have been characterized over the 20 years since 1985. The structure of the TTR gene from three vertebrate species, that is, human, mouse, and rat, has been characterized, and the entire TTR genomic sequences are highly conserved among these species. They have two intronic open reading frames (ORFs) but neither is productively expressed as an independent polypeptide. The proximal and distal promoters with several regulatory elements necessary for transcription and tissue-specific expression of the TTR gene are located at the 50 -flanking region of the transcription initiation site. The polypeptide coding region of TTR has been studied for a variety of species from fish to human. TTR cDNA from up to 24 vertebrates have been isolated and analyzed to date. The nucleotide sequence and primary structure of TTR are highly conserved, in particular among eutherian, marsupial, bird, reptile, amphibian and fish species. All positions in the central channel, in particular, those involved in the binding interaction with THs have not been altered since at least 400 million years ago. However, mutations of TTR mostly occur in the 50 -terminal region of the TTR gene, which corresponds to the N-terminal segment of TTR polypeptide, during evolution of vertebrates. Shortening and change in hydropathy of the N-terminal regions of TTR occurred and have been demonstrated to influence the binding affinities to THs. The molecular mechanism underlying these phenomena is a series of single
P. Prapunpoj Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_3, # Springer‐Verlag Berlin Heidelberg 2009
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base mutations that leads to a stepwise progressive movement of the 30 splice site, but not the 50 splice site, of the TTR intron 1. It is apparent that a rare molecular mechanism of positive Neo-Darwinian evolution has occurred during the evolution of TTR gene. Keywords cDNA, Evolution, Gene, Structure, Transthyretin
3.1 3.1.1
Genomic Structure of the Transthyretin Gene General
The transthyretin (TTR) gene is located on different chromosomes in different species. While the TTR genes from human and rat are located on chromosome 18 (Wallace et al. 1985; Remmers et al. 1993), those from mouse are located at the C6-D1 region of chromosome 4 (Qiu et al. 1992) and those from Sus scrofa and Bos taurus are on chromosome 6 (Arachibald et al. 1996) and 24 (Larsen et al. 1996), respectively. The TTR genes are varied in length spanning from 6.9 kb (in human) to 7.3 kb (in rat), and are composed of four exons and three introns. Until now, the genomic structure of the TTR gene from only three vertebrate species, that is, human, mouse and rat, has been characterized by nucleotide sequences of the 50 and 30 flanking regions, intron–exon borders, and part of introns being sequenced. The entire sequences of all exons, the 50 proximal region, and the flanking region of the exon–intron borders are highly conserved between human and rodent TTR genes (Tsuzuki et al. 1985; Wakasugi et al. 1985, 1986). The 50 -flanking region of the human and mouse TTR genes and the 30 end region of the first intron of the mouse TTR gene contain sequences homologous to the glucocorticoid receptor (GCR)binding site sequences (Sasaki et al. 1985; Tsuzuki et al. 1985). Moreover, the latter TTR gene also contains an enhancer sequence that is present in the immunoglobulin kappa-chain joining-constant kappa intron (Wakasugi et al. 1986).
3.1.2
Genomic Structure of Human TTR
Each human TTR monomer is encoded by a single-copy gene (Sparkes et al. 1987) that was located by Southern hybridization to the long arm of chromosome 18 (Wallace et al. 1985), in particular at the 18p11.1-q12.3 region, and most likely at the cen-q12.3 (Jinno et al. 1986). The TTR gene spans about 6.9–7.0 kilobases and contains four exons and three introns (Sasaki et al. 1985; Tsuzuki et al. 1985). Exon 1 encodes 23 amino acid residues, which comprises 20 residues of a signal peptide and the first three residues of the mature protein; exons 2, 3, and 4 encode for 44, 45, and 35 amino acid residues, respectively, of the mature protein. The 50 -untranslated region (50 -UTR) (26 bases) and the 30 -untranslated region (30 -UTR) (139 bases) are located in exon 1 and exon 4, respectively. There are three introns of 934, 2,090,
3 Evolution of Transthyretin Gene Structure
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and 3,308 bp, and the GT/AG splicing consensus sequence was identified at all exon/intron borders of the gene (Sasaki et al. 1985). Two independent ORFs, ORF1 and ORF2, with putative regulatory sequences for transcription in the same transcription direction as that of TTR locate in the first and the third intron of the TTR gene (Tsuzuki et al. 1985). The nucleotide sequence of ORF1 is conserved among vertebrate species and codes for polypeptides of 60 and 37 amino acid residues. ORF2 also has two putative initiation codons and codes for a polypeptide of 49 amino acid residues (Tsuzuki et al. 1985). The transcriptional process under these TTR intronic ORFs occurs in liver, pancreas, and brain. However, these transcripts were produced as part of larger transcripts rather than as gene-specific mRNAs (Soares et al. 2003). Two copies of the Alu sequence with opposite directions, which are possibly involved in TTR gene regulation, were found in introns 2 and 3. Several regulatory signals including the Goldberg–Hogness sequence, CAAT boxlike sequence (GTCAAT), two glucocorticoid responsive elements (GRE), and liver-specific nuclear factors HNF-1, 3, 4, and C/E BP locate in the 50 -flanking region of human TTR gene (Sasaki et al. 1985, 1989). The cis-elements that are required for developmental tissue-specificity and for level of expression were also demonstrated at 0.6 to 6.0 kb of the gene (Nagata et al. 1995). The 30 -UTR of the human TTR gene contains polyadenylation signal located at 123 bp downstream from the coding region or at 23 bp upstream from the polyadenylation site (Tsuzuki et al. 1985). In addition, three enhancer core sequences were found in introns and 30 -UTR (Sasaki et al. 1985).
3.1.3
Genomic Structure of TTR from Rodents
The chromosomal structures of the TTR genes from other vertebrates that have been characterized till now are from mouse (Wakasugi et al. 1986) and rat (Fung et al. 1988). These rodent TTR genes are single copies, and the nucleotide sequences of the genes are highly conserved in comparison to human TTR. The genes contain four exons, three introns, TATA-box, and CAAT-box sequences, and the highly conserved regions are in the exons, 50 -flanking region, and 30 end region of intron 1, where only one ORF rather than two as identified in human TTR is present (Wakasugi et al. 1986). Introns of the mouse TTR gene are 0.9, 3.4, and 3.6 kb in length, but have low homology (up to 49% homology) to the corresponding human TTR introns. Two GCR-binding sites and one JkCk enhancer, which are found in immunoglobulin kappa chain gene, are located in the 30 end region of intron 1, similar to that found in the human TTR gene. In addition, two mouse mammary tumor virus (MMTV) GCR-binding site sequences are in the 50 -flanking region (Wakasugi et al. 1986). The proximal promoter (108 to 150 nucleotides) and the distal enhancer element (1.6 to 2.15 kb) of mouse TTR gene possess several binding sites for nuclear factors (Costa et al. 1986). Of the three binding sites present in the enhancer, two are responsible for the cell-specificity and the third is required for the general activation mechanism (Costa et al. 1988). Several nuclear
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factors have been identified so far, including the activation protein one (AP-1), HNF-1 (binding site is 130 to 116 bp), HNF-3 (strong binding site is 106 to 94 bp while weak binding site is 140 to 131 bp), HNF-4 (binding site is 151 to 140 bp), HNF-6 (binding site is 106 to 94 bp), and CCAAT/enhancer binding protein (C/EBP; binding sites are 195 to 177 bp and 135 to 112 bp) (Costa et al. 1989, 1990; Grayson et al. 1989; Samadani and Costa 1996; Samadani et al. 1996). Cooperation between these factors to the TTR promoter and enhancer regions is required for the tissue-specific expression of the gene (Costa and Grayson 1991), such as nuclear factors NHF-1, 3, and 4 and C/EBP are required for TTR gene expression in the hepatocytes (Costa and Grayson 1991). The rat TTR gene spans to 7.3 kb (Fung et al. 1988). The nucleotide sequence of the proximal 50 -flanking region (position 51 to 89 bp), which corresponds to the binding sites for hepatocyte nuclear factors in mouse TTR (Costa et al. 1986), is highly conserved in comparison to human (93% identity) and mouse (97% identity) TTR genes. However, the homology significantly decreases with the nucleotide sequence further upstream from this region (Fung et al. 1988). The rat TTR gene has dual promoters, similar to those human and mouse TTR genes (Motojima and Goto 1989, 1990). A major proximal promoter is mainly used in liver and a minor distal promoter is mainly used in kidney and significantly in brain. Both the TATA-box like sequence (TATATAA sequence; at 30 to 24 bp) and the CAAT-box like sequence (GTCAAT; at 101 to 96 bp) are in the upstream region of the proximal promoter; however, neither is in the distal promoter. An additional transcriptional start site for the specific expression of rat TTR gene in the brain was located at least 65 bp upstream of the normal start site (Motojima and Goto 1989). The AATAAT sequence is present upstream of the region and this region is expressed only in the brain tissue.
3.2
Evolution of the TTR Gene Structure and the Stepwise Shift of the Splicing Mechanism
In the evolution of the TTR gene structure, two types of mutation patterns can be distinguished. The first type consists of random mutations found throughout the polypeptide chain of the human TTR subunit, and more than 80 positions of this mutation type are related to the medical disorder called amyloidosis (see details in chapters on TTR amyloid mutations). The second type consists of mutations occurring near the N-terminal region of the TTR subunit, which lead to changes in length and hydropathy of the N-terminal regions during evolution. Up to date, the primary structures of TTRs from many vertebrate species have been characterized. Although some were obtained directly from the amino acid sequencing, most were derived from nucleotide sequencing of the TTR cDNAs. Up to 24 vertebrate species of TTR cDNAs have been isolated and characterized. These include eutherians (human, hedgehog, shrew, rat, mouse, pig, cow, sheep, and rabbit), metatherians (wallaby, kangaroo, dunnart, opossum, and sugar glider),
3 Evolution of Transthyretin Gene Structure
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bird (chicken), reptiles (crocodile and lizard), amphibians (bullfrog and xenopus), and fishes (sea bream, pacific bluefin tuna, and two genera of lamprey, Petromyzon marinus and Lampetra appendix). Messenger RNA of the vertebrate TTR subunit is generally composed of a 50 -UTR, a coding region, and a 30 -UTR preceding the poly (A) tail, similar to other mRNAs of eukaryotes. The nucleotide sequence in the coding region of these TTR genes is highly conserved, in particular, among eutherians, marsupials, birds, reptiles, and amphibians. This coding region corresponds to TTR presegment, 18–20 amino acid residues, and the functional subunit, which is varied in length depending on animal species, that is, from 125 amino acid residues in hedgehog TTR to 136 amino acid residues in lamprey TTR (Fig. 3.1). Alignment of the TTR primary structures, either obtained directly from amino acid sequencing or derived from the nucleotide sequence of TTR cDNA, from 28 vertebrates including mammals (small eutherians, insectivores, marsupials), birds, reptilians, amphibians, and fish shows that residues in all positions in the central channel of TTR including those involved in the binding interaction with thyroid hormones (THs) (Blake and Oatley 1977; Blake 1981; Wojtczak et al. 1996) are conserved and have not evolved since primitive fish (Fig. 3.1). Homology of the amino acid sequences is high, in particular, among eutherians, marsupials, birds, and reptiles (Table 3.1). On the contrary, most mutations of TTR that occurred during evolution are located only in the 50 -terminal region of the TTR gene that corresponds to the first ten amino acids (based on the amino acid numbering of human TTR) from the N-terminal region of the TTR subunit. A systematic change of the N-terminal region of the TTR subunit from longer and relatively hydrophobic to shorter and more hydrophilic is observed in the evolution of TTRs from eutherians from their common ancestors. Analysis and systemic comparisons between nucleotide sequences of TTR cDNAs and genomic DNAs isolated from eutherian, marsupial, and birds revealed that the shortening of the TTR polypeptide chain at the N-terminus occurred at the border between exon 1 and exon 2 (Aldred et al. 1997). Comparing the nucleotide sequences of genomic DNA and cDNA at the border between exon 1 and exon 2 showed that the position of the 30 splice site of exon 1 and the 50 splice site of intron 1 of all studied TTRs remained unchanged. The consensus recognition sequences for splicing at the 50 end of the intron are typical but well agreed with that found in other vertebrate genes (Moore et al. 1993), that is, the nucleotides U or C rather than A is preferred for the consensus sequence in the exon 1 region flanking the splice site. However, all changes occurred in the region of 30 end of intron 1 and the 50 end of exon 2. Position of the 30 splice site of the TTR intron 1 shifted to the 30 direction in successive steps during evolution of eutherians from their ancestral TTR genes similar to that in fish, leading to a successive shortening of the 50 end of exon 2 (Fig. 3.2). The mechanism underlying this splice site shift possibly occurred by a series of single base mutations that converted codons for amino acids into a new splice recognition site. According to the fact that the splice site recognition sequence at the 30 end of introns is relatively short (Moore et al. 1993), codons for amino acids, in particular, glutamine, CAA, and histidine, CAC or CAU, can be converted into the 30 splice site recognition sequence CAG by a single base change,
P. Prapunpoj
50
-------
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------>
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Human Hedgehog Shrew Rhesus Monkey 1 Rhesus Monkey 2 Pi g Sheep Bovine Rabbit Rat Mouse Tammar Wallaby Grey Kangaroo Brush-tail Possum Sugar Glider Wombat Stripe-faced Dunnart Grey Opossum Virginia Opossum Chicken Pigeon Emu Ostrich Crocodile Lizard Bullfrog Xenopus Sea Bream Lamprey
MASHRLLLLC LAGLVFVSEA GPT ******F*** ******M*** *** ***R****** *****L*T** *** *** *** ***Y****** ********** **A ***F****** ********** S*A ***F**F*** ********** *SV **V ***L**F*** ****I*A*** **G ***L**F*** ********** **A **F*S***** ****A****T AAV **F*S***** ****A****T AAV V **F*S***** ****L***** **V A*E **F*S***** ****L*L*** **V **F*S****G **S*L***D* A*V A*V **F*ST**VF ******L*** A*L A*L A*L A*L **F*SM**VF ******LT** A*L *G*SS***V* ***M*YLT** A*L **YYNT*A*L TIFIFSGAFH RAQ ***FKSF** **L*AI**** A*P *LQPLHC**L ASAVLCNTAP T** *--RF*C**V *VASSSLLCR ADD
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HVFRKAADDT WEPFASGK K**K****E* ******** R**K****E* ********
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*F**V*E**S *F**I*E*** SD**I*N*A* SD**I*N*** AT****N*I* KT**S*H*IS
30
40
50
60
20
10 -strand -----G----
-strand -----F----
-- -helix-
GTGESKC *Q*** ***Q*** *VD**** *ID**** *A***** *A***** *A**P** ***D*** *A***** *A***** EGEH*** ESEH*** *GEH**X *GED*** *GDDX*X *AED*** *AED*** *AEDX*X *SVD*** *SVD*** *SVD**X *SVD**X *SID*** *SID*** *EAD*** *EAD*** *GSDTR* *GVKDS*
*QE**A** *KE**N** *DLIS**T **QIT*** *TQI*T*V *KEV*T*V
-strand ----H---
VVFTANDS ******** ******** ** ** ** ** ******** ******** ******** ******** ******** *******A *******A *******A *******A ***K***A ******** ******** ***S**** *******A *******A ***D*HPE ***M*HGA
GPRRYTIAAL *Q******** *K******** * R* H*** * ** *L*H****** ***H****** *H*S****** * H *H *** ** * *H*H****** *H*H****** *H*H*****Q *H*H*****Q *H*H*****Q *H*H****** *H*H****** *H*H****** *H*H****** *H*H**T*V* *H*Q****V* *HGH**L*L* *HKH*H*PM*
LSPYSYSTTA ********** ********** ********** ********** ********** ***F****** **** **** ** ********** *****F**** *****F**** *****F**** ***F*F**** ********** ***F****** ***F****** ***F****** *T***F**** *T***F*S** ***F**T*** ****FFA*G*
VVTNPKE L*SD*** L*SD*** L *SS***GAL L*SS*** L*SS**A **S**Q* **S**QN **S**QN I*S**T* I*S**T* I*S**T* **S***D **S***D **SD*Q* **SD*Q* **SD*** **SDV**AHV I*SE*HDDL **SSVH* I*VDG*GH
100
110
120
130
Fig. 3.1 Comparison of the amino acid sequences of vertebrate TTRs and derived amino acid sequences from 28 animal species (20 complete and 8 N-terminal sequences) are aligned. Those residues in other species with identical amino acids to those in human TTR are indicated by asterisks. First residues in the mature polypeptide are in bold. Features of secondary structure of human TTR are indicated above the sequences. The numbering of residues is based on human TTR: negative numbers, residues in the presegment; positive numbers, residues of the mature protein; a, b, g, d, e, z, Z, y; and i, positions of residues in noneutherian species. Residues in the core and the central channel of the human TTR subunit, according to Blake et al. (1974, 1978), are singly and doubly underlined, respectively. Gray shading, amino acids in the central channel those are completely conserved; arrows, positions of exon borders. Sources of TTR sequences: Human (Mita et al. 1984; Tsuzuki et al. 1985); Hedgehog and Shrew (Prapunpoj et al. 2000a); Rhesus monkey 1 and Rhesus monkey 2 (van Jaarsveld et al. 1973); Pig (Duan et al. 1995a); Sheep (Tu et al. 1989); Bovine (Schreiber et al. 1993); Rabbit (Sundelin et al. 1985); Rat (Dickson et al. 1985; Duan et al. 1989; Fung et al. 1988); Mouse (Wakasugi et al. 1985, 1986); Tammar Wallaby (Brack et al. 1995); Grey Kangaroo (Aldred et al. 1997); Brush-tail Possum, Sugar Glider, and Wombat (Richardson et al. 1994); Stripe-faced Dunnart and Grey Opossum (Duan et al. 1995b); Virginia Opossum (Richardson et al. 1996); Chicken (Duan et al. 1991); Pigeon, Emu, and Ostrich (Chang et al. 1999); Crocodile (Prapunpoj et al. 2002); Lizard (Achen et al. 1993); Bullfrog (Yamauchi et al. 1993); Xenopus (Prapunpoj et al. 2000b); Sea Bream (Santos and Power 1999); Lamprey: Petromyzon marinus (Manzon et al. 2007)
Sea bream 45 Xenopus 36 46 Lizard 38 51 56 Crocodile 40 51 58 80 Chicken 40 54 60 79 90 Opossum 45 48 58 72 73 74 Dunnart 41 48 59 72 74 75 82 Sugar 45 48 60 71 74 73 84 89 Glider Kangaroo 40 47 58 71 73 73 82 84 92 Wallaby 41 48 59 71 72 73 83 83 92 98 Mouse 42 47 57 70 75 75 74 75 74 72 72 Rat 44 49 61 68 74 75 73 73 74 71 72 93 Sheep 44 46 57 68 71 75 74 74 75 73 74 82 82 Shrew 42 44 53 68 71 71 69 68 70 67 68 78 79 83 Hedgehog 40 45 60 68 73 76 72 73 73 71 72 85 86 88 90 Human 42 45 53 67 71 74 71 71 72 69 70 81 82 85 85 87 Identity (%) of amino acid sequences were calculated using multiple sequence alignment of ClustalW program (Thompson et al. 1994). Accession number for the sequences: Human, AAA73473; Hedgehog, AAF70058; Shrew, O46375; Sheep, P12303; Rat, P02767; Mouse, P07309; Wallaby, P42204; Kangaroo, Q29616; Sugar Glider, P49142; Dunnart, P49143; Opossum, P49141; Chicken, P27731; Crocodile, O55245; Lizard, P30623; Xenopus, BAA77579; Sea Bream, AAC26108; Lamprey, ABI93605
Table 3.1 Comparison of amino acid sequences of TTRs from 17 vertebrate species Lamprey Sea bream Xenopus Lizard Crocodile Chicken Opossum Dunnart Sugar glider Kangaroo Wallaby Mouse Rat Sheep Shrew Hedgehog
52
P. Prapunpoj ---- Presegment----
Mature Protein------> ---------disordered---------------
Human Hedgehog Shrew Rat Mouse Tammar Wallaby Grey Kangaroo Sripe-faced Dunnart Grey Opossum Chicken Crocodile Lizard Xenopus Lamprey
MASHRLLLLC ******F*** ***R****** ***L**F*** ***L**F*** **F*S***** **F*S***** **F*S***** **F*S****G **F*ST**VF **F*SM**VF *G*SS***V* ***FKSF** *--RFLC**V
-20
-10
LAGLVFVSEA ******M*** *****L*T** ****I*A*** ********** ****A****T ****A****T ****L*L*** **S*L***D* ******L*** ******LT** ***M*YLT** **L*AI**** *VASSSLLCR
GPT *** *** **G **A AAV AAV **V A*V A*L A*L A*L A*P ADD
-1 +1 3
V V V G H A D H K S H E S
H H A I S S S S H
H H H H H H H H E
GTGESKC *Q*** ***Q*** *A***** *A***** EGEH*** ESEH*** *AED*** *AED*** *SVD*** *SID*** *SID*** *EAD*** *GVKDS*
- - - - - - - - - +4
10
-strand ----B---
-strand -----A---
PLMVKVLDAV ********** ********** ********** ********** ********** ********** ********S* ********** ********** ********** ********** ********** *****AI*S*
RGSPAINVAV *****V**** Q****V**** *****VD*** *****VD*** **R**V**D* **R**V**D* *****V**D* *****V**N* *****A**** *****A***I **R**TSI** **I**A*LL* Q*K**AG*KL
HVFRKAADDT K**K****E* R**K****E* K**K*T**GS K**K*TSEGS K**K*TEEQ* K**K*TEEQ* K**K*TEEQ* K**K*SEEQ* K**K****G* K**K*TS*GD K*SKMSEEGD N****TNSGK S*MK*QD*AS
20
30
40
Fig. 3.2 Comparison of intron 1 splice sites of TTRs from 14 vertebrate species The sequences of the first 40 amino acid residues including the presegment of TTR from 13 species are aligned with that of human TTR. An arrow indicates the position of the splice site at the exon 1/exon 2 border. Bold letters are the first amino acid at the N-terminus of the mature TTR polypeptide. Sources of splice site data: Human (Tsuzuki et al. 1985); Rat (Fung et al. 1988); Tammar Wallaby, Grey Kangaroo, Stripe-faced Dunnart, Grey Opossum, Chicken, and Lizard (Aldred et al. 1997); Hedgehog, Shrew, Mouse, Crocodile, Xenopus, and Lamprey (as referenced in Fig. 3.1)
that is, from A, C, or U to G. This conversion apparently has occurred at the 50 end of exon 2 of several TTR genes during evolution of eutherians. The histidine codon CAU in marsupials is possibly converted into the consensus recognition sequence CAG by a single base change from U to G, and, at least one other single base substitution, G to U or C, has occurred to inactivate the former 30 splice site recognition sequence operating in marsupial. A valine codon in position g apparently changed into the 30 splice site recognition sequence CAG, by either three base changes or deletion of the valine codon, during the evolution of the TTR gene in marsupial from its ancestor gene similar to that in bird and reptile. In addition, the conversion of the histidine codon CAU (in position e) or the histidine codon CAC (in position i) to the consensus sequence CAG occurred during evolution of bird and reptile TTR from the amphibian-like ancestor and that of amphibian from the fish-like ancestor TTR, respectively (Fig. 3.3b). These changes during evolution of the 50 end of exon 2 of the TTR gene resulted in a progressive movement of the 30 splice site, but not the 50 splice site of intron 1 (Fig. 3.3a), in the 30 direction and successive steps from fish to amphibian, reptilian and avian, marsupial and, finally, eutherian species, resulting in a progressive shortening of the 50 end of exon 2 and increase in hydrophilicity of the N-terminal region of the TTR subunit.
3.3
Influence of Changes in Gene Structure on Function of TTR
The binding affinities of THs to TTRs from several animal species were revealed (Chang et al.1999; Yamauchi et al. 1998 Prapunpoj et al. 2000b, 2002; Morgado et al. 2008). Affinity to T4 increased while that to T3 decreased during evolution of eutherians from their ancestors. The crystal structure of TTR tetramer revealed that
3 Evolution of Transthyretin Gene Structure
a
AG -8
-7
-6
-5
-4
-3
-2
-1
+1
2
53
guaagu g
3
Human
GGA CUG GUA UUU GUG UCU GAG GCU GGC CCU ACG Gly Leu Val Phe Val Ser Glu Ala Gly Pro Thr
Hedgehog
GGA CUG GUA UUU AUG UCU GAA GCU GGC CCU ACG Gly Leu Val Phe Met Ser Glu Ala Gly Pro Thr
**********gau**uau**aaucc*agu*cca*gucuccu*u*uaccucagcug*aguag*gggu
Shrew
GGA CUG GUG CUC GUC ACC GAG GCA GGC CCA ACG Gly Leu Val Leu Val Thr Glu Ala Gly Pro Thr
*********aaau*cugugaagucc*u*c***cc*uuuccugacuag*cga*ccag**gugacu**
gugaguguuucugugacaucccauuccuacauuuaagauucacgcuaaaugaaguagaagugacuc
Rat
GGA CUG AUA UUU GCG UCU GAA GCU GGC CCU GGG Gly Leu Ile Phe Ala Ser Glu Ala Gly Pro Gly
*******g*c******guga**caga*auggcagguagac*uuagau**agg***gccucccu*ca
Mouse
GGA CUG GUA UUU GUG UCU GAA GCU GGC CCC GCG Gly Leu Val Phe Val Ser Glu Ala Gly Pro Ala
*******a*c******gcgau*caga*auggcaguuagac*uuagau**a****a**ugccuu**u
Wallaby
GGC CUG GCG UUU GUG UCU GAG ACU GCA GCU GUG GLY Leu Ala Phe Val Ser Glu Thr Ala Ala Val
**a***u*ggu*acugaaagua**c*u*uug*gguuagc*u**ucau*ucc*uagu*g*caagg*a
Kangaroo
GGC CUG GCG UUU GUG UCU GAG ACU GCA GCU GUG Gly Leu Ala Phe Val Ser Glu Thr Ala Ala Val
**a***u*ggu*acuga*aguag*c*u*uug*gguuagc*ucuca**uucau***g**caa*guaa
Dunnart
GGC CUG GUG UUC CUG UCU GAG GCU GGA CCU GUG Gly Leu Val Phe Leu Ser Glu Ala Gly Pro Val
******u*ggu*acuga*aguag***u*cug*ggu*agccucuca**uccau***u**caa*guaa
Opossum
AGC CUG CUG UUU GUG UCU GAU GCU GCU CCU GUG Ser Leu Leu Phe Val Ser Asp Ala Ala Pro Val
******u*ggu*ccuga*aaguca*u**uuug*gguugacucuca**u**aucccuagcagacaag
Chicken
GGA CUG GUA UUU CUC UCC GAA GCU GCA CCA CUG Gly Leu Val Phe Leu Ser Glu Ala Ala Pro Leu
******c***uaaa
Crocodile
GGA CUG GUA UUU CUG ACU GAA GCU GCC CCA CUG Gly Leu Val Phe Leu Thr Glu Ala Ala Pro Leu
**a**c*c*****cauuuagggggcaga*uu*ga*g**c*guaaaau*uaau*aa**u*agc*uaa
Lizard
GGA AUG GUG UAC CUC ACU GAA GCU GCA CCA CUG Gly Met Val Tyr Leu Thr Glu Ala Ala Pro Leu
**a**ca**g*ac**g**aua**g*ggcua*c*agga*aaa*aaugc*g**cucacac*uga*gca
Xenopus
CUA CUG GCA UUU GUC UCA GAG GCU GCA CCA CCG Leu Leu Ala Phe Val Ser Glu Ala Ala Pro Pro
******a*g*ug*g*gu*c*uguaa*aacuca*gcuuuccuuuuaauug*agcacua*g*cuga*g
Lamprey
GCA UCC AGC UCG CUA CUC UGC AGG GCU GAC GAU Ala Ser Ser Ser Leu Leu Cys Arg Ala Asp Asp
**a****caca*caucgucau***cgucg
b
cncuga u u a
c
polypyrimidine tract -
Human
accacaaagaauaaauccuuucacucugaucaauuuuguuaa
-
-
-
cuu cuc acg ugu
-
-
cuu cuc
cag u uac
-
-
G 4
5
6
7
8
9
10
acc cag
↓GGC ACC GGU GAA UCC AAG UGU Gly Thr Gly Glu Ser Lys Cys
Hedgehog
*aaga**ga**a*****a***g*****ugc********c*g*
*** g** *uu ***
**c ***
**u
g** ***
↓--- --- GGU CAA UCC AAG UGU Gly Gln Ser Lys Cys
Shrew
cuag**cu*c*a*g*auaga*guuaaga*cuugccaau**ug
u*g acu u*u ca*
u*g **u
cuu
ug* ***
↓GGC ACU GGU CAG UCC AAG UGU Gly Thr Gly Gln Ser Lys Cys
Rat
***gg******c**gc**a**u*******c*c*c**cac*g*
*a* *** u*u ***
**c ***
**u
g** ***
↓GGU GCU GGA GAA UCC AAG UGU Gly Ala Gly Glu Ser Lys Cys
Mouse
***gg******c**gc*gg**u*******c*c****cac*g*
*a* u** u*u ***
**c ***
*gu
g** ***
↓GGU GCU GGA GAA UCC AAA UGU Gly Ala Gly Glu Ser Lys Cys
Wallaby
g*acuugccu**cuca*u*cag*u*ucc*a**ccccac*g*g
a** u** ugu **c
*** *ca
**g
↓CAC CAU GAA GGU GAG CAU UCC AAG UGC His His Glu Gly Glu His Ser Lys Cys
Kangaroo
ggacuuggcu**cuca*u*cag*u*ucc*a**ccccac*g*g
a** u** ugu **c
*** *ca
c*g
↓CAC CAU GAA AGU GAG CAU UCC AAG UGC His His Glu Ser Glu His Ser Lys Cys
Dunnart
uaacuugccc*gcuca*u**agcuaucc*au*ccccgc*g*g
u** u** ugu **c
*** *ca
c*g
↓GCC CAU GGA GCU GAG GAU UCC AAA UGC Ala His Gly Ala Glu Asp Ser Lys Cys
Opossum
**ucaugccug*cuca*u*cag*ugucc*a**uccccc*g*g
*** u** ugu **c
*** *ca
c*g
↓AUC CAU GGA GCU GAA GAU UCC AAA UGC Ile His Gly Ala Glu Asp Ser Lys Cys
Chicken
gag*agguuuu*gc*aggc*agugaacagcucc***acaacc
uc* *cu cgu *uc
gc* *ag
↓GUC UCC CAU Val Ser His
GGC UCU GUU GAU UCC AAA UGC Gly Ser Val Asp Ser Lys Cys
Crocodile
gauguggu*****gcc**agcgcg*u*ac*gc***ag*cau*
u** u** cu* cu*
ucc aag
↓GUU UCC CAU Val Ser His
GGU UCU AUU GAU UCC AAA UGC Gly Ser Ile Asp Ser Lys Cys
Lizard
uuu*ug*uc**g****ggg**uuag*a*gcauu****ccccc
*cc *c* cac cu*
a** aag
↓GUU UCA CAU Val Ser His
Xenopus
uaaua*u*au*accuaa***c**gca*c**u*ucca*cc*cu
u** g** u*a *ag
↓GGA CAU GCU Gly His Ala
Lamprey
*u*ggugcucgcu**cagccagcgcg**cg*cccacgcac*g
↓GAC CAC AAG AGC CAC GAA TCC CAC GAG GGA GGC GTC AAG GAC TCG TGC Asp His Lys Ser His Glu Ser His Glu Gly Gly Val Lys Asp Ser Cys
UCC CAU Ser His
GGC UCC AUU GAU UCC AAG UGU Gly Ser Ile Asp Ser Lys Cys GGA GAA GCC GAC UCC AAG UGU Gly Glu Ala Asp Ser Lys Cys
Fig. 3.3 Comparison of nucleotide and amino acid sequences of TTRs at the (a) exon 1/intron 1 border and (b) intron 1/exon 2 border. The 50 and 30 splice sites of intron 1 of TTR precursor mRNAs from 13 vertebrate species are aligned with those from human. The splice sites are indicated by arrows. The consensus recognition sequences for splicing (Moore et al. 1993) are indicated in bold above the position of the splice sites in human TTR precursor mRNA. Nucleotides identical with those in the consensus sequence for the 30 splice site branch point are underlined. Nucleotides in exons are in upper case; those in introns are in lower case. The amino acid residues at the N-terminus, determined by Edman degradation of the mature proteins or of the recombinant TTR, and their corresponding codons are represented in bold. For longer nucleotide sequences of intron 1, see Prapunpoj et al. (2002)
54
P. Prapunpoj
amino acid residues that are components in the binding cavity and involved in the binding with THs (Blake et al. 1974, 1978) are conserved. In addition, no amino acid residues of the N-terminal segment of TTR subunit is involved with or is a part of the binding sites. Instead, the N-terminal segment forms a radon-coil structure with high conformational flexibility (Hamilton et al. 1993; Wilce et al. 1995) on the surface of each TTR monomer, located at the entrance of the tunnel containing the TH-binding sites of the TTR tetramer. The direct involvement of these segments in the binding of THs had been, therefore, neglected. However, an indirect involvement has nevertheless been postulated. The influence on the binding of THs perhaps by interfering with the access of THs to the binding sites in the interior of the TTR molecule was demonstrated using a series of chimeric TTRs. By using the heterologous expression system of Pichia pastoris, a methylotrophic yeast, the recombinant native TTRs and the chimeric TTRs in which the N-terminal region was altered or truncated were successfully synthesized (Prapunpoj et al. 2002, 2006). Binding affinities to T4 and T3 of the recombinant TTRs including two native TTRs, that is, from human and saltwater crocodile (Crocodylus porosus), and three C. porosus chimeric TTRs, that is, one with the N-terminal sequence replaced by that of Xenopus laevis TTR (xeno/crocTTR), one with the N-terminal sequence replaced by that of human TTR (human/crocTTR), and the other with the N-terminal segment removed (truncated crocTTR), were determined using a highly reproducible and sensitive method (Chang et al. 1999). Changes in the affinities to T3 and T4 of C. porosus TTR were clearly observed when the nucleotide sequence at the 50 -terminal region of the TTR gene was either altered or truncated (Prapunpoj et al. 2006). The Kd values for T3 and T4 of the recombinant human TTR were 53.26 3.97 and 19.73 0.13 nM, respectively, while those of the recombinant C. porosus TTR were 33.03 0.42 and 55.69 7.69 nM, respectively (Prapunpoj et al. 2002). However, the affinity of human/ crocTTR for T4 (Kd was 22.75 1.89 nM) was higher than that of both human and C. porosus TTRs for T4, but the affinity to T3 (Kd was 5.40 0.25 nM) was similar for C. porosus TTR, which led to a Kd T3/T4 ratio of 0.24. In addition, the truncated crocTTR had a similar affinity for both T3 (Kd was 57.78 5.65 nM) and T4 (Kd was 59.72 3.38 nM), with a Kd T3/T4 ratio of 0.97. These led to the postulation that the N-terminal region has a role in determining the affinities of T3 and T4 to TTR. This hypothesis has been further confirmed by experiments with fish TTR. The affinity for T4 of the sea bream TTR decreased when the first six amino acid residues were removed (Morgado et al. 2008), suggesting a possibility that some residues in the N-terminal segment influence the binding to T4.
3.4
Conclusion
THs are involved in many regulatory aspects of metabolism in vertebrates. Because of its high lipid solubility, binding to TH-binding proteins is a necessity to prevent accumulation of THs in membranes and to ensure the appropriate extracellular and
3 Evolution of Transthyretin Gene Structure
55
intracellular distribution of the hormones. One of the most important and widely known functions of TTR, which acts in a network with other plasma TH-binding proteins, is to ensure an even distribution of THs (synthesized in and secreted from the thyroid gland) to target tissues throughout the body. Among vertebrates, the requirements of THs (in particular, the biologically active form, T3) are varied depending on many factors, including complexity of organs and a particular period of life, for example, during the developmental period, that requires higher concentrations of the hormones circulating in the blood. Differences in the action of THs are principally regulated via the conversion of the more biologically active T3 from the less active T4 by the deiodinases in the specific target tissues. This could also be achieved via influencing distribution of THs during transport in the blood-stream. Although the basic molecular structures of TTRs at both the genomic and the protein levels in all studied vertebrates are similar, for example, all TTR genes comprise four exons and three introns, TTR monomers are rich in b-pleated sheet structure and four monomers form the tetrameric protein. The affinities in binding to T3 and T4 differ quite dramatically during evolution of the vertebrates, that is, the affinity to T4 increased while the affinity to T3 decreased during the evolution of mammalian TTRs from common ancestoral TTRs. These changes in function are influenced by the N-terminal region of the TTR subunit that changed from longer and relatively hydrophobic to shorter and more hydrophilic during evolution. The stepwise shift of the 30 splice site of the TTR intron 1 in the 30 direction has been proven to be the underlying mechanism that led to the systemic change of the N-terminal region of each TTR subunit. The organization of the genome into exons and introns could speed evolution, as changes in one or a few bases occurring near splice sites could result in the deletion or addition of whole sequences of amino acids (Gilbert 1978). Therefore, with the splicing mechanism, novel or functionally improved proteins could be generated during evolution. The single base mutations near splicing sites that can lead to stepwise directional gradual changes in a protein have been demonstrated in TTR gene. It is most likely that during evolution, selection pressure has operated on the length and composition of the 50 end of exon 2 of the TTR gene, leading to the shorter and more hydrophilic N-termini during the evolution of eutherian TTRs from their ancestors. Such shifts could produce the stepwise changes in the functional properties of TTRs, in particular, the changing affinities observed for THs. This stepwise ‘‘improvement’’ of protein function could be the point of operation of selection pressure and it is an example of a molecular mechanism of positive Neo-Darwinian evolution.
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Aldred AR, Prapunpoj P, Schreiber G (1997) Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 39 splice site of intron 1). Eur J Biochem 246:401–409. Arachibald AL, Couperwhite S, Jianq ZH (1996) The porcine TTR locus maps to chromosome 6q. Anim Genet 27:351–353. Blake CCF, Oatley SJ (1977) Protein-DNA and protein-hormone interactions in prealbumin: a model of the thyroid hormone nuclear receptor? Nature 268:115–120. Blake CCF, Geisow MJ, Oatley SJ, Re´rat B, Re´rat C (1978) Structure of prealbumin: secondary, ˚ . J Mol Biol tertiary and quaternary interactions determined by Fourier refinement at 1.8 A 121:339–356. Blake CC (1981) Prealbumin and the thyroid hormone nuclear receptor. Proc R Soc Lond B Biol Sci 211:413–431. Brack CM, Duan W, Hulbert AJ, Schreiber G (1995) Wallaby transthyretin. Comp Biochem Physiol 110B:523–529. Blake CCF, Geisow MJ, Swan ID, Rrat C, Rrat B (1974) Structure of human plasma prealbumin at 2.5 A resolution. A preliminary report on the polypeptide chain conformation, quaternary structure and thyroxine binding. J Mol Biol 88:1–12. Chang L, Munro SLA, Richardson SJ, Schreiber G (1999) Evolution of thyroid hormone binding by transthyretins in birds and mammals. Eur J Biochem 259:534–542. Costa RH, Grayson DR (1991) Site-directed mutagenesis of hepatocyte nuclear factor (HNF) binding sites in the mouse transthyretin (TTR) promoter reveal synergistic interactions with its enhancer region. Nucleic Acids Res 19:4139–4145. Costa RH, Lai E, Darnell JE (1986) Transcriptional control of the mouse prealbumin (transthyretin) gene: Both promoter sequences and a distal enhancer are cell specific. Mol Cell Biol 6:4697–4708. Costa RH, Lai E, Grayson DR, Darnell JE (1988) The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor (s) at two other sites. Mol Cell Biol 8(1):81–90. Costa RH, Grayson DR, Darnell JE (1989) Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and a1-antitrypsin genes. Mol Cell Biol 9:1415–1425. Costa RH, van Dyke TA, Yan C, Kuo F, Darnell JE (1990) Similarities in transthyretin gene expression and differences in transcription factors: live rand yolk sac compared to choroid plexus. Proc Natl Acad Sci USA 87:6589–6593. Dickson PW, Howlett GJ, Schreiber G (1985) Rat transthyretin (prealbumin): molecular cloning, nucleotide sequence, and gene expression in liver and brain. J Biol Chem 260:8214–8219. Duan W, Cole T, Schreiber G (1989) Cloning and nucleotide sequencing of transthyretin (prealbumin) cDNA from rat choroids plexus and liver. Nucleic Acids Res 17:3979. Duan W, Achen MG, Richardson SJ, Lawrence MC, Wettenhall REH, Jaworowski A, Schreiber G (1991) Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin. Implications for the evolution of structure and function of transthyretin in vertebrates. Eur J Biochem 200:679–687. Duan W, Richardson SJ, Ko¨hrle J, Chang L, Southwell BR, Harms PJ, Brack CM, Pettersson TM, Schreiber G (1995a) Binding of thyroxine to pig transthyretin, its cDNA structure, and other properties. Eur J Biochem 230:977–896. Duan W, Richardson SJ, Babon JJ, Heyes RJ, Southwell BR, Harms PJ, Wettenhall REH, Dziegielewska KM, Selwood L, Bradley AJ, Brack CM, Schreiber G (1995b) Evolution transthyretin in marsupials. Eur J Biochem 227:396–406. Fung WP, Thomas T, Dickson PW, Aldred AR, Milland J, Dziadek M, Power B, Hudson P, Schreiber G (1988) Structure and expression of the rat transthyretin (prealbumin) gene. J Biol Chem 263:480–488. Gilbert W (1978) Why genes in pieces? Nature 27:501. Grayson DR, Costa RH, Darnell JE (1989) Regulation of hepatocyte-specific gene expression. Ann NY Acad Sci 557:243–256.
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Hamilton JA, Steinrauf LK, Braden BC, Liepnieks JJ, Benson MD, Holmgren G, Sandgren O, Steen L (1993) The X-ray crystal structure refinements of normal human transthyretin and the ˚ resolution. J Biol Chem 268:2416–2424. amyloidogenic Val30! Met variant to 1.7 A Jinno Y, Matsumoto T, Kamei T, Kondoh T, Maeda S, Araki S, Shimada K, Nukawa N (1986) Localization of the human prealbumin gene to 18p11.1-q12.3 by gene dose effect study of southern blot hybridization. Jpn J Human Genet 31:243–248. Larsen NJ, Womack JE, Kirkpatrick BW (1996) Seven genes from human chromosome 18 map to chromosome 24 in the bovine. Cytogenet Cell Genet 73:184–188. Manzon RG, Neuls TM, Manzon LA (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen Comp Endocrinol 151:55–65. Mita S, Maeda S, Shimada K, Araki S (1984) Cloning and sequence analysis of cDNA for human prealbumin. Biochem Biophys Res Commun 124:558–564. Moore MJ, Query CC, Sharp PA (1993) Splicing of precursors to mRNA by the spliceosome. In: Gesteland RF, Atkins JF (eds) The RNA World. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp 303–357. Morgado I, Melo EP, Lundberg E, Estrela NL, Sauer-Eriksson AE, Power DM (2008) Hormone affinity and fibril formation of piscine transthyretin: The role of the N-terminal. Mol Cell Endocrinol 295(1–2):48–58. Motojima K, Goto S (1989) Brain-specific expression of transthyretin mRNA as revealed by cDNA cloning from brain. FEBS Lett 258:103–105. Motojima K, Goto S (1990) Dual promoters and tissue-specific expression of rat transthyretin gene. Biochem Biophys Res Commun 173:323–330. Nagata Y, Tashiro F, Yi S, Murakama T, Maeda S, Takahashi K (1995) A 6-kb upstream region of the human transthyretin gene can direct development, tissue-specific, and quantitatively normal expression in transgenic mouse. J Biochem 117:169–175. Prapunpoj P, Richardson SJ, Fumagalli L, Schreiber G (2000a) The evolution of the thyroid hormone distributor protein transthyretin in the order Insectivora, class Mammalia. Mol Biol Evol 17:1199–1209. Prapunpoj P, Yamauchi K, Nishiyama N, Richardson SJ, Schreiber G (2000b) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol 279:R2026–R2041. Prapunpoj P, Richardson SJ, Schreiber G (2002) Crocodile transthyretin: structure, function, and evolution. Am J Physiol 283:R885–R896. Prapunpoj P, Leelawatwatana L, Schreiber G, Richardson SJ (2006) Change in structure of the Nterminal region of transthyretin produces change in affinity of transthyretin to T4 and T3. FEBS J 273:4013–4023. Qiu H, Shimada K, Cheng Z (1992) Chromosomal localization of the mouse prealbumin gene (Ttr) by in situ hybridization. Cytogenet Cell Genet 61:186–188. Remmers EF, Goldmuntz EA, Zha H, Crofford LJ, Cash JM, Mathern P, Du Y, Wilder RL (1993) Linkage map of seven polymorphic markers on rat chromosome 18. Mamm Genome 4:265–270. Richardson SJ, Bradley AJ, Duan W, Wettenhall REH, Harms PJ, Babon JJ, Southwell BR, Nicol S, Donnellan SC, Schreiber G (1994) Evolution of marsupial and other vertebrate thyroxinebinding plasma proteins. Am J Physiol Regulat Integr Comp Physiol 266:R1359–R1360. Richardson SJ, Wettenhall REH, Schreiber G (1996). Evolution of transthyretin gene expression in the liver of Didelphis virginiana and other American marsupials. Endocrinology 137:3507–3512. Samadani U, Costa RH (1996) The transcriptional activator hepatocyte nuclear factor 6 regulates liver gene expression. Mol Cell Biol 16:6273–6284. Samadani U, Qian X, Costa RH (1996) Indentification of a transthyretin enhancer site that selectively binds the hepatocyte nuclear factor-3 beta isoform. Gene Expr 6:23–33. Santos CRA, Power DM (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinology 140:2430–2433.
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Sasaki H, Yoshioka N, Takagi Y, Sasaki Y (1985) Structure of the chromosomal gene for human serum prealbumin. Gene 37:191–197. Sasaki Y, Yoshioka K, Tanahashi H, Furuya H, Sasaki H (1989) Human transthyretin (prealbumin) gene and molecular genetics of familial amyloidotic polyneuropathy. Mol Biol Med 6:161–168. Schreiber G, Pettersson TM, Southwell BR, Aldred AR, Harms PJ, Richardson SJ, Wettenhall REH, Duan W, Nicol SC (1993) Transthyretin expression evolved more recently in liver than in brain. Comp Biochem Physiol 105B:317–325. Soares ML, Centola M, Chae J, Saraiva MJ, Kastner DL (2003) Human transthyretin intronic open reading frames are not independently expressed in vivo or part of functional transcripts. Biochim Biophys Acta 1626:65–74. Sparkes RS, Sasaki H, Mohandas T, Yoshioka K, Klisak I, Sakaki Y, Heinzmann C, Simon M (1987) Assignment of the prealbumin (PALB) gene (familial amyloidotic polyneuropathy) to human chromosome region 18q11.2-q12.1. Hum Genet 75:151–154. Sundelin J, Melhus H, Das S, Eriksson U, Link P, Tra¨gargh L, Peterson PA, Rask L (1985) The primary structure of rabbit and rat prealbumin and a comparison with the tertiary structure of human prealbumin. J Biol Chem 260:6481–6487. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. Tsuzuki T, Mita S, Maeda S, Araki S, Shimada K (1985) Structure of the human prealbumin gene. J Biol Chem 260:12224–12227. Tu G-F, Cole T, Duan W, Schreiber G (1989) The nucleotide sequence of transthyretin cDNA isolated from a sheep choroid plexus cDNA library. Nucleic Acids Res 17:6384. van Jaarsveld PP, Edelhoch H, Goodman DS, Robbins J (1973) The interaction of human plasma retinol-binding protein and prealbumin. J Biol Chem 248:4698–4705. Wakasugi S, Maeda S, Shimada K, Nakashima H, Migita S (1985) Structure comparisons between mouse and human prealbumin. J Biochem (Tokyo) 98:1707–1714. Wakasugi S, Maeda S, Shimada K (1986) Structure and expression of the mouse prealbumin gene. J Biochem (Tokyo) 100:49–58. Wallace MR, Naylor SL, Kluve-Beckerman B, Long GL, McDonald L, Shows TB, Benson MD (1985) Localization of the human prealbumin gene to chromosome 18. Biochem Biophys Res Comm 129:753–758. Wilce JA, Craik DJ, Ede N, Jackson DC, Schreiber G (1995) 1H NMR studies of peptide fragments from the N-terminus of chicken and human transthyretin. Biochem Mol Biol Int 36:1153–1159. Wojtczak A, Cody V, Luft JR, Paugborn W (1996) Structure of human transthyretin complexed ˚ resolution. ˚ resolution and 30 , 50 -dinitro-N-acetyl-L-thyronine at 2.2 A with thyroxine at 2.0 A Acta Crystallogr D52:758–765. Yamauchi K, Kasahara T, Hayashi H, Horiuchi R (1993) Purification and characterization of a 3,5,30 -L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinology 132:2254–2261. Yamauchi K, Takeuchi H, Overall M, Dziadek M, Munro SLA, Schreiber G (1998) Structural characteristics of bullfrog (Rana catesbeiana) transthyretin and its cDNA: comparison of its pattern of expression during metamorphosis with that of lipocalin. Eur J Biochem 256:287–296.
Chapter 4
Evolutionary Insights from Fish Transthyretin Deborah M. Power, Isabel Morgado, and Joa˜o C.R. Cardoso
Abstract Thyroid hormones (THs) synthesised and released by the thyroid follicles in fish circulate bound to TH distributor proteins (THDPs). Transthyretin (TTR) is a tetrameric THDP with a conserved structure in vertebrates and functions as a high affinity T4 binder in tetrapods and as a dual T3/T4 binder in fish. The difference in TH affinity appears to be linked to the longer N-terminal region in piscine TTR. Relatively few studies exist on gene regulation in fish, but those that do indicate that it may be similar to those described in mammals. Recently, a family of transthyretin-like proteins was identified and shown to correspond to the enzyme 5-hydroxyisourate hydrolase (HIUase) of purine metabolism in zebra fish. A combination of in silico and laboratory-based approaches indicate that TTR has a widespread distribution but is most abundant in liver, while HIUase expression seems to be mainly in the liver. Gene transcripts encoding TTR and HIUase genes are described in representatives of the orders Cypriniformes, Salmoniformes, Pleuronectiformes, Perciformes, Tetraodontiformes, Beloniformes, Gasterosteiformes, Gadiformes and Petromyzontiformes. The evolution of TTR and HIUase genes in fish is discussed. Keywords Evolution, Gene structure, Gene linkage, Regulation, Tissue distribution
Abbreviations RBP THs
Retinol binding protein Thyroid hormones
Deborah M. Power (*) Comparative and Molecular Endocrinology Group, Centre for Marine Sciences (CCMAR), Universidade do Algarve, Campus do Gambelas, 8005-139 Faro, Portugal e-mail: dpower @ualg.pt
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_4, # Springer‐Verlag Berlin Heidelberg 2009
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THDP T4 TBG TSH TTR TLP T3
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Thyroid hormone distributor protein Thyroxine Thyroxine-binding globulin Thyroid stimulating hormone Transthyretin Transthyretin-like proteins Triiodothyronine
Introduction
Fish are the most successful group of extant vertebrates with an estimated 25,000 species, which may increase in number when molecular methods for classification are applied. Fish are part of the subphylum Vertebrata (possessing a cranium and vertebrae) and include the ‘ancient’ jawless Agnatha and modern jawed fish such as the cartilaginous Chondrichthyes (which includes sharks and rays) and the bony Osteichthyes (which includes ray-finned fish, Actinopterygii and lung fish). The fish are of considerable scientific interest because of their basal position in the evolution of vertebrates the existence of a variety of genome sizes and ploidy levels, the specific genome duplication proposed to have occurred in the teleost lineage (Ohno 1970; Jaillon et al. 2004; Taylor et al. 2003), their adaptation to a wide range of aquatic habitats throughout the World, their extreme biological diversity and the numerous examples of active speciation. In fact, fish have been crucial in establishing current models of vertebrate genome evolution (Jaillon et al. 2004; Ventakesh 2003). Despite their diversity and even taking into account the very limited number of species studied, there appears to have been a general conservation of an important regulatory network, the endocrine system. The thyroid system is no exception and most of the elements of this endocrine axis are conserved from fish to mammals. The present review will focus on one of the thyroid hormone distributor proteins (THDP), transthyretin (TTR), in fish. The recently identified family of genes encoding TTR-like proteins (TLP) will be touched upon in the context of TTR evolution in fish, as these recently identified proteins are dealt with in detail in other chapters of the present book. A number of reviews exist about TTR in vertebrates and provide an integrated picture of evolution and function of this protein across diverse classes of animals (Richardson 2007; Schreiber and Richardson 1997), and so this will only briefly be considered. The thyroid axis will be overviewed for readers unfamiliar with fish endocrinology and an update of the status of TTR in fish, which was first reviewed in 2000 will be given (Power et al. 2000). Some unpublished data will be introduced to highlight new and pertinent information about piscine TTR gene evolution and function.
4 Evolutionary Insights from Fish Transthyretin
4.2
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Thyroid Axis
The thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3), are produced by thyroid follicles, which in most fish are not encapsulated in a gland but instead are scattered throughout the connective tissue of the lower jaw usually aggregated around the ventral aorta (reviewed by Bentley 1998; Leatherland 1994). The basic structural unit of the thyroid is the follicle, which is composed of a single layer of epithelial cells surrounding a central colloid-filled cavity. The thyroid appears to have the longest phylogenetic history of any endocrine gland and its basic follicular unit has been conserved in all vertebrates and homologous tissues have been identified in protochordates, including amphioxus (Cephalochordata) and ascidians (Urochordata) (Barrington 1962). The central components of the thyroid axis have largely been conserved across vertebrates, and in fish, thyroid function from TH biosynthesis to TH action seem to fit the mammalian model. Thyroid stimulating hormone (TSH) stimulates T4 secretion from the thyroid, which is converted in the periphery to T3 by deiodination. A family of selenoprotein enzymes, the deiodinases (I, II and III), activate and inactivate the THs in fish, including agnathans, chondrichthyes and teleosts (Orozco and Valverde-R 2005). THs bring about their action by binding to nuclear thyroid receptors (TRs) and activate or repress gene expression (Chin and Yen 1997; Zhang and Lazer 2000). The cytosolic TH-binding proteins and plasma membrane TH transporters identified in mammals still remain to be characterised in fish. In contrast to mammals, where plasma T4 largely exceeds T3 levels (50–100:1), plasma T3 levels in teleosts may exceed that of T4, suggesting a strong 50 -monodeiodination activity, and peripheral deiodination may be the primary control of teleost thyroid function (reviewed in Eales and Brown 1993; Power et al. 2008). In teleosts, as in mammals, more than 99% of the THs do not circulate in the free form (Eales and Shostak 1985; Weirich et al. 1987) but are bound to plasma proteins including lipoproteins (Babin 1992). In fish, TH transport by plasma proteins is poorly explored and so far only two of the three THDPs present in the blood of mammals have been reported, albumin (Richardson et al. 1994) and TTR (Kawakami et al. 2006; Santos and Power 1999; Yamauchi et al. 1999; Manzon et al. 2007).
4.3
Thyroid Hormones Distributor Proteins
The relative abundance and hormone binding properties of THDPs are not conserved in vertebrates. In a series of studies, reviewed by Richardson (2002), the presence of THDPs in serum samples from representatives of different vertebrates (mammals, birds, reptiles, amphibians and also fish, (Richardson et al. 2005)) was analysed. As albumin is the only THDP identified in all species studied, it is
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proposed to be the evolutionarily oldest TH carrier protein. The same studies suggest an apparent absence of TTR in the plasma of adult amphibians, reptiles and fishes, although the presence of gene transcripts and protein is well established for the early stages of development (Funkenstein et al. 1999; Kawakami et al. 2006; Richardson et al. 2005; Santos and Power 1996, 1999; Yamauchi et al. 1993, 1998, 1999), which are characterised by a surge in TH blood levels (Hulbert 2000). The recent development of specific anti-sera for piscine TTR its presence by revealed by both western blot and ELISA blood of juvenile as a tetramer in juvenile (5 months), young adult (13 months) and adult (26 months) sea bream (Sparus aurata, Morgado et al. 2007b) and several other perciform species (authors’ observations). These data raise questions about why TTR was not detected in adult blood in the previous studies that used binding assays followed by native electrophoresis (Richardson et al. 2005). In the study by Morgado et al. (2007b), a specific ELISA was developed and revealed that the concentration of TTR in sea bream plasma is 4 mg ml1, which is 100 times lower than that in human plasma (0.3 mg ml1) (Ingenbleek et al 1972; Purkey et al. 2001; Sachs and Bernstein 1986), and may explain the failure to detect it in binding assays. In fact, it has been estimated that in humans the concentration of plasma TTR largely exceeds that of plasma THs (0.05-0.12 mg ml1), and less than 1% is estimated to be involved in TH transport (Robbins and Bartalena 1986). In this context, it has been proposed that the large excess of plasma TTR is associated with its second important function, to bind and transport all-trans-retinol complexed with retinol binding protein (RBP) and thus reduce glomerular filtration of retinol (Kanai et al. 1968; Monaco et al. 1995; Peterson 1971; Van Jaarsfeld et al. 1973). In the 1970s, RBP was isolated from the plasma of several fish species (Shidoji and Muto 1977) and rainbow trout (Oncorhynchus mykiss) and was shown to interact weakly with mammalian TTR (Berni et al. 1992). The subsequent cloning and isolation of TTR (Santos and Power 1996, 1999; Yamauchi et al. 1999; Kawakami et al. 2006; Manzon et al. 2007) and RBP (Bellovino et al. 2001; Sammar et al. 2001) from fish permitted binding studies with the piscine proteins, which revealed that TTR has a very low affinity for RBP (Folli et al. 2003). More recently, it has been clearly demonstrated that they do not form a complex (Zanotti et al. 2008). We hypothesise that the high circulating levels of plasma TTR and its tendency to form a TTR-RBP complex in mammals and birds (Heller 1976; Kopelman et al. 1976; Blaner 1989; Blomhoff et al. 1990), but not in fish, may indicate that the role of TTR in retinol transport may be an innovation in tetrapods. Further studies will be required to clarify when TTR emerged as an RBP binder in vertebrates and the evolutionary pressure that favoured the molecular modifications in the gene(s) which led to this function. Confirmation of whether RBP exists or not in lamprey (Petromyzon marinus, Lampetra appendix) and studies of its interaction with recently cloned lamprey TTR (Manzon et al. 2007) should contribute to resolve this issue. Nonetheless, preliminary analysis of the amino acids in TTR important for the stabilization of the TTR-RBP complex in humans (Folli et al. 2003)
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reveals that they are more conserved in lamprey (5 out of 8 amino acids) than in sea bream (2 out of 8 amino acids).
4.4
Structure and Hormone Binding Characteristics
Cloning of sea bream TTR cDNA coupled with homology modelling using human (Hamilton et al. 1993) (PDB ID: 1tta), rat (Wojtczak et al. 2001) (PDB ID: 1gke) and chicken (Sunde et al. 1996) (PDB ID: 1tfp) TTR predicted that the overall topology of sea bream TTR is preserved, although its surface potential, especially at the TH binding site, is much more electronegative in chicken and sea bream (Power et al. 2000). Subsequent crystallographic studies confirmed the conserved structure of sea bream TTR with that of mammalian and avian TTRs (Folli et al. 2003; Eneqvist et al. 2004) and revealed that it is a tetramer that contains two high affinity sites to which L-T4 may bind. The TH binding site is highly conserved, although Ser 117 (human sequence) is substituted by Thr in sea bream TTR (Folli et al. 2003). The affinity of fish TTR for THs is ambiguous and initial qualitative binding studies indicated that T3 appeared to have greater affinity than T4 for sea bream TTR. However, the development of a specific binding assay using recombinant sea bream TTR revealed that it binds T4 and T3 with a similar affinity (9.8 0.97 and 10.6 1.7 nM, respectively; Morgado et al 2008). Interestingly, truncation of the N-terminal region of the protein modified its affinity for T4 but did not significantly affect its affinity for T3 (Morgado et al. 2008). The results of this study substantiate the idea that the N-terminus of TTR plays an important role in determining hormone affinity (Prapunpoj et al. 2006). Recent studies have revealed that in common with what is observed in other vertebrates (Brouwer et al. 1999; Cheek et al. 1999; Ishihara et al. 2003a, b; Lans et al. 1993; McKinney and Waller 1994; Rickenbacher et al. 1986; Yamauchi et al. 2000), piscine TTR has a high affinity for a number of pollutants commonly found in the environment (Morgado et al. 2007a). Interestingly, however, despite the relatively high overall sequence and structural conservation of vertebrate TTR, the binding profile of chemical contaminants varies, for example, diethylstilbestrol (DES) and pentachlorophenol are powerful inhibitors of [125I] T3 binding to chicken, bullfrog, Xenopus, Rana and Japanese quail TTR, but have poor affinity for piscine TTR (Ishihara et al. 2003a, b; Morgado et al. 2007a; Yamauchi et al. 2000). It will be of interest in the future to establish the structural basis of this differing affinity and such studies may help to better define tetramer/ligand interactions. Moreover, understanding the biological consequences for the organism of the high affinity of some pollutants for TTR represents a significant challenge because of the importance of THs at different stages of the life cycle. The tendency of pollutants to accumulate in aquatic ecosystems (from industrial residues, land run-offs and acid rains) and the consequent continuous exposure of aquatic organisms to such pollutants make studies in aquatic vertebrates such as fish pertinent.
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Evolution TTR Tissue Distribution and Function
TTR has been identified in most vertebrate classes, including placental mammals, marsupials, birds, reptiles, amphibians and fish (Dickson et al. 1985; Duan et al. 1991, 1995; Larsson et al. 1985; Manzon et al. 2007; Mita et al. 1984; Prapunpoj et al. 2000a, 2002; Richardson et al. 1994, 1997; Santos and Power 1996, 1999; Tsuzuki et al. 1985; Yamauchi et al. 1993, 1999), and their overall amino acid sequence similarity is relatively high. TTR tissue expression and secretion is variable in the vertebrates in which it has been studied, and on the basis of the existing data, its specific expression in the liver or choroid plexus has been proposed to be class- and stage-dependent (reviewed in Richardson 2007). To explain life-stage-specific variations in the expression of TTR and other THDPs, it has been hypothesised that an augmented THDP network occurs at times of elevated TH in blood, such as, during development (Richardson et al. 2005). As mentioned earlier, fish are extremely diverse, and representatives from only a fraction of the superorders have been studied. However, considerably more examples from fish are required before a clear picture of the evolution of TTR, and its tissue-specific expression or life-stage-specific variations can be established. In Cyclostomata (e.g. lamprey), RT-PCR has revealed moderate TTR expression in the liver and heart and low expression in the brain gills, gut, kidney and blood of larvae (Manzon et al. 2007). Analysis of TTR was restricted to the liver and brain in adult fish, where the expression appeared to be much lower than that of the larval and juvenile stages. Analysis of TTR expression in other fish is restricted to a salmoniform (masu salmon, Oncorhynchus masu) and two perciformes (sea bream and Pacific bluefin tuna, Thunnus orientalis). In these species, TTR appears to be expressed during ontogeny (Funkenstein et al. 1999; Santos and Power 1996, 1999) and in juveniles, principally in the liver, although in the sea bream it is also detected at appreciable levels in the intestine and heart and to a lesser extent in the kidney, skin and brain (Power et al. 2000). Despite repeated attempts to identify TTR protein by immunohistochemistry (IHC) in the liver of juvenile and adult sea bream, sea bass (Dicentrarchus labrax) and Atlantic halibut (Hippoglossus hippoglossus), it has not been possible (unpublished observations). Western blot of adult sea bream liver extracts also revealed relatively low concentrations of TTR protein in spite of fairly constant circulating concentrations (4 mg ml1) and generally high hepatic transcript abundance. We hypothesise that such observations are consistent with the constitutive release of TTR (Morgado et al. 2007b). In addition, the detection of TTR in adult plasma suggests that it is very important as a THDP during development in fish. Relatively few physiological studies of TTR have been carried out on fish and its regulation and response to changing conditions remain largely unknown. In the sea bream injection of the sex steroid, estradiol-17 (5 mg g1 body weight) causes a reduction in hepatic TTR transcripts (Funkenstein et al. 2000), although it remains
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to be established if this is a normal physiological response during reproduction. Food restriction for several weeks in sea bream (Power et al. 2000) causes downregulation of liver TTR mRNA expression, suggesting that fish may be similar to mammals in which TTR is a marker of malnutrition or stress (Ingenbleek et al. 1972; Ingenbleek and Young 1994, 2002; Ingenbleek and Berstein 1999; Beck and Rosenthal 2002; Brugler et al. 2002). When plasma T3 or T4 are increased in fish, there is a significant increase in the circulating levels of TTR, although hepatic transcription is unchanged (Morgado et al. 2007b). Further studies of hepatic gene regulation and protein turnover are required to elucidate the way in which TTR contributes to TH homeostasis and whole animal physiology in fish.
4.5.2
TTR and TLP Evolution in Fish
The integration of data about TTR gene sequence, protein structure and hormone affinity across vertebrates has led to a general evolutionary model. TTR is proposed to have changed from a T3 binder in ‘lower vertebrates’ to a T4 binder in eutherians as a result of changes in the length of the N-terminal region. In vertebrates, the TTR gene is composed of four coding exons, and the longer hydrophobic N-terminus in fish and the shorter hydrophilic region in mammals are a consequence of a shift in the intron1/exon2 splice site during evolution (Aldred et al. 1997; Prapunpoj et al. 2000a, b, 2002). In masu salmon and bluefin tuna, the affinity of TTR is greater for T3, and in sea bream, TTR has similar affinity for T3 and T4 and the N-terminus of the fish proteins is several amino acids longer than mammalian TTR (Kawakami et al. 2006; Morgado et al. 2008; Yamauchi et al. 1999). The affinity of lamprey TTR for THs has not yet been determined, but analysis of the 50 region of the genomic DNA is consistent with the proposed evolutionary model and the N-terminus is nine amino acids longer than mammalian TTR (Manzon et al. 2007). The increase in available sequence data for fish in recent years makes evolutionary studies relevant, although a more comprehensive analysis still requires the inclusion of sequences from far more species. A family of transthyretin-like proteins (TLPs) or transthyretin-related proteins (TRPs, Eneqvist et al 2003) has recently been identified in prokaryotes and eukaryotes and shown to be enzymes (Lee et al. 2005; Ramazzina et al. 2006). The enzyme 5-hydroxyisourate hydrolase (HIUase) of purine metabolism in zebra fish (Danio rerio, Q06S87) is a TLP, and duplication of this gene is proposed to have given rise to TTR in vertebrates (Zanotti et al. 2006). To conduct phylogenetic analysis of TTR and TLP in fish, extensive searches in Genebank (www.ncbi.nlm.nih.gov) and Ensembl (www. ensembl.org) (October 2008) were carried out and specific cDNA, ESTs and predicted genes were retrieved (Table 4.1). It was possible to identify gene transcripts encoding TTR and HIUase genes (TLP) in 18 species, which included representatives of the orders Cypriniformes, Salmoniformes, Pleuronectiformes, Perciformes, Tetraodontiformes, Beloniformes, Gasterosteiformes, Gadiformes and Petromyzontiformes. The ESTs encoding TTR were identified in a range of
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Table 4.1 Accession numbers of the TTR and HIUase (TLPs) proteins, predicted genes and ESTs clones identified by searching public databases (available from NCBI and Ensembl) using the mature precursor sequences of published teleost TTR sequences with the tBlastn algorithm (sequences with e-value smaller than 105 were selected) Accession numbers Observations ACTINOPTERYGII (ray-finned fishes) Beloniformes Oryzias latipes (Medaka) TTR Not identified HIUase1 AM156132 BJ516106, BJ010835, BJ024650, DK059860, DK047918, DK070190, DK164988, DK141970, DK082087, DK145206, DK168247, DK170492, DK147440 BJ910632, DK261436, DK261403, DK242464, DK261679, DK242707, DK242998, DK261968, DK229688, DK248571 DC247390, DC256322 ENSORLG00000009326 HIUase2 ENSORLG00000000779 Gasterosteiformes Gasterosteus aculeatus (Three-spined stickleback) TTR Not identified HIUase1 DN731287, DW668813, DW668812 ENSGACG00000015936 HIUase2 ENSGACG00000008507 Perciformes Sparus aurata (Seabream) TTR CB184881, CB184737, CB184929 AAC26108 HIUase
AM950811, AM952219, AM952201, AM951997 Dicentrarchus labrax (Seabass) TTR CV186278 HIUase FM000902, FM000585 FM019182 Pleuronectiformes Platichthys flesus (Flounder) TTR DW985862 ABU54858 HIUase DV566090, EC378613 Psetta maxima (Turbot) TTR Not identified HIUase EY454781
Neurula Embryo
Liver
Fin Predicted Predicted
Brain Predicted Predicted
Liver and embryos Santos and Power (1999) Liver
Liver Liver Kidney
Liver Goetz et al. (unpublished) Liver
Liver (continued )
4 Evolutionary Insights from Fish Transthyretin Table 4.1 (continued) Accession numbers Tetraodontiformes Tetraodon nigroviridis (Green spotted pufferfish) TTR CR642656, CR635654, CR650478, CR649397, CR636486, CR651447, CR651621, CR652101, CR656042 ENSTNIG00000010473 HIUase SCAFFOLD_14581 Takifugu rubripes (Japanese pufferfish) TTR ENSTRUG00000006085 HIUase1 SCAFFOLD_2288 HIUase2 SCAFFOLD_9318 Cypriniformes Danio rerio (Zebra fish) TTR CN167258 EH532041, EH505861,EH515185, EH488817 AAI64894, XP_001919368 HIUase BC090440, BC097189 Q06S87 ENSDARG00000068644 Cyprinus carpio (Common carp) TTR CA966004, CA964904 CAD66520 HIUase Not identified Carassius auratus (Goldfish) TTR ABY40363 HIUase Not identified Gobiocypris rarus (Rare minnow) TTR ACE74244 HIUase EE394013 Gadiformes Gadus morhua (Cod) TTR ES240035, ES238908, ES239200, ES240301 HIUase ES471358 Salmoniformes Oncorhynchus mykiss (Rainbow trout) TTR BX886606, CX256523,BX878043, BX878043, CU073679, CX256522, CA377780 CX152238,CX148258,CX139266,CX138173, CX138172 CB497711 HIUase CU066583 BJ494178 Salmon salar (Atlantic salmon) TTR CK888905, BE518610, AM397609
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Observations
Liver
Predicted Predicted Predicted Predicted Predicted
Liver Gut/Internal Organs Predicted Embryo Zanotti et al. (2006) Predicted Liver Apreda et al. (unpublished)
Wiens et al. (unpublished)
Li et al. (unpublished) Liver
Liver Liver
Multi-tissues Juvenile Steelhead Multi-tissues Embryo Liver (continued )
68 Table 4.1 (continued) Accession numbers CK887387 CK895595 CK990828 HIUase EG904021, EG904019 Thymallus thymallus (Grayling) TTR FF846145, FF846144 HIUase Not identified CEPHALASPIDOMORPHI (Lampreys) Petromyzontiformes Petromyzon marinus (Sea lamprey) TTR ABI93605 HIUase Not identified Lampetra appendix (American brook lamprey) TTR ABI93606 HIUase Not identified
Deborah M. Power et al.
Observations Kidney Swim bladder Multi-tissues Multi-tissues Multi-tissues
Manzon et al. (2007)
Manzon et al. (2007)
different piscine tissue libraries, which included liver, gut, kidney, swimbladder, whole juvenile and embryo. In contrast, HIUase cDNAs were principally identified in liver, although they were also identified in whole embryo and trunk kidney cDNA, suggesting this gene may have a more restricted tissue distribution than TTR, although gene expression studies are required to confirm the in silico results. An alignment of piscine TTR and HIUase amino acid sequences (Fig. 4.1) revealed that they are highly conserved. A novel zebra fish HIUase gene (HIUase2) was identified and duplicate HIUase genes were also found in stickleback (Gasterosteus aculeatus), Takifugu (Takifugu rubripes) and medaka (Oryzias latipes) genomes (Fig. 4.1). In common with the gene structure of teleost TTR, four coding exons were also identified for HIUase genes and sequence comparison suggests that in some species N-terminal splicing of the HIUase gene probably occurs and accounts for shorter transcripts lacking exon 1, which are similar in size to the mouse homologue. In the other teleosts for which sequence data exists, a single copy gene for HIUase was identified and the presence of a duplicate copy remains to be established. The teleost TTR and HIUase genes share conserved splice consensus regions and the signature motifs identified in prokaryotes and eukaryotes such as the N-terminal VKILDAV and C-terminal GHRHY in TTR and DGRCPG in TLPs are also present (Hennebry et al. 2006). The C-terminal TLP motif, YRGS, was present in nearly all piscine HIUase, with the exception of stickleback HIUase2 (ENSGACG00000008507, contig_1506) in which R has been substituted by T (YTGS) and in medaka HIUase2 (ENSORLG00000000779, chromosome 6) where the C-terminal YRGS motif is substituted by HRSN. Of particular note was the large number of cDNAs identified for HIUase in the medaka, which were the product of the HIUase1 gene. However, no EST transcripts
4 Evolutionary Insights from Fish Transthyretin
SP
TTR Mouse Seabass Seabream Flounder Takifugu* Tetraodon Goldfish* Carp Zebrafish Cod Trout Salmon Grayling* Lamprey SeaLamprey
69
MASLRLFLLCLAGLVFVSEAGPA--------GAGESKC MVK MLQP-LRCLLLTSAVLLCNATPT---PTEKHGGTDTRC TVK MLQP-LHCLLLASAVLLCNTAPT---PTDKHGGSDTRC MVK -IQP-VHCLLLASAVLLCNSSPT---PTEKHGGSDTKC TVK -----------------------------AHGGSDTKC TVK MLPL-LLSLLLVSAALPSHAAPV----LKSHGGSDTTC TVK -------------------SAPV-----GIHGGSDAHC TVK MAKA-VICVLLVSLFACCRSAPV-----GIHGGSDVHC TVK MAKE-VICVLLASLFALCRSAPV-----AFHGGSDAHC TVK MMRP-LLWLLLASISLPCDTAPV-----EKHGESDTTC MVK MDSS-LLCVLLATAVLLCSAAPV-----DRHGESDTHC MVK MDSS-LLCVLLAAAVLLCSAAPV-----DRHGESDTHC MVK ------------------------------------------K MTRFLCLLVLVASSSLLCSADDDHKSHESHEGGVKDSC MVKA -MRFLCLLVLVASSSLLCRADDDHKSHESHEGGVKDSC MVKA
AVR AVK AVK AVK AVK AVK AVK AVK AVK AVK AVK AVK AVK SVR SVQ
S AVD T AGS T AGS T AGS T AGP TAAGP T AGN T AGN T AGN L AAS V AGA V AGA V AGS K AAG K AAG
A A A A A A A A A A A T A K K
K K K K N T D D D K S S S S S
F-KKTSEGS S-QKAADGG S-QKTADGA F-QKAADGA Y-QRTADGG Y-KKAADGG Y-RQEQGGT Y-RQDQGGT F-RQDQGGT S-KKGTDGE S-RRVNGMT S-RRVNEMT S-RRVNGMT M-KKQEDAS M-KKQDDAS
--------------------------------MATESS TTH TAS L AQG ---------MSTFR-LQQLKGHISPENKITTMASLE-S TTH VAM V ASN -------------------------------MSTAEVS TTQ SSD V GAR -------------------------------MAGSA-S TTH TAT V GAN ---------------------------------MMESCT TTH SVD V AAR --------MAAAESSPLTTHVLNTGDGVPAAMAAAESS TTH TGD V AAR ---------MGANR-LQQLKGHILPENKITAMAGSP-S TTH TGM V GSN ---------MSVSR-LQKLKGHILSENKSTAMAGSP-S TTH TAL I ASN -------------------------------MAAAASS TTH TGD I AAK -------------------------------------- TTH TAM I ASN -------------------------------------------------------------------------------------------S TTH TGM I ASN -----------MNR-LQHIRGHIVSADKHINMSATLLS STH IAQ V GAN ------------------------------MSSASDIS STH TGD V AQR -----------MNR-LQHIRGHIVSADKY--MAATLPS TTH TAQ V GAN ---------MSADRRLHNLSNQIVAANPCGAMAQPG-S TTH TAM V GSN ---------MSTSR-LQHIKDHLLDEYTCAEMAAPY-S TTH TGM V GAH ------------------------------MSSTLPNS TTY TGD V GAR ------------MIKFLLFLAIAAATVISNAELAVPTAS SAH ISG S AGG
C T A T A A A A A A A A T T T T A A Q
R SRLEAPCQQ R YRQDPSSKT S HRLDSELVI S HRQDPLTSA S HRLEPPLMV S HRLDSDLMI S YRQDPSTNV S SRQVPSTND S HRLDSELMI H YRQDPSTNA S HRLDSKLMI N YRKDPSNDT V HRLDPVSSA S HRLEPRITV I YRLDPISSV L YKQDSSAAA S HRMDPSTSL S HRLDSLLVI LAFILLN--NG
EPFAS TH AN TQ AT TQ AN TQ AN TQ AN EK AS EK AS EK AS TH AI AQ AS AQ AS AQ AS KE AT KE AT
K AES V AT V AT V DT M AS T AT KV IT KV MT KV MT V ET V LT V LT V LT V GKT V GKT
ELHG EIHD EIHN ESHN EIHN EIHE EVHN EVHN EVHN EVHN EVHN EVHN EVHN ESHH ESHH
78 82 82 81 57 81 62 80 80 80 80 80 44 86 85
RTSY LD NT I AD SV T EA TT I AD SL T ED SV T ED TT T DD IN T DD SI T ED KT T ED SV - ED KT T ED TT I DD TV S ED TT T ED TT I SD TT T DD TV T DD GSQF QDN
RCPG RYPG RCPG RCPG RCSG RCPG RCPG RCPG RCPG RCPG CCPG RCPG RCPG RCPG RCPG RCPG RCPG RCPG RVDW
55 76 56 55 54 79 76 76 56 49 31 50 75 57 73 77 76 57 73
HIUase Mouse Medaka1 Medaka2 Stickleback1 Stickleback2 Seabass2 Seabream1 Flounder1 Turbot2 Takifugu1* Takifugu2* Tetraodon1* Zebrafish1 Zebrafish2 Minnow1 Cod1 Trout1 Salmon2 Celegans
ME QL TM SL SL NM SL TL NM SL NM SL NI SL NL NL NL NL TN
TTR Mouse Seabass Seabream Flounder Takifugu* Tetraodon Goldfish* Carp Zebrafish Cod Trout Salmon Grayling* Lamprey SeaLamprey
TTDEK TEQQ TEQQ TEQQ TEQK PEEA TEQE TEQE AEQE TDQQ SDQD SDQD SEQD GDKD SDKD
VE PA PA SA LP PP TP TP TP PA QS QS QA TE T TE T
ELD E D E D E D D D D D E D E D E D D D E D E D E D R D R E
KS KA KA KS KS KA KS KA LT KA KA KA KA QA QQ
KTL KNE TNQ KNE KNE MNE KAE KAE KTE KSQ KSQ KSQ KSQ TKA TKT
I P S P S P S P SVP N P R P R P R P S P T P T P T P I P I P
HEFAD TANDSGHRH T AA P TAVVSNPQNHEAAD EAHTEGHRH T A -P TAVVTD-H-HEVAE DAHPEGHRH T A P TAVVSSVHEHEAAD EAHAEGHRH T A P - TAVVTDTHQHEVTN EAHSEGHRH T A P TALVTDVQHHEVTN EAHAGGHRH T A P TALVSDVPHHQL-------------------------------------HQLAD EAHAEGHRH T A P TAVVVKAHEHQLAD EAHAEGHRH T A P TAVVVKAHDHEMAE EAHGEGHLH T A P TAVVS-THQHETAE EAHAEGHRH T A P TAVVMKAHEHETAE EAHAEGHRH T A P TAVVMKAHEHETAE EAHAEGHRH T A P TAVVINAHEHEAAE MAHGAGHKH H P P F A GTIVGDAEGH HEAAE MAHGAGHKH H P P F A GAIVVDGKGH
F H R H R R H H R H R H R R R H R R V
ER AQ AS AQ GS GS VQ AQ GS AK GP AQ GK QQ GK DR GQ GQ EP
KER QES YPYVE TITKETQ-K H P P YRGS ASL D S YPYVE TINDPGQ-K H P R YRGS ESRTQ S YPYVE SISEPEQ-S H A P HRSN QSMEE C YPYVE TINDPGK-KHH P R YRGS ESR Q SLHPYVE SISDHHQ-RIH P R IYTGS ETL Q S YPYVE TISEPDQ-RVH P R YRGS ESL E C YPYVE TINNPGQ-K H P R YRGS ASI D S YPYVE TINDPGL-K H P R YRGS ESL Q S HPYVE TISSPEQ-R H P R YRGS ERI E G YPYVE TINDPGQ-K H P P YRGS ESL HIS YPYVE R---------------------------ESM Q S YPYVE IINDPGQ-K H P R YRGS DAL E C YPYVE TITNTSQ-H H P R YRGS ESL Q S YPYVE TITDVDQ-RLH P R YRGS DAL E C YPYVE TITDPSQ-K H P C YRGS GCL EES YPYVE TIRNPVD-K H P R YRGS GSL E S YPYVE TITDHSQ-K H L C R YRGS ESL HNS YPYVE TITNPNE-R H P R YRGS TAKNVES YPYVE NIRNATQ-H H P T P G YRGS
147 151 151 149 126 150 94 149 149 148 149 149 113 156 155
HIUase Mouse Medaka1 Medaka2 Stickleback1 Stickleback2 Seabass2 Seabream1 Flounder1 Turbot2 Takifugu1* Takifugu2* Tetraodon1* Zebrafish1 Zebrafish2 Minnow Cod1 Trout1 Salmon1 Celegans
TPSQIKP T TKEL TA RREA TP TTQM TS AAREELGP T SRRA AA TKET TP TKEL TP SREA IS TPQA TSD PHPD LP TSQM TS TKEN IA SRDA TP KKES VA TTREL TP TRET TPA TTKA TP SPDFTLIP T
D E E D E E E D E E E D E E E D E E I
118 139 119 118 117 142 139 139 119 112 67 113 138 120 136 140 139 120 136
Fig. 4.1 Multiple sequence alignment of the teleost and lamprey TTR and HIUase mature precursors with mouse TTR and HIUase and C.elegans TLP. The order of teleost sequences in the alignment is in agreement with Table 4.1 and their common names are indicated: Medaka (Oryzias latipes), Stickleback (Gasterosteus aculeatus), Seabass (Dicentrarchus labrax), Seabream (Sparus aurata), Flounder (Platichthys flesus), Turbot (Psetta maxima), Takifugu (Takifugu rubripes), Tetraodon (Tetraodon nigroviridis), Goldfish (Carassius auratus), Carp (Cyprinus carpio), Zebra fish (Danio rerio), Minnow (Gobiocypris rarus), Cod (Gadus morhua), Trout (Oncorhynchus mykiss), Salmon (Salmon salar), Grayling (Thymallus thymallus), Lamprey
70
Deborah M. Power et al.
TAKIFUGU Scaffold_401
TTR ZEBRAFISH Chr24
TETRAODONT Scaffold_14628
ECT2
B4GALT6 TTR
RFC4 B4GALT6
B4GALT6
B4GALT6
TTR
TTR
TTR
0,1M
0,03M
3M
0,124M STICKLEBACK Scaffold_348
MEDAKA Chr3
HIUase ZEBRAFISH Chr 18
TCF25
MVD PDCD5
HIUase1
MTHFSD9 TCF25
TCF25 HIUase1
PDCD5
PDCD5
MTHFSD
MVD FOXF1
FOXF1
MTHFSD
HIUase1 0,029M
Chr6 Chr 25
RFWD3 HIUase2 0,1M 0,4M
MOUSE Chr 7 PCDC5
0.1M Scaffold_1056
0,2M
ZNF277P
MOUSE Chr 18
ECT2 RFC4
TNN12
ZNF277P TNN12
TNN13 CACNA2D4 LRTM2
TNN13 CACNA2D4
RFWD3 HIUase2 ZNF277P
HIUase2
HIUase 9M
LRTM2 RFWD3 0.4M
Fig. 4.2 Comparison of the TTR and HIUase (TLPs) gene environments in teleosts with the mouse homologue regions. Genes are presented by open boxes and chromosomes or genome fragments by vertical lines. Only common genes are represented and are named according to HUGO annotation. TTR and HIUase genes are in bold and underlined and their respective linked genes, B4GALT6 and PDCD5 are in bold. Homologue genes were identified based upon their sequence similarity using the BLAST programme and the linkage maps were constructed based on gene annotated available from Mapviewer (http://www.ncbi.nlm.nih.gov/mapview/) and Ensembl (www.ensembl.org) databases. The figure is not drawn to scale and the size of the genome regions analysed is indicated
< Fig. 4.1 (continued) (Lampetra appendix) and Sea lamprey (Petromyzon marinus). The HIUase duplicates identified in teleosts are designated as 1 (most similar to the published zebra fish HIUase sequence) and 2. Vertical arrows indicate the localisation of exon–intron splice sites and the double arrow the homologue region of the human signal peptide sequence (SP). The degree of amino acid conservation between TTR and HIUase is indicated by shading and an asterisk (*) indicates teleost sequences that are incomplete. Specific motifs previously identified by Hennebry et al. (2006) are within open boxes and the region where amino acids are proposed to have been lost at the 50 end of exon 2 is in a dashed box. The conserved exon–intron splice sites are indicated by open arrows and the closed arrow indicates the beginning of the homologue region of the mature human TTR protein. Accession numbers of the predicted genes, ESTs and peptides used to generate the alignment are indicated in Table 4.1. The Minnow TTR sequence was not included as it was very incomplete. The accession numbers for TTR and HIUase from mouse (Mus musculus) are NP_038725 and Q9CRB3, respectively, and transthyretin-like protein (TLP) for C. elegans is ZK697
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for its paralogue or for the predicted stickleback HIUase2 gene were identified, suggesting that they may be low abundance or expressed in tissues for which few ESTs exist in the NCBI database. A further intriguing observation in the medaka and stickleback was the failure to identify a homologue of the teleost TTR gene despite the extensive number of Genebank sequences that exist for both model organisms (22,500 and 19,000, respectively, in the Unigene database, http://www. ncbi.nlm.nih.gov/), and it will be of interest to explore the significance of these observations. It is generally accepted that gene duplication has fuelled organismal complexity, and two major rounds of genome duplications (1R and 2R) are suggested to have occurred at the origin of vertebrates. Such events explain the general increase in gene number in vertebrate genomes and provide a plausible explanation as to how TTR emerged in the vertebrates. Despite the demonstrated sequence and structural conservation of TTR and HIUase (TLP) genes in vertebrates, comparison of the immediate gene environment fails to reveal homologous regions, suggesting that after gene duplication the future TTR gene was transposed to a different genome region (Fig. 4.2). However, some conservation of the flanking genes exist in teleosts and mammals and homologues of B4GALT6 map in close proximity to TTR and homologues of PDCD5 are linked to teleost HIUase1 and mouse HIUase, suggesting that the HIUase2 gene is the duplicated gene form of TLPs, which arose from a specific duplication event. The failure so far to identify a TTR paralogue in teleosts may indicate that it was either eliminated from the genome or arose as a consequence of partial genome duplication.
Acknowledgements Some of the work described was co-financed by POCI 2010 and European Social Funds attributed by the Portuguese National Science Foundation (FCT) to project POCTI/ CVT/38703/2001 and Pluriannual funding to CCMAR.
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Prapunpoj P, Leelawatwatana L, Schreiber G, Richardson SJ (2006) Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3. FEBS J 273:4013–4023 Prapunpoj P, Richardson SJ, Schreiber G (2002) Crocodile transthyretin: structure, function, and evolution. Am J Physiol 283:R885–R896 Prapunpoj P, Richardson SJ, Fugamalli L, Schreiber G (2000a) The evolution of the thyroid hormone distributor protein transthyretin in the order Insectivora, class Mammalia. Mol Biol Evol 17:1199–1209 Prapunpoj P, Yamauchi K, Nishiyama N, et al. (2000b) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol 279:R2026–R2041 Purkey HE, Dorrell MI, Kelly JW (2001) Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma. PNAS 98:5566–5571 Ramazzina I, Folli C, Secchi A, et al. (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nature Chem Biol 2:144–148 Richardson SJ (2002) The evolution of transthyretin synthesis in vertebrate liver, in primitive eukaryotes and in bacteria. Clin Chem Lab Med 40:1191–1199 Richardson SJ (2007) Cell and molecular biology of transthyretin and thyroid hormones. Int Rev Cytol 258:137–193 Richardson SJ, Bradley AJ, Duan W, et al. (1994) Evolution of marsupial and other vertebrate thyroxine-binding plasma proteins. Am J Physiol 266:R1359–R1370 Richardson SJ, Hunt JL, Aldred AR, et al. (1997) Abundant synthesis of transthyretin in the brain, but not in the liver, of turtles. Comp Biochem Physiol B 117:421–429 Richardson SJ, Monk JA, Shepherdley CA, et al. (2005) Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish. Am J Physiol 288:R1264– R1272 Rickenbacher U, McKinney JD, Oatley SJ, Blake CC (1986) Structurally specific binding of halogenated biphenyls to thyroxine transport protein. J Med Chem 29:641–648 Robbins J, Bartalena L (1986) Plasma transport of thyroid hormones. In: Thyroid Hormone Metabolism. Marcel Dekker, New York Sachs E, Bernstein LH (1986) Protein markers of nutrition status as related to sex and age. Clin Chem 32(2):339–341 Sammar M, Babin PJ, Durliat M, et al. (2001) Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues. Gen Comp Endocrinol 123:51–61 Santos CRA, Power DM (1996) Piscine (Sparus aurata) transthyretin. Ann Endocrinol 57:58 Santos CRA, Power DM (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinol 140:2430–2433 Schreiber G, Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B 116:137–160 Shidoji Y, Muto Y (1977) Vitamin A transport in plasma of the non-mammalian vertebrates: Isolation and partial characterisation of piscine retinol binding protein. J Lipid Res 18:679–691 Sunde M, Richardson SJ, Chang L, et al. (1996) The crystal structure of transthyretin from chicken. Eur J Biochem 236:491–499 Taylor JS, Braasch I, Frickey T, et al. (2003) Genome duplication, a trait shared by 22,000 species of ray-finned fish. Genome Res 13:382–390 Tsuzuki T, Mita S, Maeda S, et al. (1985) Sturcture of the human prealbumin gene. J Biol Chem 260:12224–12227 Van Jaarsveld PP, Edelhoch H, Goodman DS, Robbins J (1973) The interaction of human plasma retinol-binding protein and prealbumin. J Biol Chem 248:4698–4705 Venkatesh B (2003) Evolution and diversity of fish genomes. Curr Opin Genet Dev 13:588–592 Weirich RT, Schwartz HL, Oppenheimer JH (1987) An analysis of the interrelationship of nuclear and plasma triiodothyronine in the sea lamprey, lake trout, and rat: evolutionary considerations. Endocrinol 120:664–677
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Wojtczak A, Cody V, Luft JR, Pangborn W (2001) Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallog D 57:1061–1070 Yamauchi K, Kasahara T, Hayashi H, Horiuchi R (1993) Purification and characterization of a 3,5,30 -L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinol 132:2254–2261 Yamauchi K, Nakajima J-I, Hayashi H, et al. (1999) Purification and characterization of thyroidhormone-binding protein from masu salmon serum: A homolog of higher-vertebrate transthyretin. Eur J Biochem 265:944–949 Yamauchi K, Prapunpoj P, Richardson SJ (2000) Effect of diethylstilbestrol on thyroid hormone binding to amphibian transthyretins. Gen Comp Endocrinol 119:329–339 Yamauchi K, Takeuchi H, Overall M, et al. (1998) Structural characteristics of bullfrog (Rana catesbeiana) transthyretin and its cDNA: comparison of its pattern of expression during metamorphosis with that of lipocalin. Eur J Biochem 256:287–296 Zanotti G, Cendron L, Ramazzina I, et al. (2006) Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9 Zanotti G, Folli C, Cendron L, et al. (2008) Structural and mutational analyses of proteinprotein interaction between transthyretin and retinol-binding protein. FEBS Journal 275(23): 5841–5854 Zhang J, Lazar MA (2000) The mechanism of action of thyroid hormones. Ann Rev Physiol 62:439–466
Chapter 5
The Salmonella sp. TLP: A Periplasmic 5-Hydroxyisourate Hydrolase Sarah Hennebry
Abstract The primary, secondary, tertiary and quaternary structures of TTRs have been highly conserved during the evolution of vertebrates. This suggests that the TTR gene must have evolved prior to the divergence of vertebrates from invertebrates. Open reading frames predicted to encode a TTR homolog, transthyretin-like protein (TLP) were identified in all kingdoms. The Salmonella dublin TLP was selected as a model by which to characterise the structure, function and evolutionary relationships between TTR and TLP. The X-ray crystal structure of the S. dublin ˚ . As the first X-ray crystal structure of a TLP determined, TLP was solved to 2.5 A the results revealed the remarkable conservation of the TLP/TTR fold. The only significant differences in the structures of S. dublin TLP with TTRs were localised to the dimer–dimer interface and indicated that thyroid hormones could not be bound by TLP. Studies by others had indicated that cytoplasmic TLPs functioned in purine metabolism by hydrolysing 5-hydroxyisourate (5-HIU). It was demonstrated that a periplasmic TLP, the S. dublin TLP also hydrolysed 5-HIU. Three residues at the dimer–dimer interface of S. dublin TLP were shown to be essential for chemical catalysis and are 100% conserved in all TLP sequences but absent in TTRs. Therefore it was proposed that following the duplication of the TLP gene in early protochordate evolution, loss of these catalytic residues resulted in the formation of a deep, negatively charged channel which runs through the centre of the TTR tetramer. The results thus demonstrate the remarkable evolution of the TLP/TTR protein from a hydrolytic enzyme to a thyroid hormone distributor protein. Keywords 5-Hydroxyisourate, Periplasm, TLP
S. Hennebry Human Neurotransmitters Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
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Introduction
Studies investigating the evolution of the structure of TTR have highlighted the significant conservation of the primary, secondary, tertiary and quaternary structures of vertebrate TTRs. Alignments of the TTR subunit sequences from the organisms in which it has been identified, revealed significant sequence homology (for a review, see Power et al. (2000) and previous chapter). The most evolutionarily distant TTR sequences, those from human and sea bream, share 48% identity (67% similarity). Within the group of eutherian TTR sequences, the identity is higher, with 82% sequence identity (92% similarity) between human and rat TTR sequences. The significantly higher degree of sequence similarity among TTR sequences compared to sequence identity, suggests that the overall chemical properties of the protein have been conserved throughout its evolution (Power et al. 2000). In the previous chapter, the X-ray crystal structures of TTR from human, rat, chicken and fish were compared. The secondary, tertiary and quaternary structures of these proteins were shown to be highly conserved. Given the homology of TTR structures throughout vertebrate evolution, it was postulated that the TTR gene arose prior to the divergence of vertebrates from invertebrates. In 2000, Prapunpoj et al. (2000) announced the identification of open reading frames (ORFs) in the recently sequenced genomes of invertebrate organisms. These ORFs were predicted to encode a putative TTR homolog which was named the transthyretin-like protein (TLP). At the time, little was known about TLP other than the fact that it shared approximately 60% sequence similarity with TTR. To further characterise the distribution of TLP genes in nature, a more extensive mining of the available databases was required. Determination of the sequence characteristics of TLP and in particular, the shared sequence characteristics of TLP and TTR was also required to elucidate the evolutionary relationship, if any, between TLP and TTR. This chapter focuses on the initial characterisation of TLP structure and function using bioinformatic tools, and the subsequent in vitro characterisation of a bacterial TLP, the Salmonella sp. TLP.
5.2 5.2.1
The Identification of Non-Vertebrate TTR Homologs The Relative Distributions of TTR and TLP Genes
At the time of the first discovery of TLP ORFs, in 2000, very few genomes had been completely sequenced. The last decade has seen a considerable shift in the manner and speed with which protein phylogenies could be determined as a result of large-scale sequencing of genomes from a wide variety of organisms. Genome sequence data have increased exponentially and have enabled the completion of a more thorough analysis of the distribution of TLP genes by examining the number of organisms with TLP genes (Eneqvist et al. 2003; Hennebry et al. 2006a).
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Phylogenetic analyses have also been used to provide a powerful insight into the evolution of these two protein families (Eneqvist et al. 2003; Hennebry et al. 2006a). The results of the bioinformatic analyses have provided an important ‘first step’ in our understanding of TLP and have influenced the direction of subsequent research. To date, in excess of 200 individual TLP sequences have been identified using a combination of BLAST and HMM searches (Hennebry et al. 2006a). TLP genes are found in all kingdoms. In contrast, TTR genes are only found in vertebrate genomes. This finding has led to the hypothesis that the TTR gene may have evolved from the duplication of the TLP gene in early vertebrate evolution (Hennebry et al. 2006a).
5.2.2
Motifs Characterising TLP and TTR Sequences
In order to better define the sequence characteristics of TLP and TTR and to distinguish between the two protein families, the sequences were probed for isolated signature motifs (Hennebry et al. 2006a). The high level of similarity between TTR sequences was such that a single motif was found to span the entire protein. Therefore, a set of TTR-specific motifs was unattainable. However, five motifs were identified among TLP sequences (A–E) and were found in the arrangement (E), B, D, C, A along the length of the protein sequence (Fig. 5.1a(i)). Motif A, which corresponds to the region of greatest similarity between TLP sequences, was found at the C-terminal region of the sequences and included a consensus YRGS tetrapeptide which was 100% conserved in all TLP sequences. Motif B is the second strongest motif and was found in the N-terminal region of the protein. Motifs C and D were found in the middle of the TLP sequences. Motif E was only found in TLPs from plant species and from two alphaproteobacteria: Bradyrhizobium japonicum and Magnetospirillum magnetotacticum. Motif E is homologous to the proteins of COG 3195 (Cluster of Orthologous Groups), a group of bacterial proteins where the entire protein is made up of this single domain. Motif E has been subsequently identified as a unique protein, OHCU decarboxylase whose function relative to TLP will be discussed further in chapter ‘Clinical Implications of TTR Amyloidosis’. Three motifs were identified to be in common between TLP and TTR sequences: A’, B’ and C’ (Fig. 5.1a(ii)). Motif A’ is equivalent to Motif A of the TLP-only set: at the C-termini of the proteins. Motif A’ is the most highly conserved among the set of TLP + TTR sequences. Motif B’ in the TLP + TTR set completely overlapped with the Motif B in the TLP only set, and extended for three and five residues further toward the N- and C-termini, respectively. Motif C’ found in the TLP + TTR set was shorter (15 vs. 21 residues) but partially overlapped Motif C in the TLP only set. The absence of a Motif D’ in the TLP + TTR set implied that Motif D is specific for TLP sequences and is the region with the least similarity between TTR and TLP sequences (Fig. 5.1a(ii)).
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Fig. 5.1 Motifs identified in TLP sequences and in the set of TLP + TTR sequences. (a) Motifs identified in (i) TLP sequences and (ii) TTR + TLP sequences. (i) In the set of TLP sequences, five motifs were identified (A–E). The motifs are found in the order (E), B, D, C, A, with A being the most highly conserved. Motif E (not shown) is only found in the TLPs from plants and the a-proteobacteria Magnetospirillum magnetotacticum and Bradyrhizobium japonicum. (ii) In the set of TTR + TLP sequences, three motifs were identified. Motif A’ is equivalent to Motif A from the TLP motif set. Motif B’ is similar but extended in the N- and C-terminal regions to Motif B. Motif C’ is shorter than Motif C and its location is shifted towards the N-terminus. Motif D is specific to the TLP set of proteins. Motifs logos were generated using the MEME program (Bailey and Elkan 1994). (c) The motifs identified in the TLPs correlate to regions of structural significance. (i) TheSalmonella dublin sequence is shown with the predicted secondary structure indicated above the sequence. Motifs identified in common between TLPs and TTRs are indicated below the alignment of the S. dublin TLP sequence with that of human TTR. The experimentally determined secondary structure of human TTR is indicated above its sequence. The alignments indicate that the primary and secondary structures of TLPs and TTRs are highly conserved. (ii) The motifs identified in common between TLPs and TTRs were superimposed on the tertiary structure of sea bream TTR (Folli et al. 2003). Motif A’ lines the hydrophobic core. Motif B’ forms the dimer–dimer interface and the entrance to the hydrophobic core. Motif C’ forms the monomer– monomer interface. The program SWISS-PDB viewer was used to generate the figure (from Hennebry et al. 2006a)
5.2.3
Structural Significance of Motifs
In order to determine whether the conserved sequence motifs corresponded to regions of structural significance, a homology model of the Salmonella dublin TLP was constructed (Hennebry et al. 2006a). The S. dublin TLP was selected for this analysis as it was among the first TLP sequences identified (Prapunpoj et al. 2000). In addition, the predicted amino acid sequences from Salmonella sp. were identical, and so the S. dublin TLP would represent TLPs from this genus. The sequence motifs which were determined to be common to both TLP and TTR sequences were aligned with the predicted and experimentally determined secondary structures of TLP and TTR respectively (Fig. 5.1b(i)). Motif A’ corresponded to b-strand Gloop-strand H region of TTR. Motif B’ corresponded to b-strand A-loop-strand B region of TTR. Motif C’ corresponded to b-strand E-turn-a-helix of TTR. The equivalent regions of the S. dublin TLP were predicted to share the same secondary structure features as TTRs (Hennebry et al. 2006a). These results suggested that the
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conserved motifs might correspond to important structural features of the TTR tetramer. The motifs were then superimposed onto the crystal structure coordinates of sea bream TTR (pdb identifier: 1OO2; Folli et al. 2003) (Fig. 5.1b(ii)). The residues comprising Motif A’ (the most highly conserved motif) were found to line the dimer–dimer interface of the protein and were also potentially involved in monomer–monomer interactions. The residues comprising Motif B’ were also found to cluster at the dimer–dimer interface of the protein, this time forming the entrance to the ligand binding channel of TTR. Residues comprising Motif C’ were also found to have a structural role, potentially in monomer–monomer interactions. The finding that the sequence motifs conserved between TLP and TTR corresponded to structurally important regions of the TTR tetramer lent further weight to the prediction that TLP shares similar secondary and tertiary structures to TTR. The identification of the motifs specific to TLP and to the set of TLP + TTR revealed that the regions of greatest similarity between the proteins are in the Nand C-terminal regions and that these correspond to the b-strands (strands A, B, G and H) forming the entrance to and lining the dimer–dimer interface of the TTR tetramer. However, a close examination of the differences between the TLPspecific motifs and TLP + TTR motifs revealed important information regarding the likely ligand(s) of TLPs. The ligands of TTR (thyroid hormones and related compounds) bind deep within the central channel of the protein, formed by the dimer–dimer interface. Important residues involved in this ligand binding include: Lys15, Glu54 and Thr119. An inspection of the dimer–dimer interface in the molecular models generated for the S. dublin TLP suggested a structurally distinct binding channel. The presence of conserved histidine, arginine and tyrosine residues in all TLP sequences at the equivalent regions of Lys15, Glu54 and Thr119 in all TTR sequences suggested that the central channel would be considerably smaller and more positively charged in TLP. These observations suggested that if the dimer–dimer interface is the ligand binding site of TLP, the ligands bound are likely to be smaller than thyroid hormones and carry different distribution of charges.
5.2.4
Variations in the Predicted Subcellular Localisation of TLPs
Whilst all TLP sequences identified have been shown to contain Motifs A–D, further variations were observed in the N-terminal regions of these sequences. Careful inspection of the N-terminal regions of TLP from various organisms revealed that many TLP sequences contained likely subcellular localisation sequences or secretion peptides. On this basis, TLP sequences can be divided into four groups: (1) cytoplasmic TLPs (the majority of TLP sequences); (2) periplasmic TLPs (a small group of gram-negative bacterial TLP sequences mainly from the group Enterobacteriaceae); (3) peroxisomal TLPs (most metazoan TLP sequences and plant TLP sequences); and (4) extracellular TLPs (nematode TLP sequences). As noted, plant and metazoan TLPs are represented by both cytoplasmic and
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peroxisomal sequences. This is due to splice variation of the TLP gene in these organisms which results in the loss of the peroxisomal targeting sequence (Hennebry et al. 2006a). It is postulated that TLPs from plants and metazoan organisms are localised to both the cytoplasm and the peroxisome. In most instances, only one TLP gene was identified per haploid genome. However, several bacteria were found to possess two or three non-identical TLP genes. In these cases, the second or third TLP gene was found to encode a protein with a periplasmic localisation sequence. The functional significance of the dual localisation of TLPs in some bacteria and in plants and metazoans will be discussed later in this chapter.
5.3 5.3.1
Structural Characterisation of the S. dublin TLP Synthesis and Characterisation of Recombinant S. dublin TLP
Given that bioinformatic analysis of the S. dublin TLP suggested shared similar structural characteristics with TTR, this protein was selected for further structural and functional studies. Recombinant S. dublin TLP was synthesised using an E. coli expression system (see Hennebry et al. 2006a). Given the close evolutionary distance between E. coli and Salmonella, the periplasmic localisation signals utilised by both bacteria are identical. Thus, the N-terminal periplasmic localisation signal of the S. dublin TLP was left intact so that secretion of the recombinant protein to the periplasm could be tested in E. coli. Recombinant S. dublin TLP was successfully purified from the periplasm of host E. coli (Hennebry et al. 2006a). N-terminal sequencing of the recombinant protein confirmed that the N-terminal signal peptide had been cleaved upon translocation of the protein into the periplasm (Hennebry et al. 2006a). This indicated that it was likely that in vivo the S. dublin TLP was localised to the periplasm. Recombinant S. dublin TLP was subjected to gel filtration and comparison of its subunit molecular mass (as determined by ionising mass spectrometry) with its apparent native molecular weight suggested that the protein formed homotetramers, similarly to TTR.
5.3.2
The X-Ray Crystal Structure of the S. dublin TLP
The three-dimensional structures of TTR from various organisms have been well characterised (refer to relevant chapter in this book). The first X-ray crystal structure of a TTR to be solved was that of human TTR, in 1978 (Blake et al. 1978). The Protein Database (http://www.pdb.org) contains multiple crystal structure coordinates for human TTR (including multiple amyloidogenic forms
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and with various ligands bound to both wild-type and mutant human TTRs). The crystal structures of TTR from rat (Wojtczak 1997), chicken (Sunde et al. 1996) and sea bream (Folli et al. 2003; Eneqvist et al. 2004) have also been solved. All of these structures demonstrate the remarkable conservation of the prealbumin-like fold (as described by SCOP, http://scop.mrc-lmb.cam.ac.uk) which consists of an eightstranded b-sandwich (strands A–H) with each sheet adopting a Greek-key topology. A two-turn a-helix usually exists between strands E and F in TTR. The two TTR dimers associate via non-polar interactions between the loops joining stands G and H with the loops joining strands A and B, making the TTR tetramer a ‘dimer of dimers’. ˚ (Hennebry The X-ray crystal structure of the S. dublin TLP was solved to 2.5 A et al. 2006b) (Fig. 5.2). Comparison of the structure of S. dublin TLP with that of TTRs from various organisms has enabled a greater understanding of the structural evolution of these paralogous proteins. In the months following the publication of the S. dublin TLP, the X-ray crystal structures of the TLPs from E. coli, B. subtilis and Danio rerio (Lundberg et al. 2006; Jung et al. 2006; Zanotti et al. 2006) were solved. The structures of these proteins will be discussed in the subsequent chapters of this book. The overall structure of the S. dublin TLP strongly resembled the archetypal TTR fold (Blake and Oatley 1977; Blake et al. 1978): a prealbumin-like fold (as defined by the SCOP database: http://scop.mrc-lmb.cam.ac.uk) consisting of an eight-stranded b-sandwich with each sheet adopting a greek-key topology (Hennebry et al. 2006b). A two-turn a-helix was found between strands E and F. The asymmetric unit consisted of two protein molecules (a TLP dimer; Fig. 5.2a) which dimerise via intermolecular hydrogen bonds between H-strands to form an eight-stranded b-sheet. A similar interaction has been found in all TTR crystal structures published to date. Application of tight non-crystallographic symmetry (NCS) restraints during refinement yielded identical molecules in the asymmetric unit. Therefore, the S. dublin TLP, similarly to TTR, was determined to be a tetrameric ‘dimer of dimers’ (see Fig. 5.2b). The two dimers were found to associate via non-polar interactions between the loops joining strands G and H with loops joining strands A and B. This was consistent with the observation from gel filtration analyses, that the TLP formed tetramers in solution (Hennebry et al. 2006a).
5.3.3
Comparison of the Structure of S. dublin TLP with TTRs from Human, Chicken, rat and Seabream
5.3.3.1
Comparison of the Overall Fold of S. dublin TLP with TTR
The structure of S. dublin TLP was overlayed with structures of TTRs from human (1F41), chicken (1TFP), rat (1KGI) and seabream (1SNO) (Hornberg et al. 2000; Sunde et al. 1996; Muziol et al. 2001; Eneqvist et al. 2004). The human, rat and fish ˚ . Chicken TTR and TTR tetramers superimposed to within an RMSD of 1.0 A
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˚ crystal structure of the S. dublin TLP. (a) Dimeric structure within the Fig. 5.2 The 2.5 A asymmetric unit of the crystal, showing the interaction between the b-strands H of each monomer. Secondary structure elements are labelled. The C and N termini are marked with an asterisk (*). (b) The homotetrameric structure generated by crystal symmetry, in an orthogonal orientation to (a). Approximate positions of predicted ligand binding sites at the dimer–dimer interface (shown as a dotted line) are indicated by black circles. Figure generated using PyMol (Delano 2002) (from Hennebry et al. 2006b)
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˚ (Fig. 5.3). S. dublin TLP showed larger variation, with RMSD values up to 4.0 A The structural deviation observed between the S. dublin TLP and the TTR subunits was of the same order of magnitude to that within the set of TTR subunits. The total ˚ ) was contacting surface area between molecules within the tetramer (5,275 A ˚ ˚ ˚ ) and 6–14% less than that of fish (5,625 A), human (5,810 A), chicken (5,828 A ˚ rat (6,133 A) TTR structures. 5.3.3.2
Comparison of the Putative Active Site of S. dublin TLP with the Thyroid Hormone Binding Site of Human TTR
One of the striking features of the quaternary structure of TTR is the central channel of the protein into which the thyroid hormones and other ligands (with the exception of retinol-binding protein) bind. This central channel traverses the entire tetramer. Whilst the overall structure of S. dublin TLP and human TTR were highly homologous, examination of the dimer–dimer interface of the TLP revealed significant differences compared to human TTR. The conserved residues His6, His95 and Tyr108 at the dimer–dimer interface of S. dublin TLP and other TLPs were shown to dramatically alter the physicochemical properties of this region of the protein in comparison to human and other TTRs. Figure 5.4 shows a comparison of the dimer– dimer interface of S. dublin TLP with the equivalent region in human TTR. The Tyr108 residue in S. dublin TLP has a profound effect on the size and shape of the dimer–dimer interface compared with human TTR, dramatically reducing the depth of the central channel. The equivalent residue in human TTR is a conserved threonine (Thr119). Residue Leu99 of S. dublin TLP also adopts a conformation which plugs the base of the pocket, compared to the equivalent residue Leu110 in human TTR. Thus, the residues Leu99 and Tyr108 in the S. dublin TLP contribute to a groove-like dimer–dimer interface. This contrasts with the dimer–dimer interface of the TTR molecule, which forms a deep and funnel-shaped central channel which runs through the middle of the tetramer. Other residues at the dimer–dimer interface of S. dublin TLP which differ considerably to the equivalent interface of TTR, are several positively charged residues (Fig. 5.4), His6, Arg44 and His95. These residues are also 100% conserved among TLP sequences and contribute to a significantly more positively charged dimer–dimer interface of S. dublin TLP compared to the more negatively charged channel of human TTR (Fig. 5.4a(i) and 5.4a(ii)). The comparison of the structures of TTRs and TLPs indicates that following the duplication of the TLP gene in early vertebrate evolution, specific amino acid changes at the dimer–dimer interface of the TLP tetramer resulted in increased negative charge and an ‘opening’ of the central channel of the protein. None of the charged residues identified at the dimer-dimer interfaces of TLPs are conserved in TTR sequences. One exception is the recently identified TTR from the lamprey, Petromyzon marinus (Manzon et al. 2007). This is the only TTR sequence to have a histidine residue at the position equivalent to His95 in S. dublin TLP (which is a conserved histidine in all TLPs). Therefore, it is likely that changes to the dimer– dimer interface of TLP occurred firstly at His6, Arg44 or Tyr108 residues and lastly at His95.
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Fig. 5.3 Structural comparison of the S. dublin TLP with TTR. (a) Stereo diagram showing a superimposition of the S. dublin tetramer (magenta) with human (1F41, Hornberg et al. (2000), cyan), rat (1KGI, Muziol et al. (2001), yellow), chicken (1TFP, Sunde et al. (1996), orange) and fish (1SNO, Eneqvist et al. (2004), green) TTRs. Tetramers were superimposed using the A chain only. (b) Stereo diagram showing a superimposition of monomers of S. dublin TLP (magenta) with human (1F41, cyan), rat (1KGI, yellow), chicken (1TFP, orange) and fish (1SNO, green) TTRs. Bound thyroxine ligand in the human structure is shown as sticks. Regions of plastic deformation are labelled. Residues at the dimer–dimer interface of S. dublin TLP are shown as red sticks (from Hennebry et al. 2006b)
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Fig. 5.4 Comparison of the dimer–dimer interfaces of human TTR and S. dublin TLP. (a) (i) Residues that make up the ligand binding pocket of (i) human TTR are shown. Thyroxine is shown in stick representation, in yellow. The residues Lys15, Glu54 and Thr119 are labelled and form important interactions with thyroxine. Some elements of secondary structure are not shown, for clarity. (ii) The dimer–dimer interface of S. dublin TLP is shown (at the equivalent position to human TTR shown in part (i)). Hydrogen bonds are shown as broken cyan lines. Some elements of secondary structure are not shown, for clarity. Residues known to be important to catalysis in S. dublin TLP are indicated with an asterisk. Residues His6, His95 and Tyr108 of S. dublin TLP are equivalent upon structural alignment to Lys15, Thr106 and Thr119 of TTR, respectively. (b). Surface characteristics and interactions at the ligand binding sites of (i) human TTR and (ii) S. dublin TLP. Positively charged residues are in blue, negatively charged residues are in red. (i) The electrostatic surface potential surface of human TTR (1F41; Hornberg et al. 2000), shown with thyroxine ligand inside the hormone-binding pocket, is negatively charged. (ii) The equivalent region in the S. dublin TLP is positively charged. Figure generated using PyMol (Delano 2002) (from Hennebry et al. 2006b)
5.3.3.3
Comparison of the X-Ray Crystal Structure of S. dublin TLP to TLP Structures from E. coli, B. subtilis and D. rerio
The X-ray crystal structures of the TLP from E. coli (Lundberg et al. 2006), B. subtilis (Jung et al. 2006) and D. rerio (Zanotti et al. 2006) were published shortly after the structure of the S. dublin TLP was published. A comparison of the TLPs from the three species of bacteria with the vertebrate TLP (zebrafish) shows
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little structural divergence. Major differences between the S. dublin TLP and D. rerio TLP were found in the flexible portions of strand B and C that protrude towards the solvent and in the conformation of the loop connecting strands D and E (Zanotti et al. 2006). Greater differences were observed between the structures of TLPs from B. subtilis and D. rerio: the B-C loop was significantly shorter in B. subtilis TLP whilst the loop connecting the short a-helix to strand F was extended. The dimer–dimer interfaces of TLP from prokaryotes and eukaryotes were nearly identical. The location and orientation of the residues present in this region were well maintained, including the highly conserved residues His6, Arg44, His95 and Tyr108 (S. dublin TLP numbering). The only significant difference was found in the C-terminal serine residue (from the YRGS tetrapeptide), which assumed different orientations in the structures from S. dublin, E. coli and B. subtilis compared with D. rario. It is therefore clear that the dimer–dimer interface of TLP has been incredibly well conserved throughout evolution. This suggests that the dimer–dimer interface might be the ligand binding site of TLP.
5.4 5.4.1
The In Vitro Function of the S. dublin TLP Binding of Thyroid Hormones by the S. dublin TLP
Even before the publication of the X-ray crystal structure of a TLP, homology modelling of the likely structures of these proteins led to the hypothesis that it was unlikely that thyroid hormones were the natural ligands of TLPs. Homology modelling of the S. dublin TLP (Sect. 5.2.3) suggested that the shape and charge of the dimer–dimer interface of this protein might preclude the binding of thyroid hormones. To confirm this, experiments characterising the binding of thyroid hormones to the S. dublin TLP were performed using the method published by Chang et al. (1999). The binding of radiolabelled tri-iodothyronine (T3) and tetraiodothyronine (T4) by the S. dublin TLP could not be detected. The resultant scatchard plots suggested low levels of non-specific interactions between the protein and thyroid hormones, indicating that the affinity of the thyroid hormones for TLP was too low to be of physiological significance. This finding supported the hypothesis that it was likely that the dimer–dimer interface of TLP would be too shallow to accommodate bulky compounds bound by TTRs.
5.4.2
Examining the Role of the S. dublin TLP in Purine Metabolism
In order to gain clues as to the likely function of TLP, the genomic context of TLP genes from bacteria was examined. In bacteria, genes encoding proteins which act in a shared metabolic pathway are often found grouped on the chromosome in an
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operon. Genes encoding cytoplasmic TLP sequences were frequently found in a specific genomic context: a purine metabolism operon. Specifically, the cytoplasmic TLP genes were frequently found neighbouring the gene encoding the enzyme uricase. Indeed, a role for TLP in purine metabolism was proposed in 2001. In an effort to develop a greater understanding of purine metabolism in the gram-positive bacterium, Bacillus subtilis, Schultz et al. (2001) generated a series of insertion mutants. One of these mutations was made in the TLP gene which is located immediately downstream of the gene encoding uricase. The bacteria harbouring this mutation were characterised by a reduced rate of proliferation (compared to wild-type) on media containing uric acid as the principal source of nitrogen (Schultz et al. 2001). In gram negative bacteria, the TLP gene was also found in purine metabolism operons. However, the purine metabolism-associated genomic context of the TLP gene was only found for the TLP genes encoding cytoplasmic proteins. There was no consistent genomic context identified for TLP genes encoding periplasmic proteins. For example, the S. dublin TLP gene is immediately downstream of genes (CopR and CopS) encoding a two-component sensor-histidine kinase system for bacterial resistance to copper. Therefore it was unclear whether the S. dublin TLP would share similar functions to TLPs from other bacteria. Purines are significantly smaller compounds that thyroid hormones, whilst sharing similar chemical characteristics. Therefore, the binding of uric acid and allantoin to S. dublin TLP was assessed, using fluorescence quenching. The static quenching of the intrinsic fluorescence of tryptophans results from conformational changes in proteins upon ligand binding (Engelborghs 2001). No such conformational static quenching of the intrinsic fluorescence of the tryptophan residues of S. dublin TLP could be detected following titration with uric acid or allantoin (Hennebry et al. 2006a). These results, however, could not be taken as conclusive evidence of the lack of binding of the compounds by the TLP. Rather, the lack of quenching merely indicated a lack of significant conformational change to the protein in regions containing a tryptophan residue.
5.4.3
Hydrolysis of 5-HIU by the the S. dublin TLP
It had long been reported that that the enzyme uricase oxidises the purine uric acid directly into allantoin (e.g. Howell and Wyngaarden 1960). The enzyme reaction has since been demonstrated to be more complex. Tipton’s group showed in 1997 that uricase oxidises uric acid to a metastable compound, 5-hydroxyisourate (5-HIU) (Kahn et al. 1997). This intermediate decomposes into racemic allantoin nonenzymatically via another unstable intermediate, 2-oxo-4-hydroxy-4-carboxy5-ureidoimadolazine (OHCU) (Fig. 5.5a). The decomposition of 5-HIU was shown to occur over 30 min at pH 7, in vitro (Sarma et al. 1999). In biological systems, only (S)-allantoin is found and the enzyme which degrades this purine, allantoinase, specifically binds the (S) form of the compound (Lee and Roush 1964).
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Fig. 5.5 The hydrolysis of 5-HIU by S. dublin TLP. (a) The uricase degradation pathway showing the role of three enzymes (uricase, TLP and OHCU decarboxylase) in converting uricase to allantoin. (b) Hydrolysis of 5-HIU by wild-type S. dublin TLP and by His6Ala, His95Ala and Tyr108Phe mutant S. dublin TLPs. 0.05 units ml 1 uricase was equilibrated in 50 mM potassium phosphate buffer (pH 7.8) for 3 min. The reaction was commenced with the addition of 50 mM uric acid. The appearance of 5-HIU was monitored spectrophotometrically at 312 nm. The amount of 5-HIU peaked after approximately 3 min and began spontaneous decomposition if left unperturbed. Addition of 0.04 mM wild-type S. dublin TLP resulted in rapid hydrolysis of 5-HIU (10 mmol min 1 mg 1 TLP). Addition of equimolar amounts of His95Ala or Tyr108Phe mutants S. dublin TLPs resulted in reduced hydrolysis (1.2 mmol min 1 mg 1 TLP; activity reduced by 90%). The His6Ala mutation abolished hydrolytic activity (from Hennebry et al. 2006b)
The implication, as hypothesised by Kahn and Tipton (1998), was the involvement of at least two additional enzymes in the degradation of uric acid to allantoin: to increase the rate of decomposition and to facilitate the generation of stereo-specific allantoin. The significance of this hypothesis in the context of a possible function of TLP was not fully appreciated until recently, when it was revealed that the TLP from the gram positive bacterium, B. subtilis, hydrolysed one intermediate in the uric acid degradation pathway, 5-HIU to OHCU (Lee et al. 2005). Importantly, the same researchers established that human TTR did not possess this hydrolytic activity. Ramazzina et al. (2006) have more recently shown that mouse TLP hydrolyses 5-HIU and that the COG3195 protein was responsible for the decarboxylation of
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OHCU to S-allantoin (Fig. 5.5). Thus, the pathway of the conversion of uric acid to S-allantoin via the three enzymes, uricase, TLP (5-HIUase) and OHCU decarboxylase was revealed (Fig. 5.5a). The TLP from B. subtilis was predicted to be cytosolic and its gene was located in a purine metabolism operon. However, it was not clear whether a periplasmic TLP, such as the S. dublin TLP, would also hydrolyse 5-HIU. The recombinant S. dublin TLP was tested for its ability to hydrolyse 5-HIU, using the method published by Lee et al. (2005). One mg of recombinant S. dublin TLP was shown to hydrolyse 10 mol min 1 of 5-HIU (Hennebry et al. 2006b).
5.4.4
Characterisation of the Active Site of S. dublin TLP
In the previous section the ligand binding channel of human TTR was compared with the equivalent region of the crystal structure of S. dublin TLP. Several residues at the dimer–dimer interface of the S. dublin TLP (Fig. 5.4b) were 100% conserved across all TLP sequences but not found in any TTR sequences. Examination of the likely arrangement of these residues indicated a potential catalytic site where 5-HIU might be hydrolysed (Fig. 5.4b). With this in mind, residues which could theoretically perform a role in chemical catalysis were selected for site-directed mutagenesis to see what affect this would have on the hydrolytic activity of TLP. These residues were His6, His95 and Tyr108 (Fig. 5.4b). The mutations His6Ala, His95Ala and Tyr108Phe were introduced into the S. dublin TLP sequence (Hennebry et al. 2006b). The S. dublin TLP His6Ala, His95Ala and Tyr108Phe proteins were synthesised, purified and shown to form homotetramers, in order to rule out the potential global structural importance of the mutated residues. The hydrolytic activity of each mutated S. dublin TLPs was compared with that of wild-type S. dublin TLP. All of these mutations had significant effects on the hydrolytic activity of the protein (Fig. 5.5b). The His6Ala mutation abolished hydrolytic activity which was reduced to a negligible rate of 0.1 mol min 1 mg 1 of protein. The His95Ala and Tyr108Phe S. dublin TLPs had activities of 1.2 mol min 1 mg 1 of protein: a reduction of 90%.The His10Ala mutation completely abolished enzyme activity. The His99Ala and Tyr119Phe mutations reduced activity by more than 90% (Hennebry et al. 2006b).
5.5
Perspectives
We determined that the S. dublin TLP is most likely a periplasmic protein. The periplasm is a layer of peptidoglycan found between the inner and outer membranes of gram-negative bacteria. In these bacteria, the periplasm has a variety of important roles. Several groups of enzymes with specialised functions reside in the
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periplasm. For example, hydrolytic enzymes which can degrade foreign DNA or peptides are important part of the bacteria’s defence system. Some periplasmic enzymes, for example, superoxide dismutase and catalase detoxify oxidising compounds (free radicals) from the external environment. Proteins involved in chemotaxis and nutrient acquisition are localised to the periplasm as are numerous transport proteins which dock to membrane-bound proteins to elicit transport of their substrate into the cytoplasm (for a review, see Oliver 1996). In all bacteria studied to date, the proteins involved in purine metabolism are located in the cytosol (for a review, see Vogels and van der Drift (1976)). Therefore, a role for the S. dublin TLP in purine metabolism is unclear. In addition, Salmonellae lack the upstream enzymes required for generation of 5-HIU: no homologs of xanthine dehydrogenase, uricase or OHCU-decarboxylase have been identified in the genomes of Salmonella species. Nonetheless, import proteins and enzymes for the degradation of downstream compounds, such as allantoin are encoded in the Salmonellae genomes. If 5-HIU is the true ligand of the S. dublin TLP, then the source of 5-HIU must be the external environment, as is the case for allantoin. In vivo, 5-HIU is short-lived and is produced by the non-enzymatic oxidation of uric acid (Santos et al. 1999). 5-HIU is highly reactive and capable of reacting with free radicals, causing lipid oxidation (Santos et al. 1999). It has therefore been proposed that together, TLP and OHCU decarboxylase rapidly detoxify 5-HIU, yielding the relatively inert allantoin (Hennebry et al. 2006a; Ramazzina et al. 2006). In the case of S. dublin, an enteric bacterium, possible sources of oxidised uric acid (i.e. 5-HIU) include the gut of its host: the guts of reptilian and avian Salmonella hosts are particularly high in uric acid (Wright 1995); mammalian hosts secrete uric acid into their guts in response to bacterial infection (Vorbach et al. 2003). The data presented here demonstrate the rapid kinetics by which recombinant S. dublin TLP hydrolyses 5-HIU, a reactive compound known to generate damaging oxidative radicals. Therefore, it is possible that the role of periplasmic TLP in enteric bacteria such as S. dublin is to rapidly detoxify 5-HIU from the external environment. The investigation of the structure and function of the S. dublin TLP represented the first step in furthering our understanding of the non-vertebrate evolution of the TTR molecule. The conservation of catalytic residues at the TLP dimer-dimer interface, which have been demonstrated to be essential for enzymatic activity, indicates that is likely that all TLPs share 5-HIU hydrolytic activity. Furthermore, the distribution of TLPs in all kingdoms, but the representation of TTRs in vertebrates alone, clearly suggests that the transthyretin-like fold originally functioned in purine metabolism. Following the duplication of TLP gene, amino acid substitutions to residues at the dimer-dimer interface dramatically altered the protein’s function whilst its overall structure was unchanged. Thus, the evolution of TLP represents a remarkable case of the divergent evolution from a hydrolytic enzyme (TLP) to a thyroid hormone distributor (TTR).
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References Bailey, T.L., Elkan, C. (1994) Fitting a Mixture Model by Expectation Maximisation to Discover Motifs in Biopolymers. AAAI Press, Menlo Park, CA Blake, C.C.F., Oatley, S.J. (1977) Protein–DNA and protein–hormone interactions in prealbumin: A model of the thyroid hormone nuclear receptor? Nature 268, 115–120 Blake, C.C.F., Geisow, M.J., Oately, S.J. (1978) Structure of prealbumin: Secondary, tertiary and ˚ . J. Mol. Biol. 121, 339–356 quaternary interactions determined by Fourier refinement at 1.8 A Chang, L., Munro, S.L.A., Richardson, S.J., Schreiber, G. (1999) Evolution of thyroid hormone binding by transthyretins in birds and mammals. Eur. J. Biochem. 259, 534–542 DeLano W.L. (2002) The PyMol User’s Manual. Delano Scientific, San Carlos, CA, USA Eneqvist, T., Lundberg, E., Nilsson, L., Abagyan, R., Sauer-Eriksson, A.E. (2003) The transthyretinrelated protein family. Eur. J. Biochem. 270, 518–532 Eneqvist, T., Lundberg, E., Karlsson, A., Huang, S., Santos, C.R.A., Power, D.M., Sauer-Eriksson, A.E. (2004) High-resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J. Biol. Chem. 279, 26411–26416 Engelborghs, Y. (2001) The analysis of time resolved protein fluorescence in multi-tryptophan proteins. Spectrochim. Acta A Mol. Biomol. Spectrosc. 57, 2255–2270 Folli, C., Pasquato, N., Ramazzina, I., Battistutta, R., Zanotti, G., Berni, R. (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett. 555, 279–284 Hennebry, S.C., Wright, H.M., Likic´, V.A., Richardson, S.J. (2006a) Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins 64, 1024–1045 Hennebry, S.C., Law, R.H., Richardson, S.J., Buckle, A.M., Whisstock, J.C. (2006b) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J. Mol. Biol. 359, 1389–1399 Hornberg, A., Eneqvist, T., Olofsson, A., Lundgren, A., Sauer-Eriksson, A.E. (2000) A comparative analysis of 23 structures of the amyloidogeneic protein transthyretin. J. Mol. Biol. 032, 649–669 Howell, R.R., Wyngaarden, J.B. (1960) On the mechanism of peroxidation of uric acids by hemoproteins. J. Biol. Chem. 235, 3544–3550 Jung, D.-K., Lee, Y., Park, S.G., Park, B.C., Kim, G.-H., Rhee, S. (2006) Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc. Nat. Acad. Sci. U.S.A. 103, 9790–9795 Kahn, K., Serfozo, P., Tipton, P.A. (1997) Identification of the true product of the urate oxidase reaction. J. Am. Chem. Soc. 119, 5435–5442 Kahn, K., Tipton, P.A. (1998) Spectroscopic characterisation of intermediates of the urate oxidase reaction. Biochemistry 37, 11651–11569 Lee, K.W., Roush, A.H. (1964) Allantoinase assays and their application to yeast and soybean allantoinases. Arch. Biochem. Biophys. 108, 460–467 Lee, Y., Lee, D.H., Kho, C.W., Lee, A.Y., Jang, M., Cho, S., Lee, C.H., et al. (2005) Transthyretinrelated proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett. 579, 4769–4774 Lundberg, E., Backstrom, S., Sauer, U.H., Sauer-Eriksson, E. (2006) The transthyretin-related protein: structural investigation of a novel protein family. J. Struct. Biol. 155, 445–457 Manzon, R.G., Neuls, T.M., Manzon, L.A. (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen. Comp. Endocrinol. 151, 55–65 Muziol, T., Cody, V., and Wojtczak, A. (2001) Comparison of binding interaction of dibromoflavonoids with transthyretin. Acta Biochim. Pol. 48, 885–892 Oliver, D.B. (1996) The periplasm. In: Neidhart, F.C., Curtiss, R. III, Ingraham, I.L., Lin, E.C.C., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaecter, M., Umbarger, M.E. (eds.) Escherichia coli and Salmonella: Cellular and molecular biology. ASM Press, Washington. pp 88–103
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Power, D.M., Elias, N.P., Richardson, S.J., Mendes, J., Soares, C.M., Santos, C.R.A. (2000) Evolution of the thyroid hormone-binding protein, transthyretin. Gen. Comp. Endocrinol. 119, 241–255 Prapunpoj, P., Yamauchi, K., Nishiyama, N., Richardson, S.J., Schreiber, G. (2000) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am. J. Physiol. Reg. Integr. Comp. Physiol. 279, R2026–R2041 Ramazzina, I., Folli, C., Secchi, A., Berni, R., Percudani, R. (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat. Chem. Biol. 3, 144–148 Sarma, A.D., Serfozo, P., Kahn, K., Tipton, P. (1999) Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolising enzyme. J. Biol. Chem. 274, 33863–33865 Santos, C.X.C., Anjos, E.I., Augusto, O. (1999) Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation. Arch. Biochem. Biophys. 372, 285–294 Schultz, A.C., Nygaard, P., Saxild, H.H. (2001) Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR Transcription activator. J. Bacteriol. 183, 3293–3302 Sunde, M., Richardson, S.J., Chang, L., Pettersson, T.M., Schreiber, G., Blake, C.C. (1996) The crystal structure of transthyretin from chicken. Eur. J. Biochem. 236, 491–499 Vogels, G.D., van der Drift, C. (1976) Degradation of purines and pyrimidines in microorganisms. Bacteriol. Rev. 40, 403–469 Vorbach, C., Harrison, R., Capecchi, M.R. (2003) Xanthine oxidoreductase is central to the evolution and function of the innate immune system. Trends Immunol. 24, 512–517 ˚ resolution: First report on a Wojtczak, A. (1997) Crystal structure of rat transthyretin at 2.5 A unique tetrameric structure. Acta Biochim. Pol. 44, 505–517 Wright, P.A. (1995) Nitrogen excretion: Three end products, many physiological roles. J. Exp. Biol. 198, 273–281 Zanotti, G., Cendron, L., Ramazzina, I., Folli, C., Percudani, R., Rodolfo, B. (2006) Structure of zebrafish HIUase: Insights into evolution of an enzyme to a hormone transporter. J. Mol. Biol. 363, 1–9
Chapter 6
Vertebrate HIU hydrolase: Identification, Function, Structure, and Evolutionary Relationship with Transthyretin Giuseppe Zanotti, Ileana Ramazzina, Laura Cendron, Claudia Folli, Riccardo Percudani, and Rodolfo Berni
Abstract 5-Hydroxyisourate hydrolase (HIUase) is an enzyme widely distributed in prokaryotic and eukaryotic organisms that catalyzes the hydrolysis of HIU into OHCU in the degradation route from urate to (S)-allantoin. During early vertebrate evolution, a duplication event in the gene encoding HIUase gave rise to the thyroid hormone binding protein transthyretin (TTR). Due to the close evolutionary relationship between HIUase and TTR, these two proteins possess a high degree of amino acid sequence similarity, while performing quite different functions. The 3D structure of zebrafish HIUase compares very well with that of TTR: a highly preserved scaffold harbors distinct functional sites located in the same regions of the two proteins. The residues that are differentially conserved in HIUase as compared to TTR map in catalytic regions occupying the outer portions of the two halves of the central channel that transverses the whole tetrameric proteins. The evolution of HIUase into TTR has been accompanied by remarkable changes of the catalytic sites to give rise to a channel open at both ends, thus allowing free access to hormone molecules. Keywords 5-Hydroxyisourate hydrolase, Transthyretin, Molecular evolution, Uric acid degradation, Thyroid hormones
G. Zanotti (*) Department of Biological Chemistry, University of Padua, and ICB-CNR, Section of Padua, Viale G. Colombo 3, 35121 Padova, Italy e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_6, # Springer‐Verlag Berlin Heidelberg 2009
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Introduction
5-Hydroxyisourate (HIU) is the product of the urate oxidase reaction occurring in the ureide pathway, in which the oxidative degradation of urate to (S)-allantoin is accomplished. Urate is generated in purine metabolism, through the degradation of the nucleic acid components adenine and guanine. Depending on the organism, urate, the anion of uric acid which is predominant under physiological conditions, either represents an end metabolic product or is further degraded oxidatively, involving the enzyme urate oxidase, to produce allantoin in the ureide pathway. Specifically, humans lack a functional urate oxidase, a characteristic they share with apes, Dalmatian dogs, birds, reptiles, yeasts and various bacteria. As a consequence, the concentration of urate in human blood serum is remarkably higher than that present in the blood serum of other primates and mammals that possess urate oxidase. In the presence of hyperuricemia, a pathological situation that may affect humans, the poorly soluble urate salts can precipitate in joints, giving rise to gouty arthritis, and can produce kidney stones and renal failure. The reasons for the absence of a urate degradative pathway in humans, associated with high levels of urate in blood serum, are controversial. It should be pointed out, however, that due to its antioxidant properties urate may exert a beneficial effect by neutralizing reactive oxygen species, so that the advantage of having a large amount of this compound may compensate the potential onset of a painful disease. It has long been known that urate undergoes oxidation catalyzed by urate oxidase in a diverse array of organisms. The longstanding question as to the way by which urate in such organisms produces allantoin was not answered until recently. In particular, based on the observation that allantoin could be generated in vitro upon urate oxidase reaction, it was initially thought that allantoin might represent a product of this enzymatic reaction. More recently, it has been established that HIU is the true product of the urate oxidase reaction and that it undergoes a quite slow nonenzymatic decomposition into racemic allantoin (Kahn 1997). By contrast, (S)-allantoin is produced in living organisms, consistent with the enzymatic conversion of HIU into this allantoin stereoisomer in vivo. The two enzymes involved in the ureide pathway leading to (S)-allantoin from HIU in the urate oxidase reaction have indeed been identified. An enzyme catalyzing the hydrolysis of HIU, and therefore designated HIU hydrolase (HIUase), to form 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) was first found in soybean root nodules (Sarma et al. 1999; Raychaudhuri and Tipton 2002). Two homologous HIUases, respectively from Bacillus subtilis (Lee et al. 2005) and from mouse (Ramazzina et al. 2006), which lacked homology with the soybean enzyme, have then been heterologously expressed and identified unambiguously by assaying their catalytic activity. OHCU decarboxylase is the next enzyme in the pathway, catalyzing the decarboxylation of OHCU to produce (S)-allantoin (Ramazzina et al. 2006). Very recently, the crystal structures of OHCU decarboxylase from zebrafish (Cendron et al. 2007) and from Arabidopsis thaliana (Kim et al. 2007) have been determined, revealing features consistent with the stereoselective formation of (S)-allantoin as reaction product. The complete degradation
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Scheme. 6.1 Degradation route from urate to (S)-allantoin
route from urate to (S)-allantoin is illustrated in Scheme. 6.1. It must be considered that HIU can form allantoin nonenzymatically on a time scale of hours, while HIUase and OHCU decarboxylase accomplish this conversion stereoselectively, producing (S)-allantoin, on a time scale of seconds (Ramazzina et al. 2006). Remarkably, the aforementioned HIUases from B. subtilis and mouse have been found to belong to a family of prokaryotic and eukaryotic proteins of unknown function but similar to the vertebrate protein thyroxine binding transthyretin (TTR). The members of this protein family were therefore designated transthyretin-like proteins (TLPs) (Prapunpoj et al. 2000; Hennebry et al. 2006a) or transthyretinrelated proteins (TRPs) (Eneqvist et al. 2003). The presence of members of the TLPs/TRPs family possessing HIUase activity has also been established for the enterobacteria Salmonella dublin (Hennebry et al. 2006b) and Escherichia coli (Lee et al. 2005; Lundberg et al. 2006). We report here on the identification and structural characterization of vertebrate HIUase and on its evolutionary relationship with TTR.
6.2
Identification and Function of Vertebrate HIUase
To find the missing enzymes in the biological conversion of uric acid to allantoin, we sought proteins with a functional link to urate oxidase, the first enzyme of the pathway. Functional links between different proteins can be inferred through genome comparisons, by the identification of a relationship between the corresponding genetic elements. Three criteria were used for this identification. First, we examined genes of unknown function that are found in the neighborhood of the gene coding for urate oxidase. The rationale for this criterion is that genes that have a coordinated expression, and hence a related function, tend to be physically clustered, as observed frequently, but not exclusively, in prokaryotic genomes (Snel et al. 2002; Hurst et al. 2004). Second, we looked for cases in which urate
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oxidase is fused together with other proteins; such instances indicate that the two proteins can interact or be involved in the same metabolic pathway, even when they are encoded by distinct polypeptide chains (Marcotte et al. 1999). Third, we selected genes that show a pattern of presence and absence across a range of genomes that is similar to that observed for urate oxidase; common ‘phylogenetic profiles’ (Pellegrini et al. 1999) of different genes indicate that the different traits are under a common selective pressure and hence there is a functional relationship among them. Two gene families encoding proteins of unknown function have been found to be linked to urate oxidase in accordance with the aforementioned criteria. When the pattern of gene presence or absence across genomes was evaluated in a phylogenetic context (Barker and Pagel 2005), the link between urate oxidase and the other two gene families emerged clearly. Urate oxidase has been gained or lost several times during species evolution; these events have been accompanied by a parallel gain/loss of the other two gene families (Ramazzina et al. 2006). On the basis of the evidence obtained by genome comparison, we devised experiments to investigate the role of the two protein families in urate metabolism. Phylogenetic across-genome correlation indicated that the functional linkage with urate oxidase had been maintained from bacteria to humans (Ramazzina et al. 2006). We therefore chose to probe the activity of mammalian proteins. Genes of murine origin, designated as uraH and uraD, representatives of the two protein families, were expressed in recombinant form, by using E. coli as expression organism, and the corresponding proteins were purified. We then tested the activity of the two proteins on the conversion of HIU produced by the urate oxidase reaction. Because optically active compounds are involved, the formation of the products of the reactions occurring in the presence of the two proteins was conveniently monitored by circular dichroism. In particular, the HIU CD spectrum presents specific maxima (at about 210 and 270 nm) and minima (at about 240 and 310 nm), the OHCU CD spectrum is characterized by a maximum and a minimum at about 230 and 260 nm, respectively, while the CD spectrum of (S)-allantoin shows a maximum at about 215 nm (Ramazzina et al. 2006). The HIU hydrolase and OHCU decarboxylase activities for uraH and uraD, respectively, could thus be definitely established: formation of levorotatory OHCU from levorotatory HIU catalyzed by HIUase and formation of (S)-allantoin from levorotatory OHCU catalyzed by OHCU decarboxylase (Fig. 6.1). The recombinant HIUase orthologue from zebrafish (Danio rerio), possessing a specific activity similar to that of the corresponding murine enzyme, was then obtained in our laboratory and structurally characterized (Zanotti et al. 2006).
6.3
Molecular Evolution of the HIUase/Transthyretin Family
The oxidative degradation of purines is found in bacteria and in the main eukaryotic lineages. In this pathway, there are examples of alternative branches and of single enzymatic steps involving different genes in different organisms, with genes that
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Fig. 6.1 Enzymatic activity of HIUase and OHCU decarboxylase. Time resolved CD spectra of uric acid degradation products in the presence of urate oxidase (a), or in the presence of urate oxidase and HIU hydrolase (b), or in the presence of urate oxidase, HIUase and OHCU decarboxylase (c). Spectra were collected every 2 min. For additional details regarding the kinetics of the enzymatic reactions, see Ramazzina et al. (2006)
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appear to be of more recent acquisition (Ramazzina et al. 2008). Genomic analyses indicate, however, that the enzymatic oxidation of uric acid is always associated with HIU hydrolysis, and that this activity is always encoded by uraH. The uraH gene was thus probably present in the early evolution of the oxidative pathway of purine degradation, which could have started in bacteria after the innovation of oxygenic photosynthesis and the formation of atmospheric oxygen about 2.3 Gyr ago (Mulkidjanian et al. 2006). From bacteria, the pathway could have been horizontally transferred to eukaryotes with mitochondrial symbiosis around 1.8 Gyr ago (Hedges et al. 2001), thus explaining the lack of HIUase and the other enzymes of the pathway in archea. During evolution, most of the uraH-type genes have maintained the original role in purine degradation and a close association with those encoding urate oxidase and OHCU decarboxylase. Similar to urate oxidases and OHCU decarboxylases, HIUases are typically cytosolic proteins in bacteria and peroxisomal proteins in eukarya. However, some innovations have also taken place, as in the case of the periplasmic HIUase of enterobacteria (Hennebry et al. 2006a; Eneqvist et al. 2003) and the expansion and functional diversification of the family observed in nematodes (Jacob et al. 2007; Saverwyns et al. 2008). In vertebrates, the major evolutionary modifications of HIUases have been pseudogenization of the uraH gene, which occurred in man and other vertebrates (Ramazzina et al. 2006), and a gene duplication event that gave rise to TTR (Hennebry et al. 2006a; Zanotti et al. 2006). Consistent with a gene duplication event, in vertebrates the gene encoding HIUase and TTR have the same intron/exon structures (Fig. 6.2a). The first coding exon encodes a signal peptide in TTR proteins and a type 2 peroxisomal targeting signal (PTS) in HIUases. However, the analysis of the Espressed Sequence Tag (EST) of HIUase indicates that, in mouse and other organisms, a shorter protein corresponding to HIUase but lacking the PTS sequence is also produced. Given that HIU is an unstable substrate that should only be produced in peroxisomes, the functional significance of extra-peroxisomal HIUases is unclear. Conservation of the intron position and sequence similarity in the first exon suggest that, after gene duplication, the PTS sequence of HIUase was transformed into the TTR signal peptide by point mutations. The gene duplication event that gave origin to TTR probably occurred after the separation of earliest deuterostoma (500 Myr ago), as suggested by the absence of TTR sequences in the genomes of protochordates. On the other hand, the presence of a TTR sequence in the shark Leucoraja erinacea (GenBank CV221819) indicates that the duplication predates the separation between cartilaginous and bony fishes (400 Myr ago). Consistent with these observations, a phylogenetic reconstruction of the two families shows the branching of TTR slightly before the separation of chordates (Fig. 6.2b). During the early evolution of chordates, profound modifications of the TTR ancestor occurred to adapt the binding cavity to the two thyroid hormones 3,5,30 -triiodo-l-thyronine (T3) and 3,5,30 ,50 -tetraiodo-l-thyronine (thyroxine, T4) (see below).
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Fig. 6.2 Evolutionary comparison of HIUase and TTR. (a) Sequence alignment of the proteins encoded by the uraH genes from mouse (Acc. No: EDL17955), green monkey (Acc. No: ABE02226) and zebra fish (Acc. No: Q06S87) with mouse TTR (Acc. No: EDK96952). The two alternative starting methionines (M1 and M2), the peroxisomal targeting signal (PTS) and the position of introns (with intron phases in parenthesis) are indicated. (b) Phylogenetic relationship of HIUase and TTR proteins. The unrooted phylogenetic tree has been constructed with the maximum-likelihood method implemented in the Proml program of the PHYLIP package (Felsenstein 1981) and displayed with the TreeIllustrator program (Trooskens et al. 2005) with branch length correction for visibility enhancement. Eukaryotic sequences are indicated with the binomial name of species. The major taxonomic groups are indicated
6.4
Crystal Structure of Zebra Fish HIUase
To date, the structure of zebrafish HIUase is the only known structure of a vertebrate HIUase (Zanotti et al., 2006). It has been determined for two crystal forms, monoclinic (P21) and orthorhombic (P212121), both at a relatively good
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Fig. 6.3 (a) Stereo view of a cartoon representation of zebra fish HIUase. The view is along the twofold molecular axis that transverses the central channel of the tetrameric enzyme. (b) Amino acid sequence of zebra fish HIUase aligned with that of human TTR. Secondary structure elements for both proteins are reported (b-strand, arrows; a-helix, spirals). The residues conserved in the two proteins are boxed; those conserved in HIUases and substituted in TTR are denoted by an orange background
˚ , respectively). In the monoclinic crystal form there is resolution (1.98 and 1.70 A one monomer in the asymmetric unit, whilst two monomers are present in the asymmetric unit for the orthorhombic form. The two models are virtually identical, suggesting that the protein conformation is quite rigid and barely influenced by crystal packing. Like TTR, whose molecular model was in fact used to solve the structure by molecular replacement, HIUase is a homo-tetramer, formed by four identical subunits arranged according to a 222 symmetry (Fig. 6.3a: monomers are labeled from A to D). An analysis of a monomer performed with the software Procheck (Laskowski et al. 1993) indicates that it is composed of ten b-strands (position and numbering convention for b-strands are illustrated in Fig. 6.3b), but two of them (strands 1–2 and 8–9) can be considered the two halves of strands bA and bG. Consequently, each HIUase monomer can be described as composed of eight antiparallel b-strands, arranged in a topology similar to the Greek key bbarrel, with a short a-helix located at the end of b-strand E. As in the case of TTR, a dimer is formed through the interactions between strands F and H of a monomer with the corresponding ones of another, resulting in an eight-stranded b-sandwich. In turn, two dimers interact, mainly through hydrophobic contacts, to form the tetramer. Most of the interactions between dimers are established between monomers A and C (and their equivalent B and D), but some contacts are also present between monomers A and D (and B and C).
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Comparison of the Structures of HIUase and Transthyretin
The structure of zebrafish HIUase is very similar to that of human TTR (PDB 1F41, (Hornberg et al. 2000)): a comparison of monomers and dimers of the two proteins ˚ , respectively. Similar values are gives root mean square deviations of 1.0 and 1.2 A found for the comparison between zebrafish HIUase and sea bream (Sparus aurata) TTR (PDB 1OO2, (Folli et al. 2003)). The structures of HIUases from three bacteria (S. dubli, PDB 2GPZ, (Hennebry et al. 2006b); B. subtilis, PDB 2H0E, (Jung et al. 2006); E. coli, PDB 2G2N, (Lundberg et al. 2006)) also compare quite well with that of the zebrafish enzyme. Major differences are found in some loops that are exposed to the solvent and in some cases are flexible in human TTR. The S. Dublin HIUase (PDB 2GPZ) differs from the vertebrate enzyme mainly in the flexible portions of strands B and C that protrude towards the solvent, and in the conformation of the long loop connecting strands D and E. This situation is similar for the E. coli enzyme (PDB 2G2N). More relevant differences are present in the B. subtilis enzyme (PDB 2H0E), in which the loop B–C is shorter, whilst the loop that connects the short a-helix to strand F is longer as compared to zebrafish HIUase. In comparison with TTR, some of the b-strands in zebrafish HIUase present irregularities in the hydrogen bonding pattern. In particular, Pro-105 (residue numbering of zebrafish HIUase, Fig. 6.3b) in the middle of b-strand G hinders the formation of H-bonds with residues of nearby strands: in addition to the lack of interaction between the nitrogen of residue Pro-105 with the carbonyl of Tyr-116 of b-strand H, the carbonyls of Val-104 and Pro-105 do not form H-bonds with nitrogen atoms of His-12 of b-strand A and of Tyr-116 of b-strand H, respectively. This results in irregularities in b-strands A, G and H. However, the major difference between HIUase and TTR is found for the long channel that transverses the entire tetrameric proteins and is coincident with one of their twofold symmetry axes. In TTR, this channel harbors the binding sites for two thyroid hormone molecules, which are accommodated in the two symmetrical halves of the channel (Wojtczak et al. 2001). In HIUase this channel is still present, but is obstructed at both ends by the side chains of Tyr-116, Leu-14, and Leu-107. In particular, the comparison of the molecular surface of the catalytic cavity of HIUase with that of the corresponding region of TTR, i.e. the thyroid hormone binding cavity, shows that the cavity in HIUase is markedly shallower than that of TTR, such that it cannot host the bulky hormone molecule (Fig. 6.4a). The structural comparison also illustrates the evolutionary changes that have made possible the functional transition of the enzyme into a transport protein. In each catalytic cavity of HIUase, two Tyr-116 residues contribute substantially to the formation of the bottom of the cavity. The key molecular event has been the substitution of Tyr-116 with Thr-119 residue of TTR. In TTR, this replacement opens up the two ends of the channel, making it accessible to two hormone molecules. At the same time, all the residues of HIUase potentially relevant for catalysis (see below) are replaced in TTR: His-12/Lys-15, Asp-50/Ser-52, Arg-52/Glu-54, His-103/Thr-106, and Ser-119/Val-122 (Fig. 6.3b). Remarkably, the first three replacements appear to be relevant to allow thyroid
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hormone binding and are therefore associated with the acquisition of the novel functional properties of TTR. The internal portion of the channel, which represents a closed cavity not accessible to the solvent, is filled in the crystal structure of HIUase by five ordered water molecules. The residues lining the internal cavity are quite conserved as compared to TTR.
6.4.2
The HIUase Active Site
Taking into account the positions of conserved residues of putative HIUases from a variety of prokaryotic and eukaryotic organisms, the results of site-directed mutagenesis experiments (Hennebry et al. 2006b; Zanotti et al. 2006; Jung et al. 2006), and the crystal structures of the B. subtilis enzyme in complex with substrate analogues (Jung et al. 2006), the location of two putative catalytic regions in the HIUase tetramer, occupying the two symmetrical entrances of the central channel, can be considered definitely established with high reliability. A model of the crystal structure of zebrafish HIUase with the substrate analogue 8-azaxanthine as it is
a
Fig. 6.4 (continued)
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b
Fig. 6.4 (continued) (a) van der Waals representation of the homo-tetramers of HIUase (left) and TTR (right, PDB ID 1F41). Details of the molecular surfaces of the boxed area of the HIUase active site cavity (left) and of the corresponding area in TTR (right) are shown below van der Waals representations. The TTR-bound thyroxine molecule (stick model, PDB 1IE4, Wojtczak et al. (2001)) is also shown. Different colors correspond to different subunits of the tetrameric proteins. (b) Detail of the active site of zebra fish HIUase, located in between two subunits at one end of the central channel; the residues conserved in the enzyme family are labeled. Due to the molecular symmetry, two active sites are present on opposite sides of the tetramer. Each of them is formed by the same residues belonging to two different subunits
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bound to B. subtilis HIUase (Jung et al. 2006) superimposed reveals that several residues near the HIUase-bound substrate analogue are conserved and identically positioned (not shown), indicating that active site characteristics have been well preserved from prokaryotes to vertebrates during evolution. In particular, His-12, Asp-50, Arg-52, His-103, and Pro-105 are the residues in the area of the active site that are conserved in all HIUases, along with four residues (Tyr-116, Arg-117, Gly-118, and Ser-119) constituting the HIUase signature at its C-terminus. It should be noted that all these residues, due to the twofold molecular axis coincident with the protein central channel, are contributed twice, at each of the two ends of the channel, by two subunits (subunits A and C and subunits B and D) belonging to different dimers (Fig. 6.4b). Site-directed mutagenesis experiments have shown that the replacement of Asp-50 by Asn reduces the enzymatic activity of zebrafish HIUase to about 50%, indicating that this residue is not essential for catalysis (Zanotti et al. 2006). Similar experiments with B. subtilis HIUase (Jung et al. 2006) have shown that the presence of His-12 and of a positively charged residue (Arg or Lys) at position 52 represent absolute requirements for catalytic activity. The residues of the C-terminal signature, except for the last Ser residue, are also essential for the enzymatic activity. While confirming the essential role played by His-12, site-directed mutagenesis experiments with S. Dublin HIUase have shown that mutations replacing His-103 and Tyr-116 affect substantially the activity, without abolishing it completely (Hennebry et al. 2006b). Interestingly, the crystal structure of the E. coli HIUase has revealed Zn2+ and Br ions near the putative enzyme active sites; however, the finding of such ions bound to HIUase might have been caused by their presence in the crystal medium (Lundberg et al. 2006). In this regard, it should be noted that Zn2+ ions do not appear to be required for the catalytic activity of zebrafish HIUase (Zanotti et al. 2006). A catalytic mechanism has been proposed for B. subtilis HIUase (Jung et al. 2006), according to which the major catalytic role is played by His-12 and Arg-52. Since no evident nucleophile provided by a catalytic residue appears to be present near the active site, it has been proposed that His-12 could serve as a general base for the deprotonation of a water molecule, which would carry out a nucleophilic attack on C6 of HIU to generate an oxyanion O6, stabilized by the positively charged Arg-52. In this mechanism, a role for other residues, such as His-103 and Tyr-116, has also been proposed (Jung et al. 2006).
6.5
Conclusions
Despite the similarity with TTR, the function of vertebrate HIUase could be correctly predicted by bioinformatic analysis, through whole genome comparison. HIUase and TTR are proteins with very similar sequence and structure, yet with radically different binding properties and functions. These two families thus illustrate a clear case of functional divergence that occurred in vertebrate proteins. In protein evolution, adaptation of old proteins for new functions is a common
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phenomenon (Todd et al. 2001). This phenomenon is also observed, for example, in the case of another thyroid hormone carrier protein, thyroxine-binding globulin (TGB), which originated from the protease inhibitors serpins (Irving et al. 2000). There are some well-known examples in which a single fold supports both enzymatic and nonenzymatic functions (Todd et al. 2002), as observed in the case of TTR-HIUase. In the majority of these examples, a catalytic progenitor gave rise to a nonenzyme through the substitution of residues that are critical for catalysis and/or substrate binding. However, the substitution of a limited number of residues at the active site pocket is generally sufficient for the acquisition of new functional properties. Almost all of the residues that line the zebrafish HIUase active site have been replaced in human TTR. By contrast, the residues that are located outside the active site are often conserved in the two proteins. In this respect, HIUase and TTR represent a remarkable example of active site modification and functional divergence by accelerated evolution.
References Barker D, Pagel M (2005) Predicting functional gene links from phylogenetic-statistical analyses of whole genomes. PLoS Comput Biol 1:e3 Cendron L, Berni R, Folli C, Ramazzina I, Percudani R, Zanotti G (2007) The structure of 2-oxo-4hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase provides insights into the mechanism of uric acid degradation. J Biol Chem 282:18182–18189 Eneqvist T, Lundberg E, Nilsson L, Abagyan R, Sauer-Eriksson AE (2003) The transthyretinrelated protein family. Eur J Biochem 270:518–532 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376 Folli C, Pasquato N, Ramazzina I, Battistutta R, Zanotti G, Berni R (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett 555:279–284 Hedges SB, Chen H, Kumar S, Wang DY, Thompson AS, Watanabe H (2001) A genomic timescale for the origin of eukaryotes. BMC Evol Biol 1:4 Hennebry SC, Wright HM, Likic VA, Richardson SJ (2006a) Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins Struct Funct Bioinfo 64:1024–1045 Hennebry SC, Law RH, Richardson SJ, Buckle AM, Whisstock JC (2006b) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J Mol Biol 359:1389–1399 Hornberg A, Eneqvist T, Olofsson A, Lundgren E, Sauer-Eriksson AE (2000) A comparative analysis of 23 structures of the amyloidogenic protein transthyretin. J Mol Biol 302:649–669 Hurst LD, Pal C, Lercher MJ (2004) The evolutionary dynamics of eukaryotic gene order. Nat Rev Genet 5:299–310 Irving JA, Pike RN, Lesk AM, Whisstock JC (2000) Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 10:1845–1864 Jacob J, Vanholme B, Haegeman A, Gheysen G (2007) Four transthyretin-like genes of the migratory plant-parasitic nematode Radopholus similis: members of an extensive nematodespecific family. Gene 402:9–19 Jung DK, Lee Y, Park SG, Park BC, Kim GH, Rhee S (2006) Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc Natl Acad Sci USA 103:9790–9795
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Kahn K, Serfozo P, Tipton PA (1997) Identification of the true product of the urate oxidase reaction. J Am Chem Soc 5435–5442 Kim K, Park J, Rhee S (2007) Structural and functional basis for (S)-allantoin formation in the ureide pathway. J Biol Chem 282:23457–23464 Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) Procheck – a program to check the stereochemical quality of protein structures. J Appl Cryst 26:283–291 Lee Y, Lee DH, Kho CW, Lee AY, Jang M, Cho S, Lee CH, Lee JS, Myung PK, Park BC, Park SG (2005) Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett 579:4769–4774 Lundberg E, Backstrom S, Sauer UH, Sauer-Eriksson AE (2006) The transthyretin-related protein: structural investigation of a novel protein family. J Struct Biol 155:445–457 Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D (1999) Detecting protein function and protein–protein interactions from genome sequences. Science 285:751–753 Mulkidjanian AY, Koonin EV, Makarova KS, Mekhedov SL, Sorokin A, Wolf YI, Dufresne A, Partensky F, Burd H, Kaznadzey D, Haselkorn R, Galperin MY (2006) The cyanobacterial genome core and the origin of photosynthesis. Proc Natl Acad Sci USA 103:13126–13131 Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO (1999) Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. Proc Natl Acad Sci USA 96:4285–4288 Prapunpoj P, Yamauchi K, Nishiyama N, Richardson SJ, Schreiber G (2000) Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol Regul Integr Comp Physiol 279:R2026–R2041 Ramazzina I, Cendron L, Folli C, Berni R, Monteverdi D, Zanotti G, Percudani R (2008) Logical identification of an allantoinase analog (puuE) recruited from polysaccharide deacetylases. J Biol Chem 283:23295–23304 Ramazzina I, Folli C, Secchi A, Berni R, Percudani R (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat Chem Biol 2:144–148 Raychaudhuri A, Tipton PA (2002) Cloning and expression of the gene for soybean hydroxyisourate hydrolase. Localization and implications for function and mechanism. Plant Physiol 130:2061–2068 Sarma AD, Serfozo P, Kahn K, Tipton PA (1999) Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme. J Biol Chem 274:33863–33865 Saverwyns H, Visser A, Van Durme J, Power D, Morgado I, Kennedy MW, Knox DP, Schymkowitz J, Rousseau F, Gevaert K, Vercruysse J, Claerebout E, Geldhof P (2008) Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi – comparison with other strongylid nematodes and Caenorhabditis elegans. Int J Parasitol. doi 10.1016/j.ijpara.2008.04.004 Snel B, Bork P, Huynen MA (2002) The identification of functional modules from the genomic association of genes. Proc Natl Acad Sci USA 99:5890–5895 Todd AE, Orengo CA, Thornton JM (2002) Sequence and structural differences between enzyme and nonenzyme homologs. Structure 10:1435–1451 Todd AE, Orengo CA, Thornton JM (2001) Evolution of function in protein superfamilies, from a structural perspective. J Mol Biol 307:1113–1143 Trooskens G, De Beule D, Decouttere F, Van Criekinge W (2005) Phylogenetic trees: visualizing, customizing and detecting incongruence. Bioinformatics 21:3801–3802 Wojtczak A, Cody V, Luft JR, Pangborn W (2001) Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallogr D Biol Crystallogr 57:1061–1070 Zanotti G, Cendron L, Ramazzina I, Folli C, Percudani R, Berni R (2006) Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9
Chapter 7
Transthyretin-Related and Transthyretin-like Proteins A. Elisabeth Sauer-Eriksson, Anna Linusson and Erik Lundberg
Abstract Bioinformatics programs are highly accurate in identifying protein families directly from protein sequences, even when the sequence identity is very low. The transthyretin-related proteins (TRPs) are one example of a protein family that has been identified. Whereas transthyretin (TTR) is well-characterized both in vivo and in vitro, only recently has research focused on the TRPs. Their structural similarity to TTR has been verified, and their function as a 5-hydroxyisourate (HIU) hydrolase has been established. In this review we discuss structural aspects of TRP function. We also discuss the still unknown transthyretin-like proteins (TLPs) that are seemingly unique to nematodes. Keywords Transthyretin-related protein, HIU hydrolase, Transthyretin, Amyloid, Fibril formation
7.1
Introduction
The transthyretin-related proteins (TRPs) comprise a relatively recently identified family of proteins (Eneqvist et al. 2003). Homologues of TRP can be found in a wide range of species from bacteria, plants and animals. Extensive biochemical analysis of the TRP, in combination with structural studies, has highlighted the main structural features that govern their tetrameric assembly and function. The proteins function as hydrolases, catalyzing the second reaction which degrades purine nucleotides in the ureide pathway.
A.E. Sauer-Eriksson (*) Department of Chemistry, Umea˚ University, SE-90187 Umea˚, Sweden e-mail:
[email protected]
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Whereas the physiological relevance of TRP in prokaryotes is relatively clear, the same is not true for its function in vertebrates. Purines are major components of nucleic acids and nucleotides, and purine degradation pathways can be found in all kingdoms of life (Nygaard 1983). In most mammals, allantoin is the final product of the purine degradation pathway and it is excreted as one of the major components in urine (Fig. 7.1). However, humans and other primates lack the enzyme uricase and excrete uric acid as the final product of purine degradation. Some other species (e.g., fish and amphibians) have enzymes that further degrade allantoin to allantoic acid and finally to glyoxylic acid and urea. In prokaryotes, the purine degradation products are mostly utilized as nitrogen sources, but they may serve other functions depending on the organism. In Escherichia coli the pathway seems incomplete as allantoin is produced but not urea and glyoxylic acid (Xi et al. 2000). E. coli can utilize allantoin as a nitrogen source but only under anaerobic conditions (Cusa et al. 1999). In Bacillus subtilis on the other hand a complete purine degradation pathway is present (Fisher 1999; Schultz et al. 2001). The specific function of TRP in the purine degradation pathway is to facilitate the hydrolysis of 5-hydroxyisourate (5-HIU), the end product of the uricase reaction, and to convert it to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) (Fig. 7.1). This catalytic activity was first identified in the TRPs of B. subtilis and E. coli (Lee et al. 2005; Jung et al. 2006), but it has also been verified in Salmonella dublin (Hennebry et al. 2006a) and Mus musculus TRP (Lee et al. 2006; Ramazzina et al. 2006). The need for a 5-HIU hydrolase for this task was initially not apparent as 5-HIU can nonenzymatically convert into allantoin under in vitro conditions. However, the spontaneous conversion of 5-HIU generates a racemic mix of S- and R-allantoins, and the allantoinase, which constitutes the next enzyme in the purine catabolism pathway, is specific for S-allantoins only. From the observation that racemization of allantoin takes several hours, it became apparent that a novel enzyme might be involved (Kahn and Tipton 1998). One year later the first HIU hydrolase, which facilitated stereo-specific conversion of HIU to S-allantoin, was
Fig. 7.1 The purine catabolic pathway
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discovered in soybean (Sarma et al. 1999). By producing the less reactive compound allantoin, HIU-hydrolases like TRP could also serve an important function in the periplasm of enterobacteria, to prevent accumulation of toxic free radical species, generated by antioxidant activity of uric acid (Hennebry et al. 2006b). Occasionally, the TRPs are referred to in the literature as transthyretin-like proteins or TLPs. In addition to the TRPs, which comprise the characteristic sequence motif Y-R-G-S at their C-terminal end, there are protein sequences listed as TTR-like in the databases, with a particularly large number of them in Caenorhabditis elegans. We therefore prefer to refer to the ‘‘Y-R-G-S-motif’’ family as TRP or HIU hydrolase with a TTR-like fold (Eneqvist et al. 2003; Lundberg et al. 2006), to distinguish them from the so-far uncharacterized TLP proteins. There are 57 genes in C. elegans which are listed as TTR-1 to TTR-57 in WormBase (a genetic database for C. elegans and related nematodes; www.wormbase.org). Some of these TLP sequences were originally identified as structurally TTR-like by Sonnhammar and Durbin (1997). Of these, the TLP listed as ‘‘TTR-1’’ has been shown to influence aging in C. elegans (Hansen et al. 2005). The 57 TLP sequences present in C. elegans share low sequence identity with each other; however sequences TTR-18 to TTR-31 seem to comprise a subgroup found in other nematodes. Sequence analysis of representatives from TTR, TRP, and TLP suggests that the three protein groups are not functionally related (Fig. 7.2). Whereas residues at the ligand-binding sites are almost completely conserved in the TTR and TRP families, they are not conserved in the TLP group. In contrast, the most conserved regions in TLP can be found at sites corresponding to b-strands B and E and the a-helix in TTR and TRP. The present review will focus on structural aspects of TRP which have advanced our understanding of this protein family. Recently it has also been established that TRP can form amyloid fibrils (Lundberg et al. 2009; Santos et al. 2008). We will also present results from a bioinformatic study on TLP, and a preliminary 3D model of one TLP from the nematode C. elegans. Interestingly, the model suggests that TLP’s structural similarity to TTR and TRP might not be entirely preserved.
7.2 7.2.1
The Structure of TRP Overall Structure
The sequence identity between TRP and TTR is relatively low; E. coli TRP (ecTRP) shares 30% sequence identity with hTTR (Fig. 7.2) (Prapunpoj et al. 2000; Eneqvist et al. 2003). In addition to the C-terminal Y-R-G-S motif, four more motifs have been identified as defining TRPs, and can be used to distinguish them from TTRs (Hennebry et al. 2006b). Structures of TRP have been determined from several species, and include those from E. coli (Lundberg et al. 2006),
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B
TRP-D.rerio (2H1X) TRP-S.dublin (2GPZ) TRP-B.subtilis (2H0E) TRP-E.coli (2G2N)
* * * * * ---------------MNRLQHIRGHIVSADKHINMSATLLSPLSTHVLNIAQGVPGANMT ---------------MKRHILATVIASLVAAPAMALAAGNNILSVHILDQQTGKPAPGVE --------------------------------MSEPESLMGKLTTHILDLTCGKPAANVK --------------MLKRYLVLSVATAAFSLPSLVNAAQQNILSVHILNQQTGKPAADVT
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TTR-G.gallus (1TFP) TTR-H.sapiens (1F41) TTR-S.aurata (1SN2) TTR-R.norvegicus(1GKE)
------MAFHSTLLVFLAGLVFLSEAAPL--VSHGSVDSKCPLMVKVLDAVRGSPAANVA ------MASHRLLLLCLAGLVFVSEAGP-----TGTGESKCPLMVKVLDAVRGSPAINVA -------MLQPLHCLLLASAVLLCNTAPTPTDKHGGSDTRCPLMVKILDAVKGTPAGSVA ------MASLRLFLLCLAGLIFASEAGP-----GGAGESKCPLMVKVLDAVRGSPAVDVA
32 29 32 22
TLP-H.glycines TLP-R.similis TLP-B.malayi TLP-X.index TLP-C.elegans-TTR-30 TLP-C.briggsae-TTR-18
---MFR--FSLLLLLVPLVR----CVPNPKPLFGIGIGRKQSAGAEGKLTCAGEPLADVK MAPMFLPTVSVFLLLLVVVQQSLLVLASPKPLFGIGIGRTQSAGVEGTLLCEGKPMADVL ---MFR------LLLLLIIS-----LPIVFCAFGGLVGRVQSTGVRGTLMCNGRPAPRVL MEK---------AVLVVCVF------FCLLPVTLSSLGQRQRVIVKGRLLCGNAPASNIR --------MSSVYSCLILLT-----FIVASFCISIEIGNLQSVSVNGTLLCNDKPAKNIK ------------MHQLILVA-----LFVSTASSLPFIGSVQSVRVTGKVTCNGSPAENIK * * . . * : * . * :
35 33 29 26 26 27
B
C
D
E
a
TRP-D.rerio TRP-S.dublin TRP-B.subtilis TRP-E.coli
* * IVLHRLDPVSSAWNILTTGITNDDGRCPG--LITKENFIAGVYKMRFETGKYWDALGE-VVLEQKKDN--GWTQLNTGHTDQDGRIKA--LWPEKAAAPGDYRVIFKTGQYFESKKL-IGLKRLGES-----IMKEVYTNNDGRVDVP-LLAGEELMSGEYVMEFHAGDYFASKNMNA VTLEKKADN--GWLQLNTAKTDKDGRIKA--LWPEQTATTGDYRVVFKTGDYFKKQNL--
82 74 82 77
TTR-G.gallus TTR-H.sapiens TTR-S.aurata TTR-R.norwegicus
VKVFKKAAD-GTWQDFATGKTTEFGEIHE--LTTEEQFVEGVYRVEFDTSSYWKGLGL-VHVFRKAAD-DTWEPFASGKTSESGELHG--LTTEEQFVEGIYKVEIDTKSYWKALGI-LKVSQKTAD-GGWTQIATGVTDATGEIHN--LITEQQFPAGVYRVEFDTKAYWTNQGS-VKVFKRTAD-GSWEPFASGKTAESGELHG--LTTDEKFTEGVYRVELDTKSYWKALGI--
87 84 87 77
TLP-H.glycines TLP-R.similis TLP-B.malayi TLP-X.index TLP-C.elegans-TTR-30 TLP-C-briggsae-TTR-18
VKLYDDDRGVDTDDLMGETRTDSEGRFRLEGYTHEITTIDPKINIYHDCNDGLKPCQR-VKLYDDDRGVDTDDLMAEGKTDSKGRFSLEGYTHEFTTIDPKINIYHDCND-LLPCQR-IKLYDDDRGIDMDDFMGETKSDSQGNFELSGYIHEMSPIDPKFNIYHDCNDGIKPCQR-VKLVDEDDGPDPDDDMDDGYTNDNGEFLLDGQQTEISPIDPVLKIYHDCND-GLPCQR-VKLYEEEAILDV--LLDERFTKDDGTFEMAGSKSEVTTIDPKLNIYHKCN-YDGICVR-VKLYEKEILLDK--LLDERSTDVKGSFSLAGNKKELTAIDPHVNIYHKCN-YKGVCYK-:** :.: * : : :. .* * : * *.:.*** .:***.** * :
93 90 87 83 81 82
F
G
H
TRP-D.rerio TRP-S.dublin TRP-B.subtilis TRP-E.coli
*** * * * * *** --TCFYPYVEIVFTITNTS-QHYHVPLLLSRFSYSTYRGS------DTFFPEIPVEFHISKTN-EHYHVPLLLSQYGYSTYRGS----ADQPFLTIVTVRFQLADPD-AHYHIPLLLSPFGYQVYRGS------ESFFPEIPVEFHINKVN-EHYHVPLLLSQYGYSTYRGS-----
119 111 121 114
TTR-G.gallus TTR-H.sapiens TTR-S.aurata TTR-R.norwegicus
--SPFHEYADVVFTANDSGHRHYTIAALLSPFSYSTTAVVSDPQE --SPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE --TPFHEVAEVVFDAHPEGHRHYTLALLLSPFSYTTTAVVSSVRE --SPFHEYAEVVFTANDSGHRHYTIAALLSPYSYSTTAVVSNPQN
130 127 130 120
TLP-H.glycines TLP-R.similis TLP-B.malayi TLP-X.index TLP-C.elegans-TTR-30 TLP-C-briggsae-TTR-18
--KISIMIPDKYIASGEHPNTYYDAGTVELEGKFSGETRDCLH---KISIMIPDKYISSGKTPERYYNAGEVELEGKFKGEERDCLH---KISIMIPDGYITEAKTPRKWYNAGTIELAGKFAGETRDCIH---KWRFKIPNKYIVDPEDSSTVMNMGTWNLEPIADDEERDCIH---KISILIPTEYITNGEKPARTFNVGELNLASKFSGQSTDCFN---KLKIKIPKSFISEGETAERTFDIGELNLAGSFSGESTDCLN-* : ** :* . : . : * :* .: **::
134 131 128 124 122 123
Fig. 7.2 TRP representatives from six nematodes have been aligned with representatives from TTRs and TRPs with known 3D structures. Sequence numbering for TTR and TRP are identical to the ones in their respective structures, the first residue being marked with a yellow box. The first residue after the predicted signal sequence cleavage (PHOBIUS, Kall et al. (2004)) is marked with a black box. TRPs from D. rerio and B. subtilis are non-cytoplasmic proteins. For human and chicken TTRs, the yellow box is identical to the position of the black box, thus the black box is not shown. Identical and homologous residues within the three subgroups are marked in red and pink, respectively. Similarity was defined as amino acid substitutions within one of the following groups: FYW, IVLMF, RK, QDEN, GA, TS, and HNQ. Identical and similar residues within the TRP family are shown in dark and light green; within the TTR family in dark and light blue; and within the TLP subgroup in dark and light gray. The secondary structure elements are from human TTR (Hornberg et al. 2000). Residues lining the substrate-binding channel in TTR (Wojtczak et al. 2001) are shown in turquoise stars, and in TRP as red stars (Jung et al. 2006). The assignment (*.:) shown below the sequences is directly from ClustalW2 (Larkin et al. 2007) and refers to alignment of the six TLP sequences only
S. dublin (Hennebry et al. 2006a), Danio rerio (Zebra fish) (Zanotti et al. 2006), and B. subtilis (Jung et al. 2006). Despite their low sequence identity the structures are found to be very similar to TTR (Fig. 7.3a, b). Each monomer is composed of an
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Fig. 7.3 (a) The structure of ecTRP drawn from coordinates 2g2n (Lundberg et al. 2006). The four conserved residues that constitute the active site are shown as balls-and-sticks representation. Two 8-azaxanthine molecules are modeled in the active site (Jung et al. 2006). (b) Superposition of the E. coli structure (pdb code 2g2n, green) with the structures of S. dublin (pdb code 2gpz, gray), Zebra fish (pdb code 2hix, red), and B. subtilis (pdb codes 2h0e, blue). The positions of the b-strands A-H are indicated
eight-stranded antiparallel b-sandwich where each sheet has a Greek-key topology. The b-strands in the monomers are named A-H to follow the nomenclature used for TTR. Furthermore there is one short a-helix per monomer. Two monomers
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dimerize through main-chain-main-chain interactions involving the H-strands and the F-strands. Two of the resulting extended 8-stranded b-sheets (DAGHH0 G0 A0 D0 ), one from each dimer, interact face-to-face, which create the ligand-binding site of the TRP tetramers. The tetrameric form of ecTRP is formed mainly by hydrophobic contacts involving residues situated at the AB- and GH-loops. In addition to the structure of ecTRP solved in our lab (pdb code 2g2n (Lundberg et al. 2006)), one more structure of the same protein from E. coli has become available; however, this work has not been published (pdb code 2igl, deposited by Zuo and Malhotra). The two E. coli structures are almost identical as can be ˚ , obtained by deduced from the low root mean square (r.m.s.) deviation of 0.5 A superimposition of the main chain Ca atoms. The structures deviate mostly at their N-terminal ends, and at some loop regions. Two monomers, in the 2igl structure, A and B, have well-defined N-terminal ends in the electron density. In both of them, the His22 residue is positioned at the entrance of the active site, sandwiched against the side-chain of Arg135. The side chain of A-His22 furthermore forms a hydrogen bond with the main-chain oxygen atom of D-Glu39, and the side chain of B-His22 forms a hydrogen bond to the main-chain oxygen atom of C-Gly136. Thus it appears that the AB-His22 side chains are contributing to the tetramerization of the molecule. It should be noted however that the first four amino acids of the construct, His22-Met23, are part of the vector design and not part of the native protein. Consequently, the position of the first two visible amino acids in 2igl, His22, and Met23, is of no biological relevance. The first ordered amino acid in the 2g2n coordinates is Asn4 (Asn27 in 2igl). The 2igl structure was determined from crystals grown in the absence of zinc ions, something our lab has not been successful in doing. It seems plausible that the additional residues His22 and Met23 positioned at the entrance of the active-site cavity could provide the stability needed for crystal-formation without the presence of zinc. Additional structural discrepancies between the 2igl and 2g2n structures are present in loop regions. In particular these occur at the BC-loops, which constitute the ‘‘stirrups’’ of the upper part of the molecule that we refer to as ‘‘the saddle’’ (Fig. 7.3a). These changes are probably due to different crystal packing interactions at this site. Superposition of the E. coli 2g2n structure with the structures of S. dublin (pdb code 2gpz), Zebra fish (pdb code 2hix), and B. subtilis (pdb codes 2h0e) revealed a highly conserved core structure, with structural changes among the TRP structures at the BC-, DE-, and EF-loop regions. These loops are situated on the same side of the b-barrel (Fig. 7.3b). Rms devations of Ca atoms in the monomers ˚. varies between 0.5 and 1.2 A
7.2.2
Composition of the Active site
The localization of the gene supported a functional role for TRP involving purine catabolism linked to urate oxidase (Eneqvist et al. 2003). From modeling of the E. coli TRP structure it was apparent that the putative ligand-binding site was
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almost entirely conserved and positioned at the same site as for TTR (Eneqvist et al. 2003). The first TRP structures provided the detailed descriptions of the ligandbinding site. In parallel, functional studies verified that the protein actually was an enzyme possessing 5-hydroxyisourate (HIU) hydrolase activity. Structures of TRP in complex with substrate analogues, 8-azaxanthine and 5,6,-diaminouracil, were also determined, and revealed the details of the active site composition upon ligand binding (Jung et al. 2006). The complex studies were done on B. subtilis TRP and provided the first structural evidence that these analogues were indeed ligands of TRP. Overall, binding of substrate analogues caused no large changes in the conformation of the tetrameric proteins; r.m.s. deviation values between the apo ˚ (Jung et al. 2006). As for and the analogue-complexed structures were 0.3–0.4 A TTR, modeling and refinement of bound ligands in TRP were hampered by the twofold symmetry-axis positioned right at the ligand-binding site. Due to this twofold axis, one ligand molecule had to be built with an occupancy of 0.5 with the other orientation generated by the crystallographic twofold symmetry. This procedure resulted in a structure in which two ligands were seemingly positioned very close and on some occasions even on top of each other. The electron density map over the bound ligand displayed an average of the two conformations, which made the density harder to interpret. Consequently, the positions of molecule residuals could have quite high positional errors associated with them. In the case of the B. subtilis TRP substrate–analogue complexes, the ligands could be satisfactorily modeled in the electron density, and a plausible mechanism for HIU hydrolysis was proposed which was also supported by site-directed mutagenesis studies (Jung et al. 2006). In this catalytic mechanism, four residues seem central to the composition of the active site (E. coli numbering in coordinates 2g2n are shown in brackets): Arg49 (Arg47) and His105 (His98) are positioned at the entrance of the active site cavity; His14 (His9) makes up its central part; and Tyr118 (Tyr111) is positioned at the innermost site forming the back-wall of the cavity. In the proposed TRP mechanism, unlike other HIU hydrolases, where negatively charged residues, e.g., glutamates, function as a nucleophile, a water molecule has to be deprotonated, presumably by donating a hydrogen to His14, after which it makes a nucleophilic attack on the C6 carbon of the 5-HIU substrate (Fig. 7.4). The resulting oxyanion O6-moiety is negatively charged and neutralized by the proximity of the guanidinium group of Arg49. Subsequent electron rearrangements on the 5-HIU substrate result in ring cleavage at the C6-N1 bond, most likely by abstracting a proton from the guanidinium group of Arg49. The catalytic cycle is then completed with the transfer of the proton from His14 to Arg49 (Jung et al. 2006). Results from site-directed mutagenesis studies conclusively verified the crucial roles of His14 and Arg49. Significantly, a Tyr114Phe mutant lost its activity, suggesting that also this residue is involved in the catalysis, presumably by interacting with atom O8 from the substrate 5-HIU (Jung et al. 2006). The absolute conservation of the residues His14, Arg49, His105, and Tyr114 suggests that the same mechanism is likely to be valid for all members of the TRP family (Fig. 7.2). The ecTRP structure solved in our lab did not crystallize unless zinc ions were included in the crystallization conditions. Overall, five zinc-binding
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Fig. 7.4 The proposed catalytic mechanism for HIU hydrolysis by TRP, based on work from Jung et al. (2006)
sites are present per monomer, of which two are positioned at the active site, tetrahedrally coordinated to residues His9, His96 and His98, Ser114 and three water molecules (Lundberg et al. 2006). The side chains of these residues have locked positions that do not seem compatible with substrate binding, as judged by homology modeling based on the B. subtilis complex structures. However, in the E. coli 2igl structure, where no zinc ions were present in the crystallization conditions, these side chains have rotamers resembling those found in the substrate-analogue complexes of B. subtilis. Since we have not been able to identify any functional role for zinc-binding in the E. coli TRP protein, it is reasonable to assume that the sidechains of the active site residues in E. coli TRP can adopt the rotamer conformation required for catalytic activity as seen from the B. subtilis complex structure.
7.3
The Amyloidogenic Properties of TRP
In TTR amyloidoses, the normally folded, secreted protein cannot assemble into amyloid fibrils unless a preceding partial unfolding event occurs (Colon and Kelly 1992). How important are tertiary similarities as opposed to sequence identity for proteins with a TTR-like fold to form fibrils? In an attempt to answer this question, we and others have investigated the fibril-forming properties of ecTRP in vitro, and compared the results with those from hTTR (Lundberg et al. 2009; Santos et al. 2008). The results showed that ecTRP has the ability to form Congo red-positive
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fibrils in vitro if the temperature is high enough. The fibril heights were found to be 4.0 nm for ecTRP by atomic force microscopy (AFM) (Fig. 7.5) (Lundberg et al. 2009). This is significantly higher than the 2.8 nm height of hTTR also measured by AFM (Lundberg et al. 2009). With transmission electron microscopy (TEM) the fibril diameter size was estimated to 8.0 nm for ecTRP (Santos et al. 2008). Dimensions of molecules estimated by AFM and TEM are however not directly comparable. Interestingly, misfolded and aggregated ecTRP material has been shown to be toxic for neuroblastoma cells, although the soluble protein is not (Santos et al. 2008). Still, it is not clear if the fibril-formation properties of ecTRP have any biological implication (Lundberg et al. 2009; Santos et al. 2008).
7.4
Structural Model of TLP
The sequence identity between TLP., TTR and TRP is very low. For example, pairwise alignment identified no similarity between the C. elegans TLP named TTR-30 and hTTR, and only 22% sequence identity over 79 residues comparing TTR-30
Fig. 7.5 Atomic force microscopy images of ecTRP. The samples were incubated at 65 C for 72 h (courtesy of Anders Olofsson, sample preparation as described in Lundberg et al. (2009)). The white scale bar is 500 nm
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and ecTRP. Comparison of sequence-aligned TLP, TTR, and TRP proteins with the 3D structures of TTR and TRP showed that insertions and deletions are situated exclusively at the N- and C-terminal ends, the surface exposed BC- and DE-loops, and the a-helix (Fig. 7.2). The AB- and GH-loops comprising the dimer–dimer interface at TTR and TRP are well conserved in length although the GH-loop is not well conserved in sequence. Secondary structure prediction with JPRED (Cole et al. 2008) suggested a slightly modified TTR-like fold (Fig. 7.6). Whereas TTR and TRP have only a short three-residue long D-strand, the program predicts an extended D-strand comprising nine residues in TLP. Furthermore, there seems to be no a-helix in the TLP structure. Two comparative models of the C. elegans. protein TTR-30 based on the X-ray crystallographic structures of human TTR (pdb code 1f41) and E. coli TRP (pdb code 2g2n) were made using MOE software (Molecular Operating Environment, Version 2007.09, Chemical Computing Group Inc., www.chemcomp.com) (Fig. 7.7a). The resulting models were very similar to each other, but do not seem to have converged into reasonable structures. We have not attempted however to
A Pred: hTTR:
B
---CCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCEEEEEEECC ---MASHRLLLLCLAGLVFVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAA-37
Pred: ------CCHHHHHHHHHHHHHHHHHHHCCCCCCCCEEEEEECCCCCCCCCCEEEEEECCC ecTRP: ------MLKRYLVLSVATAAFSLPSLVNAAQQNILSVHILNQQTGKPAADVTVTLEKKAD-31 Pred: -----CCHHHHHHHHHHHHHHHHHHHHHHCCCCEEEEEEEEEECCCCCCCCEEEEEECCC TTR-30 -----MSSVYSCLILLTFIVASFCISIEIGNLQSVSVNGTLLCNDKPAKNIKVKLYEEEA-34
C Pred: hTTR:
D
E
F
CCCEEEEEEEECCCCCCCCC--CCCCCCCCCCEEEEEEEEHHHHHCCCCCCCCCEEEEEE DDTWEPFASGKTSESGELHG--LTTEEQFVEGIYKVEIDTKSYWKALGISPFHEHAEVVF-95
Pred: C-CCEEEEEEECCCCCCCCC--CCCCCCCCCCEEEEEEEHHHHHHCCCCCCCCCEEEEEE ecTRP: N-GWLQLNTAKTDKDGRIKA--LWPEQTATTGDYRVVFKTGDYFKKQNLESFFPEIPVEF-88 Pred: CCCC-CCCCCECCCCCEEEEEEEEECCCCEEEEEEEEEECC-CCCCCCEEEEEEECCCCC TTR-30 ILDV-LLDERFTKDDGTFEMAGSKSEVTTIDPKLNIYHKCN-YDGICVRKISILIPTEYI-92
G Pred: hTTR:
H
EEECCCCCCEEEEEEECCCCCCEEEEEECCCC TANDSGPRRYTIAALLSPYSYSTTAVVTNPKE-127
Pred: EEECCC-CCEEEEEEECCCCCCCCCCC----ecTRP: HINKVN-EHYHVPLLLSQYGYSTYRGS------114 Pred: CCCCCCCCEEEEEEEEEECCCCCCCEECCC-TTR-30 TNGEKPARTFNVGELNLASKFSGQSTDCFN---122
Fig. 7.6 Prediction of secondary structure of hTTR, ecTRP, and. TLP (TTR-30) by the program JPRED (Cole et al. 2008). The symbols E, H, and C indicate high probability for b-strands, ahelices, or coiled structures, respectively. The predicted b-strands and a-helices are highlighted in green and yellow boxes, whereas structurally verified b-strands and a-helices are marked in blue and red boxes
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make an extensive refinement of the model, because our purpose was solely to elucidate whether or not TLP structures would have a TTR-like fold. In the TLP models, the hydrophobic core in the monomers is not preserved and includes four buried charged residues. In agreement with the predicted secondary structure, the models also show that the amino acid substitutions at the BC-loop, i.e., ‘‘the stirrups’’ lead to a structural collapse of this b-hairpin and a number of surface-exposed hydrophobic amino acids. Furthermore, conserved amino acids within the TLP family are mostly situated on the surface of the proteins, and in particular at the area we refer to as the ‘‘saddle’’ (Fig. 7.7b). In the saddle, the secondary structure is not predicted to be a-helical, and we note that the a-helix is more distorted in the TLP models. Combined, these features in the TLP models suggest that TLPs have structures not identical to the TTR-like fold. Inconsistencies are present and in particular the saddle-stirrup area most likely deviates from the TTR and TRP proteins. It is plausible that members of the TLP group/family are targeted for ligands other than thyroid-hormones and 5-hydroxyisourate, and therefore their ligand-binding site is not at the dimer-dimer interface. Interestingly, hTTR has been shown to possess a cryptic protease activity that could be inhibited with serine protease inhibitors (Liz et al. 2004). The TTR-mediated proteolysis was not inhibited by thyroid-hormone binding, suggesting that the protease active site is not situated at the hormone-binding site (Liz et al. 2004). The residues lining the hormone/5-hydroxyisourate-binding site in TTR and TRP are not conserved in TLP. However, the four residue stretch D-C-L/F-H at the C-terminus of TLP seems distinctive for its family, or at least for a sub-group of them. These residues are positioned three residues downstream of the Y-R-G-S motif in TRP. In hTTR, this region is unstructured (Hornberg et al. 2000). Another interesting feature of the TLP models, with possible structural implications unrelated to ligand binding, is that two symmetry-related Cys17 residues are modeled right at the dimer-dimer interface. The distance between the symmetry˚ , which indicates that if the protein retains some features of related Sg atoms is 4 A the TTR-like fold, we would expect these two symmetry-related cysteines to form a disulfide-bond. Furthermore, Cys73 and Cys79, positioned in the a-helices of the TRP and TTR structures, might also form a disulphide bond, especially if the a-helix has another conformation in TLP, as indicated by secondary structure prediction.
7.5
Conclusions
Recent structural work on the TRPs and studies of their catalytic process provide a new understanding of how these enzymes work. Structural similarities between TRP and TTR suggest that the TRP gene at some point duplicated in the first primitive vertebrates to produce the TTR gene (Prapunpoj et al. 2000; Eneqvist et al. 2003). The fact that TRP and TTR retain their overall structures points to a stable fold ideally suited to function as a rigid scaffold for differently evolved functions. This is supported by the presence of TLPs, nematode-specific protein
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Fig. 7.7 (a) Model of TLP from C. elegans (TTR-30) based on hTTR (pdb code 1f41, Hornberg et al. (2000)). The sequences were aligned as seen in Fig. 7.2. Water molecules, ions, and metal ions were deleted from the template pdb-files before the modeling procedure. Coordinates of nonidentical side chains were selected from a rotamer library generated from high-resolution PDB data and the energy minimizations of final comparative models were made using Amber99 force field. (b) Visualizing the conservation of the three-dimensional structure. Identical and homolgous residues of six aligned TLP sequences shown in Fig. 7.2 are drawn in black and gray, respectively
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sequences. Bioinformatic analysis and structural modeling suggest that TLPs have, at least in part, a TTR-like fold, but with unrelated function to TRP and TTR. Further investigations of TLPs are needed to determine their functional and biological role, and their structural relationship to TTR and TRP.
Acknowledgements The authors thank Uwe H. Sauer and Tobias Hainzl for valuable discussions, and Terese Bergfors for critical reading of the manuscript.
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Liz MA, Faro CJ, Saraiva MJ, Sousa MM. 2004. Transthyretin, a new cryptic protease. J Biol Chem 279:21431–21438. Lundberg E, Backstrom S, Sauer UH, Sauer-Eriksson AE. 2009. The transthyretin-related protein: structural investigation of a novel protein family. J Struct Biol 155:445–457. Lundberg E, Olofsson A, Westermark GT, Sauer-Eriksson AE. 2009. Fibril-formation properties of human and piscine transthyretin, and E. coli transthyretin-related protein. FEBS J 276 (7):1999–2011. Nygaard P. 1983. Utilization of preformed purine bases and nucleosides. In: A. Munch-Petersen (ed.), Metabolism of nucleotides, nucleosides and nucleobases in microorganism. Academic Press, London, UK p. 27–93. Prapunpoj P, Yamauchi K, Nishiyama N, Richardson SJ, Schreiber G. 2000. Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin. Am J Physiol Regul Integr Comp Physiol 279:R2026–R2041. Ramazzina I, Folli C, Secchi A, Berni R, Percudani R. 2006. Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat Chem Biol 2:144–148. Santos SD, Costa R, Teixeira PF, Gottesman M, Cardoso I, Saraiva MJ. 2008. Amyloidogenic properties of transthyretin-like protein (TLP) from Escherichia coli. FEBS Lett 582:2893– 2898. Sarma AD, Serfozo P, Kahn K, Tipton PA. 1999. Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme. J Biol Chem 274:33863–33865. Schultz AC, Nygaard P, Saxild HH. 2001. Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J Bacteriol 183:3293–3302. Sonnhammer EL, Durbin R. 1997. Analysis of protein domain families in Caenorhabditis elegans. Genomics 46:200–216. Wojtczak A, Cody V, Luft JR, Pangborn W. 2001. Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallogr D Biol Crystallogr 57:1061–1070. Xi H, Schneider BL, Reitzer L. 2000. Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage. J Bacteriol 182:5332–5341. Zanotti G, Cendron L, Ramazzina I, Folli C, Percudani R, Berni R. 2006. Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363:1–9.
Chapter 8
The Transthyretin–Retinol-Binding Protein Complex Hugo L. Monaco
Abstract Retinol-binding protein (RBP), the specific transporter in plasma of retinol and transthyretin (TTR), transporter of thyroid hormones, form in higher vertebrates under physiological conditions, a macromolecular complex that prevents glomerular filtration of the low molecular mass RBP. The crystal structures of two RBP–TTR complexes have shed light on many aspects of the interaction between the two transporters and explained observations made with other experimental methods. Since higher levels of RBP are associated with insulin resistance in obesity and type 2 diabetes it has been suggested that dissociation of the complex with the consequent renal elimination of RBP might be a way to treat the disease. Keywords Transthyretin, TTR, Retinol-binding-protein, RBP, RBP4, Retinol, Insulin resistance, Stra6
8.1
The Complex
Vertebrates obtain vitamin A from the diet either as carotenoids or as long chain fatty acid esters of retinol which, prior to absorption, are in both cases converted to free retinol, the form of the vitamin taken up by the intestinal mucosa. After cellular uptake the free retinol is reesterified in the mucosal cell by long chain fatty acids and incorporated with other lipids into chylomicrons that are absorbed in the lymphatic system. Chylomicrons transport retinyl esters to the liver, their main storage organ, and there the vitamin is laid away mainly in the stellate cells (Harrison 2005; Senoo et al. 2007). Mobilization of retinol from the liver to the
H.L. Monaco Biocrystallography Laboratory, Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_8, # Springer‐Verlag Berlin Heidelberg 2009
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peripheral target tissues takes place after the esters are hydrolysed and the free retinol is bound to plasma retinol-binding protein (plasma RBP or RBP4), the specific protein that solubilises, carries, and protects the vitamin in vertebrate plasma (Kanai et al. 1968). RBP is synthesized mainly in the liver, secreted bound to retinol with a 1:1 stoichiometry and is normally found in plasma forming a macromolecular complex with transthyretin (TTR) (Ingenbleek and Young 1994; Hamilton and Benson 2001, see also the relevant chapters of this book). The complex with TTR can be formed by both apo- and holo-RBP; however, the dissociation constant of the former is significantly higher. This observation supports the proposal of a delivery mechanism of the vitamin in which the stable complex containing retinol is retained in plasma, while the single low-molecular-weight apoRBP is cleared from circulation by glomerular filtration (van Bennekum et al. 2001). The normal RBP4 concentration in human plasma is about 2 mM and that of TTR about 4.5 mM and therefore there is an excess of the latter, and as a consequence virtually all of the holo-RBP molecules present in plasma are complexed to TTR. Each TTR tetramer with a molecular mass of 54,000 binds one RBP molecule with a molecular mass of 21,000 and therefore the molecular mass of the complex present in plasma is about 75,000. If the complex is formed in vitro, this stoichiometry may vary and the crystallographic work on the complex has definitively established that this stoichiometry cannot include more than two RBP molecules per TTR tetramer (Monaco 2000). This result was confirmed by mass spectrometry (Rostom et al. 1998) which was also used to estimate the dissociation constants of the first and the second RBP molecules in the complex. The values found with this method are 1.5 107 M for the first RBP molecule bound and 3.5 105 M for the second, and they are in agreement with the results that were determined with other techniques. The different values of the dissociation constants support an earlier proposal that binding of the first RBP molecule induces cooperative effects that decrease the affinity of TTR for the second RBP molecule. Although it has been shown that RBP has an affinity for other retinoids, the naturally occurring complex contains exclusively all-trans retinol bound to RBP. When the affinity for TTR of RBP bound to different ligands is examined in vitro, it is observed that the formation of the complex is impaired or even prevented when there is a bulky group exposed on the surface of the RBP molecule, confirming the role of retinol in conferring specificity to the interactions between the two macromolecules (Berni and Formelli 1992; Malpelli et al. 1996). The complex has been studied in several different organisms, and cross-reactivity among RBP and TTR from different species, was demonstrated many years ago (Kopelman et al. 1976). The values of the dissociation constants were comparable among the homologous complexes belonging to different species and were also very similar for the chimeric complexes formed by RBP and TTR of different origin. The two proteins that participate in the formation of this complex are very well characterized and their properties have been strongly preserved during evolution. A detailed discussion of the structure of TTR and of its complexes with different ligands is given in the appropriate chapters of this book. Here I will briefly summarize the structural elements needed for our discussion of the structure of the complex with RBP. The three-dimensional structures of human (Blake et al. 1978),
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rat (Wojtczak et al. 2001), chicken (Sunde et al. 1996) and sea bream (Folli et al. 2003; Eneqvist et al. 2004) TTR are known, as well as the structure of the complexes with several compounds of pharmacological interest (Wojtczak et al. 1993). TTR, the first human plasma protein whose structure was determined by X-ray diffraction, is a tetramer of four identical subunits, each 127 amino acids long, in which each subunit is constructed as a mostly antiparallel b-structure. In the tetramer, the four monomers do not occupy equivalent positions but are organized as a dimer of dimers. Each monomer presents two extensive b-sheets, each composed of four strands that are all antiparallel with one exception (Blake et al. 1974). Two monomers form then a very stable dimer by extending their two b-sheets through hydrogen bonding that involves the four strands (two from each monomer) at the edges of the two subunits. The two dimers of the tetramer are separated by a channel and in contact through symmetrically related loops. The channel, about 10 ˚ in diameter, is the ligand-binding site. The three orthogonal molecular twofold A axes of TTR have been designated x, y, and z (Blake et al. 1974), the latter being coincident with the ligand-binding channel of the molecule. Since TTR is one of the proteins that forms amyloid fibrils in many severe human diseases (Hou et al. 2007), a very large number of mutants that give rise to the abnormal protein deposits have been characterized. In particular, many mutants were characterized by X-ray diffraction and a comparative study of 23 different structures has revealed that for most mutants the conformational changes observed in the crystalline state are quite subtle and indistinguishable from the changes observed in two independently determined non-amyloydogenic molecules (Ho¨rnberg et al. 2000). The TTR fold is not exclusive of this transporter. In recent years the transthyretinrelated protein (TRP) family, a group of proteins predicted by their amino acid sequence similarity to vertebrate TTRs to posses the TTR fold, has been investigated by several groups (Lee et al. 2005 and the appropriate chapters in this book). The biological activity of the enzymes formerly called the TRP related protein family is that of the 5-hydroxyisourate hydrolases and they play an important role in the purine degradation pathway. Several 5-hydroxyisourate hydrolase X-ray structures have confirmed that these enzymes indeed possesses the TTR fold. Their source is bacterial: Escherichia coli (Lundberg et al. 2006), Bacillus subtilis (Jung et al. 2006) and Salmonella dublin (Hennebry et al. 2006) but the enzyme is also present in vertebrates and the structure of zebrafish 5-hydroxyisourate hydrolase has also been solved (Zanotti et al. 2006).
8.2
Plasma Retinol-Binding Protein (RBP4)
Plasma retinol-binding protein was first isolated from human plasma in 1968 (Kanai et al. 1968) and subsequently from the serum of a number of other vertebrates: including birds and fish (Berni et al. 1992). Although its major site of synthesis and secretion is the liver, other tissues are known to express the protein, in particular the adipocyte (Soprano and Blanner 1994). In the liver, the availability of retinol plays a critical role in the secretion of RBP: its secretion is specifically blocked during retinol deficiency and restored upon retinol repletion (Ronne et al. 1983).
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Retinol-binding protein belongs to the super family of proteins known with the name of lipocalins, a group of molecules which do not have a very high similarity in amino acid sequence but are highly similar in tertiary structure and share in many cases the common function of hydrophobic ligand binding in an internal cavity. The name is derived from the Greek words ‘‘lipos,’’ meaning fat, and ‘‘calyx,’’ meaning cup due to the fact that their structure has the shape of a calyx and their ligands are mostly hydrophobic (Pervais and Brew 1987). Human RBP is the first lipocalin whose three-dimensional structure was determined by X-ray diffraction analysis of single crystals (Newcomer et al. 1984). Following this work many other X-ray structures of different RBP4s were solved (Newcomer and Ong 2000; Zanotti and Berni 2004). In particular the three-dimensional structure of human (Cowan et al. 1990; Zanotti et al. 1993a) and bovine (Zanotti et al. 1993b) holo- and apo-RBP, as well as chicken RBP (Zanotti et al. 2001) and the higher resolution structure of porcine RBP (Zanotti et al. 1998) have been extensively described. The main feature of all these structures, highly similar to each other, is an eight-stranded up-and-down beta barrel which delimits the cavity followed topologically by a short a-helical segment close to the C terminus of the molecule. The retinol molecule binds into the calyx with the b-ionone ring buried and with the alcohol moiety pointing to the outside of the surface of the molecule. Examination of the model of bovine RBP4 at different values of pH, determined at the highest resolution attained up to now, has revealed the subtle details of ligand binding to the protein (Calderone et al. 2003). The main conclusions of this study regarding the conformation of the bound retinol at physiological pH are that: (a) the cyclohexenyl ring is in the half-chair conformation, (b) the polyene chain is fully extended and planar and, (c) the methyls and hydroxyls are not in the polyene plane. In this model three water molecules are present in the interior of the mostly hydrophobic ligand binding cavity and a network of other solvent molecules surrounds the hydroxyl moiety of retinol exposed on the surface of the protein. The other important contacts of the ligand and the protein, that have been extensively described for the refined model of human RBP4, are hydrophobic (Cowan et al. 1990). Figure 8.1 is a ribbon representation of the molecule showing the bound retinol as a space-filling model. The secondary structure assignments for human RBP4 are for the beta strands the following: strand A, residues 22–30; B, residues 39–47; C, residues 53–62; D, residues 68–78; E, residues 85–92; F, residues 100–109; G, residues 114–123; and H residues 129–138. The alpha helix spans residues 146– 158. The open end of the barrel is delimited by four loops joining strands A–B (residues 31–38), C–D (residues 63–67), E–F (residues 93–99), and G–H (residues 124–128). The first three are close to the retinol molecule and amino acids present in them participate in the contacts with TTR, the fourth is far from the ligand and is not involved in contacts with TTR in the complex. The loop connecting strands C and D has been found to be disordered in several X-ray structures which indicates a certain degree of flexibility which might be related to retinol release. It has been proposed that release of retinol by RBP4 involves a transition of the protein to the molten globule state triggered by a decrease of the pH near the cell membrane, a mechanism postulated for the ligand release of other lipocalins as well
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Fig. 8.1 Ribbon representation of the plasma RBP (RBP4) molecule. The bound retinol is represented as a CPK model with its OH exposed to the solvent on the surface of the protein. The figure was prepared with the program CCP4 mg (Potterton et al. 2002)
(Ptitsyn el al. 1993; Bychkova et al. 1998). A biased molecular dynamics simulation method has been used to explore the conformation of the partially folded structures of RBP in the molten globule state (Paci et al. 2005). In these simulations significant changes which result in an opening in the ligand binding cavity were observed in beta strands E and F and the loop connecting them, i.e., in residues 85–104. Though it was initially thought that removal of the retinol molecule from the calyx would result in major conformational changes leading to a collapse of the beta barrel structure, X-ray crystallographic studies of human and bovine RBP (Zanotti et al. 1993a, b) have shown that the transition from holo- to apoprotein involves only very subtle modifications. The most important is a conformational change in the loop connecting strands A and B, in particular in the region extending from amino acids 33–37. In this part of the molecule Leu 35 and Phe 36 move from their position in the holoprotein to very well defined positions in the apoprotein. When
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retinol is removed the space left empty by the vitamin is filled, in both human and bovine RBP, by the aromatic ring of Phe 36 and solvent molecules, and the movement of the Phe side chain drags the nearby amino acids into positions which are different from those adopted in the holoprotein. In particular Leu 35, which in the holo protein points towards the binding site moves out and points to the exterior of the RBP molecule. These results, obtained with apo RBP prepared from the holoprotein by removing retinol by organic solvent extraction or hydrophobic interaction chromatography was confirmed by the X-ray structure of recombinant apo RBP (Greene et al. 2001). Apo RBP prepared in vitro can be complexed with several natural and synthetic retinoids, i.e., derivatives of retinol. The interest in these compounds stems from their clinical applications that include treatment of skin disease and cancer (Moise et al. 2007). Co-crystals of RBP with different retinoids have been studied by X-ray diffraction (Zanotti et al. 1993c, 1994). The main conclusion of these studies is that the selected retinoids bind assuming the conformation of bound retinol, the natural ligand and with an overall protein structure that is virtually identical to that of the natural protein–ligand complex. Important differences are found at the entrance of the beta barrel where in some cases bulky groups protrude from the cavity into the solvent and as a consequence either reduce or abolish the interaction of RBP with TTR. The first evidence for the existence of specific cell surface receptors for plasma retinol-binding protein came from the study of the binding of 125I-labeled RBP to bovine pigment epithelium cells (Heller 1975). Both human and bovine RBP were found to bind specifically and completely in about one minute to the bovine epithelium cells. The binding was a linear function of the number of binding sites and was saturable with respect to the labeled RBP. The vitamin entered the cells but the labeled protein did not. Apo RBP was also found to bind to these cells but with a significantly higher dissociation constant. Following this work other putative RBP receptors were identified in different tissues: mucosal epithelial cells from the monkey’s small intestine (Rask and Petersson 1976), placenta (Sivaprasadarao and Findlay 1998), testis (Shingleton et al. 1989), retinal pigment epithelium (Ba˚vik et al. 1991, 1992), choroid plexus (Smeland et al. 1995), hepatocytes (Yamamoto et al. 1997) and macrophages (Hagen et al. 1999). Characterization of the receptor at a molecular level was difficult because of its fragility and the transient nature of its interaction with RBP. Using photo-crosss-linking, high affinity purification and mass spectrometry, the receptor was finally identified as Stra6, a 74 kDa, multitransmembrane protein whose function was previously unknown and whose sequence is not similar to that of any other protein of known structure (Kawaguchi et al. 2007; Wolf 2007; Blaner 2007). The amino acid sequence and hydropathy profile indicate that stra6 is a largely hydrophobic molecule with 11 predicted transmembrane domains. Analysis of the accessibility of a tag inserted in different positions in the topology of the molecule combined with a novel lysine accessibility technique has revealed which are the extracellular and which the intracellular domains of the receptor and identified the regions of the molecule implicated in RBP binding (Kawaguchi et al. 2008a). By studying a large number of random mutants and systematically analyzing their properties a domain
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which plays an essential role in RBP binding was identified. Three residues whose mutation completely abolishes the binding of RBP to stra6 were identified in this domain (Kawaguchi et al. 2008b). Mutations in Stra6 cause a series of very severe disorders in humans: anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation which highlights the crucial role played by this receptor in retinol metabolism (Golzio et al. 2007; Pasutto et al. 2007). Several mutations causing severe birth defects were also found to correlate with vitamin uptake from RBP. In addition, it is worth emphasizing that the uptake of retinol by stra6 has been shown to occur efficiently when cells are in the presence of both holo RBP alone and holo RBP complexed to TTR, i.e., the situation encountered in vivo (Kawaguchi et al. 2007). Adipose tissue is an important endocrine organ that secretes into circulation several signaling proteins that influence energy metabolism. These cell-to-cell signaling molecules are called adipocytokines or adipokines, and they include leptin, adiponectin, resistin, tumour necrosis factor-, and interleukin-6 (Henry and Clarke 2008). A recent addition to this list is plasma RBP which, in addition to the liver, is also synthesized in adipose tissue, and was found to behave as an adipokine, a marker of insulin sensitivity and adiposity in both animal models and humans. Systemic insulin resistance is associated with a decreased expression of the glucose transporter 4 (Glut4) in adipocytes, one of the grave defects of obesity and type 2 diabetes. Glut4 knockout mice show insulin resistance and present increased circulating levels of plasma RBP due to an enhanced expression in the adipose tissue of the gene encoding for the protein (Yang et al. 2005). Furthermore it was also observed that increasing the plasma level of RBP4 correlated with insulin resistance whereas lowering it led to insulin sensitivity enhancement. In humans, plasma RBP concentrations are elevated in insulin resistant patients with obesity impaired glucose tolerance or type 2 diabetes (Graham et al. 2006). These findings have led to the proposal that the measurement of the RBP4 levels in plasma might be a simple method to assess the risk of type 2 diabetes and cardiovascular disease (van Dam and Hu 2007). Although the exact intervention of RBP4 in clinical metabolic disease is not completely defined there appears to be no doubt that in addition to retinol delivery, this protein plays an important role in adipocyte lipid metabolism as well (von Eynatten and Humpert 2008).
8.3
Crystal Structures of the Complex
The first structure of a TTR–RBP complex determined by X-ray diffraction analysis of single crystals was that of a chimeric complex prepared in vitro with two molecules of chicken RBP per tetramer of human TTR (Monaco et al. 1995). These crystals diffracted to a slightly better resolution than those of the homologous complex human TTR-human RBP that were solved a few years later (Naylor and Newcomer 1999). The chimeric complex could only be prepared by imposing a stoichiometry of two molecules of RBP per TTR tetramer (Monaco et al. 1994), and an even higher ratio of 2.5–3 molecules of RBP per tetramer was used to prepare
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the crystals of the homologous human complex. In both cases the species found in the crystals was a hexamer TTR(RBP)2 but the quaternary structure of the two hexamers is different. Figure 8.2a is a ribbon representation of the chimeric hexamer in which the model is viewed looking down the z axis of the TTR tetramer as defined by Blake
a
b
Fig. 8.2 Quaternary structure of the TTR-(RBP)2-TTR hexamers. (a) Structure of the chimeric complex chicken RBP-human TTR (Monaco et al. 1995, PDB code 1RLB). The TTR molecule is oriented so that this is the view down the z axis of the TTR tetramer as defined by Blake et al. (1974). The x and y axes are in the plane of the figure, the first is horizontal and the second vertical. They intercept in the center of the TTR channel, the hormone-binding site, which is represented empty in the figure. (b) Structure of the homologous human TTR-RBP complex (Naylor and Newcomer 1999 PDB code 1QAB). In this case the twofold axis which is preserved in the complex is the z axis, i.e., the axis normal to the plane of the figure running through the TTR channel. The figure was prepared using the programs MOLSCRIPT (Kraulis 1991) and Raster3D (Merritt and Bacon 1997)
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and coworkers (Blake et al. 1974). The x and y axes are in the plane of the figure. The x axis is horizontal and separates the two TTR dimers that together form the tetramer. The vertical y axis is, in the chimeric complex, the twofold axis that relates the two RBP molecules as seen in the figure. Therefore, in this complex the two RBP molecules are found to interact most extensively with the same TTR dimer. Figure 8.2b is an analogous ribbon representation of the homologous human complex. In the crystals of this complex, the twofold axis which is preserved is the z axis instead, i.e., the axis perpendicular to the plane of the figure, and thus the two RBP molecules are found to interact most extensively with the opposite dimers of the TTR tetramer as shown in the figure. The reason why two different twofold axes are preserved in the hexamers in these two crystal forms is not known. More recently, the macromolecular complex of human TTR, human RBP and an antiRBP Fab was crystallized with a stoichiometry assumed for the triple complex in the crystals of 2:2:1 for the Fab, RBP, and TTR respectively, i.e., these new crystals preserve the 2:1 RBP–TTR stoichiometry (Malpelli et al. 1999). Therefore it appears that without the presence in the complex of at least one of the TTR twofold axes crystals cannot be formed. Thus, although the species purified from plasma is the pentamer containing only one RBP molecule per TTR tetramer, the artificial preparation of the hexamer seems to be a requirement to facilitate the packing of the molecules in the crystal lattices. In both the chimeric and the human homologous complexes, the two RBP molecules interact with the two monomers of one TTR dimer and a third monomer of the other dimer and this precludes the possibility of binding of a third and fourth RBP molecule to form a heptameric or octameric complex. In the chimeric hexamer in which the y axis is the preserved twofold axis, the two RBP molecules bind to the TTR tetramer along an axis parallel to the x axis (Fig. 8.2a) and is sufficiently close to it to hinder the potential binding of the two other RBP molecules that would be required to satisfy this symmetry element in an octamer. Something analogous happens in the homologous human hexamer in which the two RBP molecules bind on the opposite sides of the x axis but again too close to it to allow octamer formation (Fig. 8.2b). Thus the two crystal structures are consistent with a stoichiometry of either one or a maximum of two RBP molecules per TTR tetramer. Figure 8.3 is a ribbon representation of a pentamer, the species circulating in plasma, with one RBP molecule bound to a TTR tetramer. Note that the RBP molecule interacts with the two components of a dimer and a third monomer of the other TTR dimer. It is this last interaction that rules out the possibility of binding a second RBP molecule to the second dimer on the same side of the tetramer. The main amino acids involved in the contacts between RBP and TTR are listed in Table 8.1 along with the relevant distances between residues in contact. Note that, in addition to the retinol OH, the RBP residues participating in the contacts are clustered in three areas. The residues listed are Leu 35, which is found in different conformations in the apo and holo protein, Trp 67, in the loop connecting strands C and D and in the area at the end of strand E and in its connection to strand F. The last one is the region where most of the RBP amino acids participating in the contacts are located. The interacting regions of TTR with RBP are found in the following
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Fig. 8.3 Ribbon representation of the pentameric complex RBP–TTR, the species normally circulating in plasma. The retinol molecule is represented as a CPK model and the side-chains of the amino acids participating in the contacts as ball- and stick-models and colored in the case of TTR, according to the monomer to which they belong. The TTR tetramer is oriented as in Fig. 8.2 and the figure was prepared using the same programs
areas of the three monomers: monomer A, the loop joining strands F and G with Asp 99 and Ser 100, monomer B, residues 82–85 connecting the TTR alpha helix to strand F and Tyr 114 (in the loop joining strands G and H) and monomer C, the loop joining the first two strands of beta structure as well as Ile 84. An examination of the distances in Table 8.1 leads to the conclusion that the two X-ray structures are in reasonable agreement with one another. There is, however, one major significant difference, not evident from the Table, between the two models. Chicken RBP lacks the last five amino acids present in mammalian RBPs and those are found to interact with the TTR tetramer in the homologous human complex in one of the two RBP molecules present in the hexamer. Therefore in this complex, the two RBP molecules do not have exactly equivalent interactions with the TTR tetramer and the z twofold axis of symmetry is not strictly obeyed. The physiological significance of this additional interaction is discussed by Naylor and Newcomer but the reasons for this asymmetry in the binding of the two RBP molecules in the case of the human homologous complex are not known. Figure 8.4a, taken from the paper describing the chimeric complex (Monaco et al. 1995), is a cartoon representation of the most important interactions between an RBP molecule and the TTR tetramer. Note that the RBP molecule interacts with three different TTR monomers and there is one amino acid, Ile 84, present in two
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Table 8.1 Significant contacts in the TTR–RBP complex in the two structures determined by X-ray crystallography. The third column gives the distance in the chimeric complex (PDB code 1RLB) and the fifth column gives the distance in the homologous human complex (PDB code 1QAB) RBP chain identity TTR chain Distance TTR chain Distance Amino acid and amino identity chimeric identity homologous number TTR ˚) acid number Chimeric complex homologous complex (A ˚) complex (A complex PDB code PDB code 1RLB 1QAB E - 35 (Leu) [CD2] E - 67 (Trp) [CZ2] E - 67 (Trp) [CD2] E - 89 (Lys) [NZ] E - 89 (Lys) [NZ] E - 91 (Trp) [NE1] E - 95 (Ser) [OG] E - 96 (Phe) [CO] E - 96 (Phe) [N] E - 96 (Phe) [CB] E - 96 (Phe) [CE1] E - 97 (Leu) [CO] E - 99 (Lys) [NZ] E - 99 (Lys) [N] E - (Retinol) [OH]
B C C A A A B B B B C B A B B
3.41 4.20 3.45 2.73 4.54 4.07 3.09 2.69 3.38 3.37 3.44 2.80 2.70 3.87 3.15
D A A C C C D D D D A D C D D
4.24 5.03 4.04 3.57 4.92 3.68 2.71 3.25 3.46 3.30 3.25 3.59 5.47 4.33 3.48
83 (Gly) [CA] 20 (Val) [CG1] 84 (Ile) [CD1] 99 (Asp) [CO] 99 (Asp) [OD2] 100 (Ser) [CO] 114 (Tyr) [OH] 85 (Ser) [N] 114 (Tyr) [OH] 84 (Ile) [CG2] 21 (Arg) [CG] 85 (Ser) [OG] 99 (Asp) [OD2] 85 (Ser) [OG] 83 (Gly) [CO]
different TTR monomers (B & C) participating in the interaction, one in each dimer. The TTR region in the contact area is a hydrophobic zone situated between the two dimers in the tetramer, whereas the RBP area where most of the amino acids participating in the interactions are located is the end of strand E and the loop connecting it to strand F at the entrance of the barrel. These amino acids are represented in Fig. 8.4b, c. Note in the diagrams the prevalence of hydrophobic contacts. The retinol hydroxyl group, which is exposed on the surface of the protein, ˚ from the polypeptide chain of a TTR has its oxygen at a distance of about 3–3.5 A monomer. In uncomplexed TTR, there is a fairly large hydrophobic patch which becomes buried upon binding of an RBP molecule, which is a major driving force for complex formation by the two plasma proteins. Of the total RBP surface ˚ 2), 736 A ˚ 2 (about 8%) are buried when the accessible to the solvent (9,356 A molecule is in contact with TTR in the complex. It was already mentioned that the loop connecting strands C and D has been found partially disordered in some RBP X-ray structures but the electron density in this area is clear and continuous in the crystals of the chimeric complex. Therefore, in order to examine possible movements in this important area, the model of RBP in the complex can be superimposed over that of the uncomplexed holoprotein whose structure is known at high resolution (Zanotti et al. 2001). Using the program
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a
Leu E 35
Trp E 91
OH Lys E 89 Å NH3
O
O
C
C
C
Gly B 83
Asp A 99
Ser A 100
Ser E 95
o -
O
NH
C
Ser B 85
O
o
* OH
NH
He B 84
Å NH3 O Lys E 99
C Leu E 97
H-O
O
Tyr B 114
C
Trp E 67
NH
Phe E 96
N
Å
N
NH
* IIe C 84
N Arg C 21
Val C 20
Fig. 8.4 (a) The main protein–protein interactions found in the chimeric complex. An asterisk identifies Ile 84 which is present in the contact area of two different TTR monomers, B and C, that are part of the two separate dimers of the tetramer. (b) Diagram representing as ball and stick models the RBP amino acid side chains in the region 88–92 in contact with TTR. The TTR residues that make hydrophobic contacts are represented as arches in a different color. (c) The same as (b) but covering the RBP region 94–100. (d) Superposition of the models of chicken RBP bound in the complex (Monaco et al. 1995, PDB code 1RLB) and unbound (Zanotti et al. 2001, PDB code 1IIU). The RBP molecule in the complex is represented red and that isolated green. The TTR molecule is represented blue. The two RBP models were superimposed using the program LSQKAB (Kabsch 1978). Figures (b) and (c) were prepared using the visualization program LIGPLOT (Wallace et al. 1995). Figure (d) with the program CCP4 mg (Potterton et al. 2002)
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Fig. 8.4 (continued)
LSQKAB (Kabsch 1978) the coordinates of the two models were superimposed. The area of contact is represented in Fig. 8.4d where a shift in the position of Asn 65 and Trp 67 is clearly observed. Figures 8.5a, b compare the amino acid sequences of human, other five mammalian and chicken TTR and RBP aligned using the program CLUSTAL W (Thompson et al. 1994). The amino acids participating in the contacts in the complex are
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b
Fig. 8.5 Sequence alignment of six vertebrate (a) RBPs and (b) TTRs of the same species. The sequences were aligned using the program CLUSTAL W (Thompson et al. 1994). The six digit code preceding the name of the species in each sequence is the ExPASy code. The last column on the right hand side gives the percentage identity of each sequence and that of the human protein. A black dot identifies in each case a residue involved in the contacts in the complex
indicated with a back dot and the percentage identity of the sequences with that of human RBP and TTR are given in the last column. Note the very high percentage identity among the sequences and also that the residues participating in the contacts are clustered in three areas in the case of RBP and four in the case of TTR.There are a total of 31 amino acid differences in the primary structure of chicken and human TTR and interestingly six of those are found in the N terminal region of the molecule, which is disordered in the electron density maps of the complex, and three are in the C terminal region of the molecule. The sequences of human and chicken RBP differ in 24 amino acids. When the amino acids participating in the contacts listed in Table 8.1 are inspected in Fig. 8.5 only two amino acids in the TTR sequence are found to be different in human and chicken TTR. They are amino acids number 114, which is a Tyr in human TTR and a Phe in chicken TTR and, quite interestingly, amino acid number 84, which is an Ile in human TTR and a Leu in chicken TTR. A similar comparison of the two RBPs reveals that in this case no amino acids in the list are different in Fig. 8.5, although there is one amino acid present in the contact region of the homologous human complex, residue 64, which is a Leu in human RBP and a Phe in the chicken protein. Thus, the regions of protein-protein contact in the complex are strictly conserved for these species as expected given the physiological importance of this protein complex in animal physiology.
8 The Transthyretin–Retinol-Binding Protein Complex
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Experimental Evidence that Supports the X-Ray Models
The two X-ray models of the complex have allowed the rationalization of many experimental observations that preceded the X-ray work and with also of others made to test the X-ray structures. The effect of the ligand bound to the two protein carriers is totally different: thyroxine binding to TTR does not have any influence on complex formation (van Jaarsveld et al. 1973), whereas retinol binding plays a crucial role and the affinity of apo-RBP for TTR is drastically reduced. In addition, the binding of retinoids with bulky moieties exposed on the surface of the protein molecule can impair or prevent the formation of the macromolecular complex. This very different behavior of the two ligands with regard to the two carrier proteins has received a very simple explanation from the X-ray models. Thyroxine binds in a channel that does not participate in any way in the contacts with RBP, whereas retinol bound to RBP interacts with TTR through its exposed hydroxyl group on the surface of the RBP molecule. In addition, the models of the complex are consistent with the X-ray structural studies that have shown that in the holo to apo transition in RBP the most significant change involves amino acids 34–37 (Zanotti et al. 1993a, b). This region of the molecule, and in particular Leu 35, is present in the protein-protein contact area in both X-ray models. Another observation that concerns the stability of the complex is that the affinity of RBP for TTR is enhanced at high and reduced at low ionic strength (van Jaarsveld et al. 1973) which is in agreement with the participation of a hydrophobic area in the intermolecular contacts. Site-directed mutagenesis has confirmed the role of Leu 35 and has also shown that in the double mutant in which Leu 63 and Leu 64 are changed to Arg and Ser, the ability to bind to TTR is reduced (Sivaprasadarao and Findlay 1994). Deletion of the loops 92–98 results in complete loss of the ability to interact with TTR and this ability can be transferred to the epididymal retinoic acid-binding protein (that normally does not bind to TTR) by inserting the three RBP loops seen to participate in the intermolecular interactions in the X-ray models (Sundaram et al. 2002). Structural analysis of the chimeric complex using mass spectrometry has led to conclusions that are in complete agreement with the X-ray models (Rostom et al. 1998). Chemical modification of the tryptophan residues of isolated RBP and in the complex has shown that there is at least one Trp which reacts in the isolated protein but is protected upon complex formation, which implies that it is located in the area where the protein-protein contacts are established (Horwitz and Heller 1974). The identity of this residue was not established chemically but the two X-ray models of the complex have the same two Trp residues in the contact area: Trp 67 and 91. Acetylation of holo-RBP with N-acetylimidazole (using conditions under which both Lys and Tyr residues react) disrupts its binding to TTR and deacetylation of the tyrosines with hydroxylamine fails to produce RBP with a normal affinity for TTR (Heller and Horwitz 1975). This result indicates that there is at least a Lys and that there are no Tyr present in the RBP area that participate in the protein-protein contact. As Table 8.1 shows, the two models have Lys 89 and 99 on the surface in contact with TTR.
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The macromolecular complex RBP–TTR does not form in the plasma of fish although both proteins are known to be present in circulation (Folli et al. 2003). If the amino acid sequences of sea bream RBP and TTR are compared with those of other vertebrates in which the complex is known to form, significant differences are found in the regions participating in the contacts. In particular the differences found in sea bream TTR are Ile84Ser, Asp99Pro and Ser100Glu and Lys99Thr in RBP, which as Table 8.1 shows participate in important contacts in the complex. Among the many pathological mutations described in TTR that are associated with different types of amyloidosis (Hou et al. 2007) there is one in which it was found that the affinity of TTR for RBP was drastically reduced (Benson and Dwulet 1983), a reduction that can be convincingly explained by the structural models of the complex. The disease is called Indiana-type hereditary amyloidosis and the mutation consists in the substitution Ile84Ser. When the dissociation constant of the complex of this mutant with normal RBP was determined, it was found that the affinity of the altered TTR for normal RBP was negligible (Berni et al. 1994). Both X-ray models of the complex place this residue, Ile 84, twice at the interface with RBP, and the monomers to which the residues belong are in different TTR dimers, which explains the remarkable influence of this single amino acid on the complex stability. The plasma RBP concentration of several other individuals with single mutations in the TTR gene was examined by Waits et al. (Waits et al. 1995) and the only other case in which the RBP concentration was found to be significantly lowered was the Ile84Asn mutant. Interestingly one of the mutants examined was the Tyr114Cys, i.e., an amino acid involved in the contacts in the complex (see Table 8.1) suggesting that a single mutation might not be enough to lead to dissociation of the complex. Elevated RBP4 levels are correlated with insulin resistance and glucose intolerance and the complex has been identified as a target for the treatment of this widely diffused disease (Mody et al. 2008). Disrupting the stability of the complex might be a way of lowering the plasma RBP levels since it is known that uncomplexed RBP is eliminated by glomerular filtration. In the development of a molecule to implement the strategy to treat insulin-resistant individuals through dissociation of the complex, it may therefore be necessary to disrupt more than one contact in the macromolecular complex.
Acknowledgements
I thank Dr. Massimiliano Perduca for his help in preparing the figures
of this chapter
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Chapter 9
TTR and RBP: Implications in Fish Physiology Sancia Gaetani and Diana Bellovino
Abstract Transthyretin (TTR), a homotetrameric protein of 54 kDa, is one of the carriers of thyroid hormone and, as a macromolecular complex with retinolbinding protein 4 (RBP4), is involved in the transport of vitamin A in blood, preventing kidney glomerular filtration of the small, 21 kDa RBP4. TTR cDNA has been cloned in many vertebrates (man, rat, chicken, etc.) and more recently also in several teleost fishes. Fish TTR shows a strong homology with the same protein of other vertebrates, with similar monomer–monomer and dimer–dimer interfaces and a conserved tetrameric structure. RBP4 was also isolated from several teleosts and characterized. The expression of the two proteins was investigated during embryonic development and in adult tissues. TTR is expressed in adult fish liver and is therefore present in their serum, even though RBP4 and TTR have never been detected in the blood as a complex. The lack of RBP4–TTR complex formation in fish has been explained, since the residues specifically involved in the interaction between the two proteins in mammals are different from the corresponding residues in fish. Keywords Transthyretin, Retinol-binding protein, Thyroid hormones, Fish retinoids transport
Abbreviations TTR RBP4 ER THs T4-Thyroxine T3
Transthyretin Retinol-binding protein 4 Endoplamic reticulum Thyroid hormones 30 -50 -3-5-Triiodothyronine 30 -3-5-Triiodothyronine
S. Gaetani and D. Bellovino National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178, Roma, Italy e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_9, # Springer‐Verlag Berlin Heidelberg 2009
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THBP TBPA LRAT STRA6 RAR FAP
9.1
Thyroid hormone binding protein Thyroxine-binding prealbumin Lecithin:retinol acyltransferase Stimulated by retinoic acid 6 gene Retinoic Acid Receptor Familial Amyloid Polyneuropathy
Vitamin A Transport
In teleost-fish, as in higher vertebrates, retinoids exert a key role in embryonic development and in several cellular functions throughout life (Chambon 1996; Niederreither and Dolle 2008), as well as in the visual cycle (Blomhoff and Blomhoff 2006). Retinoids introduced via the diet, mainly as carotenoids and retinyl esters, undergo a series of metabolic changes to the final active form, retinoic acid, which interacts with specific transcription factors and modulates the expression of hundreds of genes (Schug et al. 2007). The form involved in the visual cycle is 11-cis-retinal (Thompson and Gal 2003). A schematic description of vitamin A transport and utilization is outlined in Fig. 9.1. As in higher vertebrates, the liver of fish is the organ where vitamin A is stored. In mammals the two proteins involved in the specific binding and efficient transport and targeting of circulating vitamin A (retinol) are expressed in the liver: retinolbinding protein (RBP, now universally denominated RBP4) and transthyretin (TTR). RBP4 is the specific blood retinol carrier, while TTR is involved in thyroid hormone (TH) transport and in higher vertebrates it binds holoRBP4. The formation of the holoRBP4–TTR complex, which takes place in the endoplasmic reticulum (ER) of the hepatocytes before secretion, has been demonstrated and characterized in higher vertebrates (Soprano and Blaner 1994; Bellovino et al. 1996; Melhus et al. 1991; Selvaraj et al. 2008). The secretion of RBP4 is regulated by retinol. In vitamin A deficiency, when retinol is not available in the hepatocyte, RBP4 secretion is inhibited and the protein accumulates in the ER, from where it is promptly secreted upon retinol repletion (Perozzi et al. 1991; Bellovino et al. 1999). Like the other secretory proteins, RBP4 is cotranslationally targeted to the ER where it undergoes signal peptide cleavage and oxidative folding. Secretion of RBP4 is triggered by retinol binding, that induces a conformational change from the ‘‘apo’’ to the ‘‘holo’’ form of the protein and allows its secretion from the ER. Mechanisms underlying such ligand-dependent secretion are still not fully explained. It is believed that ligand binding may relieve retention of RBP4 from the quality control machinery in the ER. However, precise interrelationships between RBP4 folding, ligand binding, TTR assembly and secretion are still not clearly understood (Selvaraj et al. 2008).
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Fig. 9.1 Outline of vitamin A metabolism. Vitamin A, from vegetable and animal sources, is introduced via the diet. Carotenoids and retinyl esthers are absorbed from enterocytes and are included in lipoprotein particles, in particular chylomicrons. Some retinol is delivered to retina where it is transformed into retinal and takes part to the visual cycle. Circulating chylomicrons are taken up into the liver, where the different components, including the retinyl-esters, are released. According to the vitamin A status of the organism, retinyl esters can be stored in the stellate cells, and/or transformed in retinol in the hepatocytes. Retinol interacts with its specific carrier protein RBP4, thus forming holo-RBP4. Holo-RBP4 forms a complex with TTR and is secreted into blood towards target tissues. Following the interaction of RBP4 with its receptor Stra6, retinol is released from RBP4 into the target cells, and is metabolized to retinoic acid. Retinoic acid is then shuttled to the nucleus, where it regulates gene expression through the activation of specific transcription factors (RARs and RXRs). CRBP-II cellular retinol binding protein II, IRBP interphoto receptor binding protein, RBP retinol binding protein, TTR transthyretin, STRA6 retinoic acid stimulated gene 6, CRBP-I cellular retinol binding protein I, CRABP cellular retinoic acid binding protein, RAR retinoic acid receptor, RXR retinoid X receptor
It is believed that formation of the holoRBP-TTR complex prevents the loss of low molecular weight RBP4 and its bound retinol through the kidney. TTR null mice were generated and were shown to be phenotypically normal and viable. In these animals, plasma level of RBP4, retinol, and THs were significantly decreased (Episkopou et al. 1993).
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Until a few years ago it was known that vitamin A was an essential nutrient for higher vertebrates and for fish, but little was known about its transport and utilization in these animals. The aim of this chapter is to describe the present knowledge of TTR and RBP4 in teleost fish, and their role in controlling the distribution and activity of essential molecules, such as vitamin A and THs, in these organisms.
9.2
Transthyretin in Fish
TTR, formerly called prealbumin for its migration faster than serum albumin during electrophoresis of whole plasma, is a homotetrameric protein forming, in the majority of vertebrates where it has been isolated, a macromolecular complex with holoRBP. It is the carrier of THs: 30 ,50 ,3,5-tetraiodothyronine (thyroxine, T4) and 30 ,3,5-triiodothyronine (T3). In higher vertebrates, TTR is expressed mainly in the liver and in the choroid plexus of the brain. In the hepatocyte, the major storage cell for retinol, both RBP4 (molecular mass 21 kDa) and TTR (molecular mass 54 kDa), are synthesized and secreted as a molecular complex in a ratio of 1:1:1 (holoRBP4-TTR) (Soprano and Blaner 1994; Bellovino et al. 1996). TTR has only been found in vertebrates, and in particular piscine TTR displays the lowest sequence identity with human TTR. In lower species only members of a TTR-related protein family were identified (Eneqvist et al. 2003; Saverwyns et al. 2008), while TTR homologues have been found among vertebrates and share high sequence identity. In adult birds, diprotodont marsupials and eutherian mammals, TTR is expressed both in the liver and choroid plexus of the brain, whereas in reptiles it is expressed only in the choroid plexus. In fish and amphibians the main production site is the liver during development (Schreiber and Richardson 1997; Power et al. 2000; Santos et al. 2002). Interestingly, piscine TTR displays a higher affinity for T3 than for T4, as do the amphibian and avian TTRs (Eneqvist et al. 2004), and in contrast to the marsupial and mammalian TTRs, that have a higher affinity for T4 (Richardson 2007). This characteristic suggests that the binding affinity of TTR changed during evolution. In mammalian TTR, transport of T4 is preferred across the blood-brain barrier, where T4 is converted by 50 -monodeiodination to the biologically more active T3. TTR evolution may correlate with the evolution of deiodinases, which generate T3 from T4 in a tissue specific action. Very recently, a paper was published (Morgado et al. 2008) where the role of the longer N-terminus of fish TTR, thought to influence TH-binding affinity and maybe TTR stability, was investigated. Recombinant wild type sea bream (sbTTRWT) and two mutants lacking the first 6 and 12 N-terminal residues (sbTTRM6 and sbTTRM12, respectively) were produced. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4, indicating that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. The authors suggest that
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sbTTRWT, less prone to dissociate into monomers than human TTR, is more resistant to fibril formation. Four TTR monomers are organized in two very stable dimers that, in turn, interact to form a tetrameric structure with a central hydrophobic channel, in which two hormone-binding sites are present (Monaco et al. 1995; Naylor and Newcomer 1999). The two RBP-binding sites have also been structurally characterized and are situated on the surface of the molecule. Previously determined structures of chicken, rat, and human TTR are very similar, with the exception of the a-helical region, which is different in the avian structure compared with the mammalian protein (Sunde et al. 1996; Wojtczak 1997). In 1985 a paper (Larsson et al. 1985) reported the binding of [125I]T4 to salmon (Salmo salar) serum proteins and the isolation of a protein which, on the basis of its specific properties, was identified as TTR. The thyroxine-binding prealbumin (TBPA), as the authors called it, from all the 15 species (human, monkey, cattle, sheep, goat, water buffalo, horse, swine, dog, cat, rabbit, rat, chicken, frog, and salmon) investigated, except salmon, showed affinity for human RBP. The presence of TBPA in all vertebrates suggests that TTR is a far more important thyroxine carrier than it was thought up to this date. However, attemps to identify TTR in serum of different fish species (Salmo trutta, Onchorhyncus mykiss, Onchorhyncus tshawytscha, Cyprinus carpio) failed (Richardson et al. 1994), and the presence of TTR in fish remained controversial for many years. In 1999, a thyroid hormone-binding-protein (THBP) from serum of masu salmon (Onchorhyncus masou) at the stage of smoltification was isolated, purified, and biochemically characterized (Yamauchi et al. 1999). In salmonids at this developmental stage thyroid hormonal profile is very similar to those found in metamorphosing tadpoles (Dickhoff et al. 1990). The authors detected a single molecular species of THBP by [I125]T3 binding assay and showed that masu salmon THBP is homologous to TTRs of other vertebrates examined. However, its binding affinity for T3 was three times higher than that for T4, and therefore opposite to the higher vertebrate hormone affinity. Santos and Power (Santos and Power 1999) demonstrated unequivocally that TTR is present in teleost fish. It is synthesized mainly in the liver and it is released into the circulation where it binds THs. In this study RNA was extracted from different organs and tissues of juvenile sea breams. A cDNA library was constructed from liver RNA, screened with a TTR cDNA probe generated by RTPCR labeled with [a32P-dCTP] and the full-length TTR cDNA cloned, sequenced and translated in vitro. TTR expression was studied in several sea bream adult tissues and larval samples. TTR was found expressed, by Southern blot analysis of the PCR products, in hatching larvae and in liver, brain, pituitary, gills, kidney, intestine, and testis (not in the ovary). However, by Northern blot hybridization TTR mRNA was detected only in the liver, as a single transcript of 0.7 kb. The authors discuss the possibility that the failure to detect piscine TTR in the past by many investigators could be attributed to the use of T4 as ligand, since their study shows that T3, which is more abundant in fish plasma (Eales and Brown 1995), binds to TTR better than T4. TTR from the amphibian Rana catesbiana (Yamauchi
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Fig. 9.2 Sequence alignment of (a) TTR and (b) RBP4 from different vertebrates. The residues identical in all the sequences presented are shaded in red. The residues identical or chemically similar in at least six sequences are denoted by red characters. The amino acid residues in the human RBP and TTR involved in forming the RBP-TTR complex interactions (Zanotti et al. 2008) are indicated by arrowheads. Sequence alignments were constructed with CLUSTAL W (Thompson et al. 1994) and annotated with Espript (Gouet et al. 1999)
et al. 1993) also showed higher affinity for T3 than for T4. Sea bream TTR is a protein of 130 amino acids with a leader peptide of 20 amino acids. Its molecular mass, estimated by SDS-PAGE migration rate of the in vitro translation product, is about 15 kDa, and it is similar to the monomers characterized in other vertebrates (Santos and Power 1999). It is highly homologous to previously isolated TTRs despite the fact that its amino acid sequence shares only 40–55% identity with the
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sequence of TTR from other vertebrates. It must be emphasized, however, that the amino acids involved in the formation of the central channel in which T3 and T4 bind, are 90% homologous to human TTR and that the amino acids involved in the binding to RBP4 in higher vertebrates are also highly conserved (Fig. 9.2a). The three-dimensional structure was generated based on the known X-ray structures from human, rat, and chicken TTRs and on the sea bream TTR amino acid sequence (Power et al. 2000). According to this model, the overall topology of sea bream TTR has been conserved and the predicted monomer-monomer and dimer-dimer interfaces and tetrameric structures are similar to those determined by X-ray crystallography of human, rat, and chicken TTRs. Funkenstein and coauthors (Funkenstein et al. 1999) used a homologous TTR probe cloned from the liver of the marine fish Sparus aurata, to investigate its ontogeny and tissue distribution. The results showed that TTR mRNA was detected in fish larvae already at the day of hatching, as determined by Northern blot analysis. The expression increased during post-hatching up to the level of the adult liver, that is the major site of TTR expression. However a low level of expression of TTR was detected in all extrahepatic tissues studied (eye, brain, gill, muscle, heart, and kidney). Interestingly, relatively high expression was found in the skin. The aim of the work was to understand the role of sea bream TTR in the transport of THs. It was reported that experimental treatment of several fish species during early stages of development with TH in appropriate doses, induced earlier hatching and accelerated yolk absorption, growth and morphological differentiation (Brown and Kim 1995; Tachihara et al. 1997). Furthermore, TH induces metamorphosis in flounder, Paralichthys olivaceus (Miwa and Inui 1987). The questions the authors wanted to answer were: how the THs are transferred from the maternal circulation to the developing oocyte and how are THs in the developing embryos and larvae transported to their target organs. Exogenous THs administered to larval fish can enter the vascular compartment via skin or gills, but how they are transported into the target organs is not clear. Moreover, the reserves of THs in the yolk will not influence development unless they also enter the circulatory system and then reach the target organs. Most THs circulating in the blood of vertebrates are bound to plasma proteins. Proteins carrying THs in higher vertebrates have been extensively studied, while in fish very little was known at the end of the 1990s. TTR seemed to be the principal carrier of TH in bony fish. In 2003 Apreda et al. (Apreda M., Morgado I., Power D., Gaetani S., Bellovino D., GenBank AJ544193) cloned a full length carp (Cyprinus carpio) TTR cDNA from adult carp liver cDNA library. The protein was translated in vitro and expressed in a bacterial system in order to immunize rabbits and produce an anti carp-TTR polyclonal antibody. Preliminary results showed that carp TTR can be detected in adult carp blood. However, it has been reported that TTR is not present in adult fish, amphibians, reptiles and polyprotodont marsupials blood, since in these vertebrates the TTR gene seems to be turned on in the liver only during specific stages of development, when there is a rise in THs level in blood (Richardson 2007). For fish, this stage is during early development, when the animals are still
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absorbing the yolk. Further research is needed to shed light on this aspect of TTR physiology in fish. A cDNA encoding TTR was then cloned from the Pacific bluefin tuna (Thunnus orientalis) (Kawakami et al. 2006). This cDNA contains a complete open reading frame encoding 151 amino acid residues. The deduced amino acid sequence is 81% and 55% identical to the gilthead seabream and common carp forms respectively, and 33–39% to mammalian, reptilian, and amphibiam forms. A 1.0-kb transcript was found in the liver and ovary, the liver being the main source of the protein. Analysis of T3 and T4 binding demonstrated that both bind to bluefin TTR, but affinity for T3 for the protein is higher than that for T4. The results indicate that bluefin tuna TTR acts as a transporter of THs in the plasma, and therefore contributes to the function of THs in target cells. In 2007, the full-length cDNAs corresponding to TTR were isolated and cloned from two genera of lamprey, Petromyzon marinus and Lampetra appendix (Manzon et al. 2007). These sequences represented the first report of TTR sequences in vertebrates basal to the teleost fishes (superorder Euteleostei). The N-terminal region of the lamprey TTR subunits is nine amino acids longer than those in mammals and four to six amino acids longer than those in other vertebrates. The authors showed that the longer N-terminal region is due to the position of the intron 1/exon 2 splice site. The lamprey TTR gene is expressed mainly in the liver during all phases of the lifecycle. Moreover, it is expressed at low levels in a variety of other tissues and, as in other vertebrates, is upregulated during lamprey metamorphosis. The deduced amino acid sequence of lamprey TTR cDNAs are between 36% and 47% identical with those from other vertebrates. However, unlike other vertebrate TTRs, which show virtually 100% conservation of amino acids lining the central channel and therefore involved in TH binding, the deduced amino acid sequence of lamprey TTR cDNAs indicates that there are five nonconservative substitutions in these amino acids. Of these five substitutions, four are predicted to be neutral with respect to protein function; only the Ser to Phe substitution at position 115 is predicted to be a disfavored substitution. The observation that the TTR gene is developmentally regulated in lampreys, consistent with observations in other vertebrates, is a paradox. In fact in lampreys, one of the most ancient living vertebrates, the elevation of TTR gene expression during metamorphosis occurs when THs levels are at their nadir, whereas in other vertebrates elevation of TTR levels correlates with, and is thought to facilitate, a developmentally important surge in TH levels.
9.3
Piscine Retinol-Binding Protein 4
As mentioned above, besides its role as a TH carrier, in higher vertebrates TTR is also involved in vitamin A transport through its interaction with RBP4. The main form of circulating vitamin A is all-trans retinol, a small hydrophobic molecule that is stored mainly in the liver as retinyl-esters droplets. RBP4, the specific retinol carrier, is essential to mobilize retinol from storage in the liver and
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to target this molecule to specific peripheric tissues (Soprano and Blaner 1994). In fact, it has been demonstrated that in RBP/ knock-out mice fed a normal diet, vitamin A transport can be carried out by other circulating plasma proteins like albumin, but under vitamin A deficiency retinol stored in the liver cannot be efficiently mobilized and retinal function in the visual cycle is impaired (Quadro et al. 1999). Moreover, the recent identification of the specific RBP4 membrane receptor STRA6 (Stimulated by retinoic acid 6 gene) (Kawaguchi et al. 2007), expressed by selected organs (as eye and placenta), demonstrates that RBP4 plays a key role in targeting retinol delivery. In teleost fish, RBP4 is synthesized mainly in the liver and, to a lower extent, in kidney and ovary. It plays essentially the same role as in higher vertebrates, since it binds and transports retinol from the liver to target tissues. In particular, in these organisms RBP4 has been proven to exert a key role in providing vitamin A during zebrafish development (Isken et al. 2008) and to ovaries in order to support retinol accumulation in eggs, and therefore embryonic development (Lubzens et al. 2003; Levi et al. 2008). RBP4 function is strictly related to the expression of the membrane receptor STRA6, that in turn is responsible for clearing holo-RBP4 from the circulation. Isken and coauthors (2008) have demonstrated that the lack of STRA6 receptor in zebrafish embryonic tissues causes an excess of RBP4-bound vitamin A and the subsequent local retinol/retinoic acid accumulation. Retinoic acid accumulation provokes impaired retinoic acid receptor (RAR) signaling and gene regulation, and therefore causes developmental abnormalities and retinoid deficiency to the eye. In particular, it has been suggested that the correct vitamin A concentration at target tissues is maintained by the coordinated action of RBP4, STRA6, and lecithin: retinol acyltransferase (LRAT) proteins. STRA6 acts as a bidirectional retinol transporter, which mediates between RBP4 retinol supply from circulation and intracellular accumulation of retinyl esters performed by LRAT. The trout ovary has been defined by Levi et al. (Levi et al. 2008) as ‘‘an important site for retinoid and carotenoid metabolism.’’ In fact, they have demonstrated that in this organ several crucial genes involved in carotenoid and retinoid metabolism, including rbp4, stra6, and lrat, are expressed and contribute to ensure ovarian follicle viability and egg quality. To date RBP4 has been identified and characterized, at amino acid and/or nucleotide level, in several fish species: yellowtail Seriola quinqueradiata (Shidoji and Muto 1977), rainbow trout (Berni et al.1992; Zapponi et al. 1992), sea bream (Funkenstein et al. 1999; Brown and Kim 1995; Tachihara et al. 1997; Miwa and Inui 1987; Kawakami et al. 2006; Manzon et al. 2007; Quadro et al. 1999; Kawaguchi et al. 2007; Isken et al. 2008; Lubzens et al. 2003; Levi et al. 2008; Shidoji and Muto 1977; Berni et al. 1992; Zapponi et al. 1992; Sammar et al. 2001), carp (Bellovino et al. 2001) and zebrafish (Tingaud-Sequeira et al. 2006). All these proteins share a high degree of homology with higher vertebrates RBP4s, in particular in the regions involved in essential functions such as retinol binding (Fig. 9.2b). RBP4 is member of the lipocalin superfamily, which includes several small soluble proteins involved in the transport of hydrophobic molecules.
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All members of this group share a overall ‘‘calix’’ structure, characterized by an hydrophobic ‘‘pocket’’ in which the ligand is embedded (Flower 1996). Three-dimensional structure prediction (Zapponi et al. 1992) and primary sequence analysis (Bellovino et al. 2002; Lubzens et al. 2003; Folli et al. 2003) suggest that the residues involved in the retinol-binding pocket and the cysteines that coordinate three disulfide bridges (residues 22, 88, 138, 147, 178, and 192 according to human RBP4 sequence in Fig. 9.2b) are highly conserved among all vertebrates studied. Disulfide bridges contribute to the organization of the tertiary structure of RBP4, that in turn is related to its specific function as a lipocalin. Residues in the binding pocket create an environment able to accomodate the hydrophobic portion of retinol, while the polyene chain and the terminal hydroxyl group of retinol are exposed to the surface of the protein, near the entrance of the hydrophobic pocket, in a region involved in the interaction with TTR. As a consequence, the ‘‘apo’’ form of RBP4 has a lower affinity for TTR compared to the ‘‘holo’’ form (Zanotti et al. 1993; Malpeli et al. 1996). This feature in higher vertebrates contributes to the stability of the circulating holoRBP4-TTR complex, while upon release of retinol to target cells through the interaction with STRA6, apoRBP4 is presumably filtered by the kidneys. Circulating fish-RBP4 transports all-trans retinol (Berni et al. 1992), even though it has been shown that 3,4-dehydroretinol (vitamin A2), abundant in fish tissues (Ganguly 1989) and in particular in the liver, in vitro is able to displace alltrans retinol from the ligand binding site on RBP4 (Shidoji and Muto 1977; Berni et al. 1992).
9.4
RBP/TTR Interaction in Fish
In past years, the crystal structure and the putative regions involved in heterologous (human TTR–chicken RBP4, (Monaco et al. 1995)) or homologous (human TTR– human RBP4 (Naylor and Newcomer 1999)) RBP–TTR interactions were analyzed (Sivaprasadarao and Findlay 1994; Berni et al. 1994; Monaco 2000). Unlike the situation in mammals, fish RBP4 does not circulate as a complex with TTR. The RBP4–TTR complex has never been isolated from fish plasma and the in vitro affinity between the two proteins, though specific, is very low (Shidoji and Muto 1977; Berni et al. 1992). In 1992 Berni and colleagues (Berni et al. 1992) purified two forms of RBP4 by gel filtration from plasma of rainbow trout (Onchorhyncus mykiss). They were very similar to mammalial RBPs, but not physiologically involved in the formation of any protein–protein complex in plasma, although capable of interacting with mammalian TTR with low binding affinity. Since the two forms of trout RBP also possessed the region that in mammalian RBP4 has a functional role in binding TTR, the authors suggested that during the phylogenetic development of the nonmammalian vertebrates, TTR was modified to acquire a binding site for RBP4.
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In 2003, the same group performed other experiments of in vitro binding between sea bream or carp TTR and human holo-RBP4 (Folli et al. 2003). The interaction between the two proteins was evaluated by analyzing the changes of the fluorescence anisotropy of RBP4-bound retinol associated with the formation of the RBP4–TTR complex, the molecular mass of which (76 kDa) is higher than that of uncomplexed RBP4 (21 kDa). When sea bream or carp TTR was added to human holo-RBP4, no significant increase in fluorescence anisotropy of the RBP4-bound retinol was revealed under conditions suitable for the formation of the human TTR– RBP4 complex. This result was also obtained when sea bream or carp TTR was added to piscine (carp) holoRBP4. This issue has been resolved by recent research by Zanotti and colleagues (Zanotti et al. 2008), in which the crystal structure of the human RBP4–TTR complex, bound to an anti-RBP4 Fab, was determined. In parallel, the TTR amino acids involved in the interaction between the two proteins have been identified by site-directed mutagenesis. In particular, some of these mutations are characteristic of amyloidogenic variants of TTR. TTR, in fact, is a potential amyloidogenic protein, since some genetic variants are able to form fibrils that accumulate in tissues (e.g., brain) and cause the related degenerative pathology (FAP) (Saraiva 2001). In particular, the Ile84Ser amyloidogenic form causes the loss of RBP4–TTR interaction, and in fact individuals carrying this mutation present an altered retinol-RBP4 plasma transport. Interestingly, it has been demonstrated that some TTR residues (Leu82, Ile84, Asp99, and Ser100), that in amyloidogenic human TTR weaken or abolish the interaction with RBP4, are present in piscine TTR. This supports and explains the lack of interaction between RBP4 and TTR in fish. For instance, human TTR Ile84 is replaced by Ser84 in seabream, while human TTR Ser100 is replaced by Glu100 in seabream, zebra fish, and trout TTR. In the same study it was shown that the RBP4 regions involved in the complex formation have limited differences between terrestrial and aquatic vertebrates, explaining why piscine RBP4 is more prone to form in vitro heterologous complex, albeit weak, with TTRs from higher vertebrates (Saraiva 2001; Folli et al. 2003). Fish RBP4s, as well as Xenopus and chicken, lack a short C-terminal aminoacid sequence that in mammals has been proposed to stabilize the RBP4–TTR interaction (Naylor and Newcomer 1999). According to Zanotti et al. (2008), this portion of the protein is not crucial for the stability of the human RBP4–TTR complex; on the other hand, chicken RBP4, lacking this part of the molecule, is able to form a complex with human TTR (Monaco et al. 1995). It is therefore generally agreed that the interaction between RBP4 and TTR, that is thought to prevent glomerular filtration of RBP4, has evolved in terrestrial vertebrates by mutations affecting a limited number of TTR and RBP4 residues. Carp RBP4 presents biochemical features that are unique among the fish RBPs characterized to date (Bellovino et al. 2001): the signal sequence is not removed (as usually happens for secretory proteins after translocation across the ER membrane) and two N-glycosylations are present at the N-terminal portion of the protein (AsnX-Thr consensus sites at residues 25 and 46). Indeed, the presence of N-glycosylation has also been detected in other two members of Cyprinidae family: zebrafish
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(Danio rerio) and goldfish (Carassius auratus) (Devirgiliis et al. 2005). While for zebrafish RBP4 the single N-glycosylation site can be predicted from the amino acidic sequence, in goldfish the modification has been revealed only through the removal of the glycan moiety by N-Glycosidase F digestion, followed by electrophoretic separation by molecular weight, since neither the amino acid sequence nor the corresponding cDNA sequence for this protein are available. The possible function of this secondary modification on carp RBP4 is not clear. An hypothesis is that the glycan moiety, rich in sialic acid, increases the apparent molecular weight and the net negative charge of the protein, slowing down its kidney filtration rate (Elger et al. 1988) and ultimately compensateing for the lack of interaction with TTR.
9.5
Conclusions
In conclusion, since many years it has been established that vitamin A is essential throughout the life of teleosts, but very little is known about its transport, metabolism, and distribution. In this chapter the results of the isolation and characterization of RBP4 and TTR in fish have been surveyed, but still very little information is available on their mechanism of action and on the implications in fish physiology. Hopefully research in this important field will soon progress rapidly and successfully.
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Chambon P (1996) A decade of molecular biology of retinoic acid receptors. FASEB J 10, 940–954. Devirgiliis C, Gaetani S, Apreda M, and Bellovino D (2005) Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane. Biochem Biophys Res Commun 332, 504–511. Dickhoff WW, and Brown CL, Sullivan CV, and Bern H (1990) Fish and amphibian models for developmental endocrinology. J Exp Zool Supplement 4, 90–97. Eales JG and Brown SB (1995) Regulation and measurement of thyroidal status in teleost fish. Neth J Zool 45, 175–180. Elger B, Ruhs H, and Hentschel H (1988) Glomerular permselectivity to serum proteins in rainbow trout (Salmo gairdneri). Am J Physiol 255, R418–R423. Eneqvist T, Lundberg E, Karlsson A, Huang S, Santos CR, Power DM, and Sauer-Eriksson AE (2004) High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine. J Biol Chem 279, 26411–26416. Eneqvist T, Lundberg E, Nilsson L, Abagyan R, and Sauer-Eriksson AE (2003) The transthyretinrelated protein family. Eur J Biochem 270, 518–532. Episkopou V, Maeda S, Nishiguchi S, Shimada K, Gaitanaris GA, Gottesman ME, and Robertson EJ (1993) Disruption of the transthyretin gene results in mice with depressed levels of plasma retinol and thyroid hormone. Proc Natl Acad Sci USA 90, 2375–2379. Flower DR (1996) The lipocalin protein family: structure and function. Biochem J 318(Pt 1), 1–14. Folli C, Pasquato N, Ramazzina I, Battistutta R, Zanotti G, and Berni R (2003) Distinctive binding and structural properties of piscine transthyretin. FEBS Lett 555, 279–284. Funkenstein B, Perrot V, and Brown CL (1999) Cloning of putative piscine (Sparus aurata) transthyretin: developmental expression and tissue distribution. Mol Cell Endocrinol 157, 67–73. Ganguly J (1989) Biochemistry of Vitamin A. Boca Raton FL: CRC Press. Gouet P, Courcelle E, Stuart D, and Metoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308. Isken A, Golczak M, Oberhauser V, Hunzelmann S, Driever W, Imanishi Y, Palczewski K, and von Lintig J (2008) RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood syndrome. Cell Metab 7, 258–268. Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, Wiita P, Bok D, and Sun H (2007) A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315, 820–825. Kawakami Y, Seoka M, Miyashita S, Kumai H, and Ohta H (2006) Characterization of transthyretin in the Pacific bluefin tuna, Thunnus orientalis. Zoolog Sci 23, 443–448. Larsson M, Pettersson T, and Carlstrom A (1985) Thyroid hormone binding in serum of 15 vertebrate species: isolation of thyroxine-binding globulin and prealbumin analogs. Gen Comp Endocrinol 58, 360–375. Levi L, Levavi-Sivan B, and Lubzens E (2008) Expression of genes associated with retinoid metabolism in the trout ovarian follicle. Biol Reprod 79, 570–577. Lubzens E, Lissauer L, Levavi-Sivan B, Avarre JC, and Sammar M (2003) Carotenoid and retinoid transport to fish oocytes and eggs: what is the role of retinol binding protein?. Mol Aspects Med 24, 441–457. Malpeli G, Folli C, and Berni R (1996) Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin. Biochim Biophys Acta 1294, 48–54. Manzon RG, Neuls TM, and Manzon LA (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen Comp Endocrinol 151, 55–65. Melhus H, Nilsson T, Peterson PA, and Rask L (1991) Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol. Exp Cell Res 197, 119–124. Miwa S and Inui Y (1987) Effects of various doses of thyroxine and triiodothyronine on the metamorphosis of flounder (Paralichthys olivaceus). Gen Comp Endocrinol 67, 356–363. Monaco HL (2000) The transthyretin-retinol-binding protein complex. Biochim Biophys Acta 1482, 65–72.
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Monaco HL, Rizzi M, and Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268, 1039–1041. Morgado I, Melo EP, Lundberg E, Estrela NL, Sauer-Eriksson AE, and Power DM (2008) Hormone affinity and fibril formation of piscine transthyretin: The role of the N-terminal. Mol Cell Endocrinol 295, 48–58 Naylor HM and Newcomer ME (1999) The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP. Biochemistry 38, 2647–2653. Niederreither K and Dolle P (2008) Retinoic acid in development: towards an integrated view. Nat Rev Genet 9, 541–553. Perozzi G, Mengheri E, Colantuoni V, and Gaetani S (1991) Vitamin A intake and in vivo expression of the genes involved in retinol transport. Eur J Biochem 196, 211–217. Power DM, Elias NP, Richardson SJ, Mendes J, Soares CM, and Santos CR (2000) Evolution of the thyroid hormone-binding protein, transthyretin. Gen Comp Endocrinol 119, 241–255. Quadro L, Blaner WS, Salchow DJ, Vogel S, Piantedosi R, Gouras P, Freeman S, Cosma MP, Colantuoni V, and Gottesman ME (1999) Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein. EMBO J 18, 4633–4644. Richardson SJ (2007) Cell and molecular biology of transthyretin and thyroid hormones. Int Rev Cytol 258, 137–193. Richardson SJ, Bradley AJ, Duan W, Wettenhall RE, Harms PJ, Babon JJ, Southwell BR, Nicol S, Donnellan SC, and Schreiber G (1994) Evolution of marsupial and other vertebrate thyroxinebinding plasma proteins. Am J Physiol 266, R1359–R1370. Sammar M, Babin PJ, Durliat M, Meiri I, Zchori I, Elizur A, and Lubzens E (2001) Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues. Gen Comp Endocrinol 123, 51–61. Santos CR, Anjos L, and Power DM (2002) Transthyretin in fish: state of the art. Clin Chem Lab Med 40, 1244–1249. Santos CR and Power DM (1999) Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisation. Endocrinology 140, 2430–2433. Saraiva MJ (2001) Transthyretin amyloidosis: a tale of weak interactions. FEBS Lett 498, 201–203. Saverwyns H, Visser A, Van Durme J, Power D, Morgado I, Kennedy MW, Knox DP, Schymkowitz J, Rousseau F, Gevaert K, Vercruysse J, Claerebout E, and Geldhof P (2008) Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi – Comparison with other strongylid nematodes and Caenorhabditis elegans. Int J Parasitol 38, 1545–1556 Schreiber G and Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B Biochem Mol Biol 116, 137–160. Schug TT, Berry DC, Shaw NS, Travis SN, and Noy N (2007) Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors. Cell 129, 723–733. Selvaraj SR, Bhatia V, and Tatu U (2008) Oxidative folding and assembly with transthyretin are sequential events in the biogenesis of retinol binding protein in the endoplasmic reticulum. Mol Biol Cell 19, 5579–5592. Shidoji Y and Muto Y (1977) Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein. J Lipid Res 18, 679–691. Sivaprasadarao A and Findlay JB (1994) Structure-function studies on human retinol-binding protein using site-directed mutagenesis. Biochem J 300(Pt 2), 437–442. Soprano D and Blaner W (1994) Plasma Retinol-Binding Protein. In The Retinoids: Biology, Chemistry and Medicine [Sporn MB, Goodman DS, editor]. New York: Raven Press. Sunde M, Richardson SJ, Chang L, Pettersson TM, Schreiber G, and Blake CC (1996) The crystal structure of transthyretin from chicken. Eur J Biochem 236, 491–499. Tachihara K, Lel-Zidbeh M, Ishimatsu A, and Tagawa M (1997) Improved seed production of gold-striped amberjack Seriola lalandii under hatchery conditions by injection of triiodothyronine (T3) to broodstock fish. J World Aquacult Soc 28, 34–44.
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Thompson DA and Gal A (2003) Vitamin A metabolism in the retinal pigment epithelium: genes, mutations, and diseases. Prog Retin Eye Res 22, 683–703. Thompson J, Higgins D, and Gibson T (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res 22, 4673–4680. Tingaud-Sequeira A, Forgue J, Andre M, and Babin PJ (2006) Epidermal transient down-regulation of retinol-binding protein 4 and mirror expression of apolipoprotein Eb and estrogen receptor 2a during zebrafish fin and scale development. Dev Dyn 235, 3071–3079. Wojtczak A (1997) Crystal structure of rat transthyretin at 2.5 A resolution: first report on a unique tetrameric structure. Acta Biochim Pol 44, 505–517. Yamauchi K, Kasahara T, Hayashi H, and Horiuchi R (1993) Purification and characterization of a 3,5,30 -L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin. Endocrinology 132, 2254–2261. Yamauchi K, Nakajima J, Hayashi H, and Hara A (1999) Purification and characterization of thyroid-hormone-binding protein from masu salmon serum. A homolog of higher-vertebrate transthyretin. Eur J Biochem 265, 944–949. Zanotti G, Berni R, and Monaco HL (1993) Crystal structure of liganded and unliganded forms of bovine plasma retinol-binding protein. J Biol Chem 268, 10728–10738. Zanotti G, Folli C, Cendron L, Alfieri B, Nishida SK, Gliubich F, Pasquato N, Negro A, and Berni R (2008) Structural and mutational analyses of protein-protein interactions between transthyretin and retinol-binding protein. FEBS J 275, 5841–5854 Zapponi MC, Zanotti G, Stoppini M, and Berni R (1992) The primary structure of piscine (Oncorhynchus mykiss) retinol-binding protein and a comparison with the three-dimensional structure of mammalian retinol-binding protein. Eur J Biochem 210, 937–943.
Chapter 10
TTR and Endocrine Disruptors Kiyoshi Yamauchi and Akinori Ishihara
Abstract In addition to binding thyroid hormones (THs), its endogenous ligands, transthyretin (TTR) can bind a large number of chemicals, including flavonoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and environmental pollutants. By binding to TTR, these chemicals have the potential to change TH homeostasis in plasma and interfere with the thyroid system, particularly in rodents and lower vertebrates. A structure–activity relationship of those chemicals that compete with TH for binding to TTR was found for several groups of chemicals. Owing to the binding specificity of these chemicals for TTR, a competitive binding assay for TH binding to TTR has been used to detect potent TH-disrupting chemicals in samples obtained from various environments. Recent advances in pharmacological and physiological research indicate that some chemicals that bind to TTR may have a chemotherapeutically beneficial effect on TTR-mediated amyloid fibril formation, whereas other chemicals that bind to TTR may have an adverse effect on retinoid homeostasis. Possible roles for the binding of chemicals to TTR are discussed. Keywords Competitive binding, Structure–activity relationship, TH-disrupting activity
10.1
Introduction
In humans, more than 99% of thyroid hormones (THs), including the precursor form l-thyroxine (T4) and the active form 3,30 ,5-triiodo-l-thyronine (T3), in plasma are bound to proteins. These proteins include thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin. TBG has a high affinity for THs and, as such, 70–80% of THs are bound to TBG, despite its low concentration in plasma K. Yamauchi (*) Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka, Japan e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_10, # Springer‐Verlag Berlin Heidelberg 2009
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(0.27 mM). In contrast, only 10–20% of THs are bound to TTR and albumin, which, compared with TBG, are found in higher concentrations in plasma (4.6 and 640 mM, respectively) (Robbins 1996). As the concentrations of T4 and T3 in plasma are 110 and 2.1 nM, respectively (Chopra 1996), 30% of TBG, 0.2–0.3% of TTR, and 0.002–0.003% of albumin are responsible for TH binding in vivo. This situation is not true in rodents, some marsupials, and lower vertebrates because they lack TBG in plasma. Studies of TH binding to TTR also revealed that eutherian TTRs have higher affinity for T4 than for T3, whereas lower vertebrate TTRs have higher affinity for T3 than for T4 (Schreiber and Richardson 1997). Furthermore, TTR interacts with retinol-binding protein (RBP) in plasma from mammals and birds but not in plasma from amphibians and fish. These comparative aspects suggest that the physiological functions of TTR and the potential for environmental chemicals to interfere with TH binding to TTR in the plasma of rodents and/or the lower vertebrates could be quantitatively and qualitatively different from those of eutherians. Humans and wildlife are exposed to many environmental chemicals that are found in food, air, and water. The plasma protein(s) to which these chemicals bind in plasma is one of the important factors that will affect the tissue distribution, metabolism, excretion, and cellular actions of these chemicals. TTR has significant binding affinity for a large number of environmental chemicals including native and synthetic flavonoids as well as medical/pharmaceutical, agricultural, industrial, and nonintentional chemicals such as dioxins and furans (Brucker-Davis 1998). If environmental chemicals, or their metabolites, displace THs from TTR in vivo, they could alter TH homeostasis in plasma by increasing the free concentration of THs. For this reason, TTR has been posited as a molecular target for thyroid disruption by these chemicals (Brouwer et al. 1998; DeVito et al. 1999; Schmutzler et al. 2007). Data accumulated over the past two decades have indicated that chemicals that have binding affinity for TTR can alter the TH levels in plasma and/or modulate cellular actions of THs in rodents or cell culture systems. However, whether chemicals that bind to TTR affect plasma TH levels, particularly in large eutherians given that TTR is responsible for binding only a small proportion of total THs in plasma, is unclear. In addition to the thyroid disruption by these chemicals, medical/ pharmacological and physiological interests in TTR research have increased. In this chapter, we will discuss how environmental chemicals can interfere with the thyroid system by binding to TTR and the possible physiological significance of this interference, as well as the application of a TTR competitive binding assay to detect TH-disrupting chemicals in environment samples.
10.2
Chemicals That Interact with the TH-Binding Sites of TTR
Flavonoids are known to affect the thyroid system, including the function of the thyroid gland, the secretion of THs from the thyroid gland, and the availability of THs for uptake into target cells, partly because of the structural similarity between
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thyroid hormones
I
O
HO
I
NH2 COOH
(I) 3’ 2’ 8
9 O 2
7
flavonoids
6
10
5
1’
8 4’
7
5’
6
6’
5
3
4
10
2
4
3
O
1’
6
2’
5
3’
4
4’
6’ 5’
O
OH
O
F F
O
H N
F
(flufenamic acid*,1.75)
OH H N
CH3
other medical drugs
8 9
O 3
1’
OH O O
6’
2
O
OH
CH3
(mefenamic acid*, 0.27) O
5’
2’
aurones (3’,5’-dibromo-2’,4,4’,6tetrahydroxyaurone) OH O
isoflavone (genistein*)
flavone (F21388, 2.5)
NSAIDs
7
9 O
4’
3’
(L-thyroxine*, 1) (3,3’,5-triiodo-L-thyronine)
(diflunisal*, 0.22)
F
F
OH
N trans-stilbene (diethylstilbestrol*, 37) (resveratrol*)
F
F environmental pollutants
2’
2
3
3
3’
6’
6
5
(Cl)n
5’
2
(Cl)n
4
(Br)n
6 5
3’
6
4’
6’ 5’
2’
3’
(Br)n
polybrominated diphenyl esters and their hydroxylated derivatives
6
CH3
6’
5’
(Cl)n or (Br)n
(Cl)n or (Br)n
2’
O
CH3
OH 5
PCBs and their hydroxylated derivatives (4-OH-3,3’,4’,5-tetrachlorobiphenyl*, 2.56)
3
2
HO
4’
4
(N-(m-trifluoromethylphenyl) phenoxazine 4,6-dicarboxylic acid*)
F
halogenated derivatives of bisphenol A (3,3’,5,5’-tetrabromobisphenol A,10.6) O OH 6 O 5 2 OH
5
3 4
phenols (triiodophenol*, 3.84) (pentabromophenol, 7.12)
4
(Cl)n
2 3
phenoxy acids (2,4-dichlorophenoxy acid)
Fig. 10.1 Thyroid hormones and chemicals with binding affinity for transthyretin (TTR). Core structures or typical examples (in parentheses) of groups of chemicals are indicated. Asterisk (*), Inhibitory activity against amyloid fibril formation (see section on TTR Amyloid). Relative affinities are given as the ratio of IC50 (T4)/IC50 (chemical) except for the value for diethylstilbestrol, which is the ratio of IC50 (T3)/IC50 (chemical). These ratios are from Ko¨hrle et al. (1988), Green et al. (2005), Ciszak et al. (1992), Munro et al. (1989), Ishihara et al. (2003), Brouwer et al. (1990), Van den Berg et al. (1991), Meerts et al. (2000), McKinney et al. (1985), Miroy et al. (1996), Peterson et al. (1998); Klabunde et al. (2000), Purkey et al. (2001), McCammon et al. (2002), Miller et al. (2004), Purkey et al. (2004), and Morais-de-Sa´ et al. (2004)
flavonoids and THs (Schro¨der-van der Elst et al. 2003; van der Heide et al. 2003; Hamann et al. 2006). Flavonoids are multihydroxylated polyphenols and have a basic structure consisting of three rings (Fig. 10.1). Among them, the flavone apigenin (van der Heide et al. 2003) and the isoflavone genistein (Green et al. 2005) have a binding affinity for TTR. These natural, plant-derived flavonoids are
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ingested from food daily, at a rate of up to 2 g per day. Even if the affinities of these flavonoids for TTR were lower than that of TH, these flavonoids are likely to compete with TH for binding to TTR because of the high concentrations of flavonoids in plasma that result from their high intake. The flavone derivatives 4,40 , 6-trihydroxyaurone (Auf’mkolk et al. 1986) and 30 ,50 -dibromo-20 ,4,40 ,6-tetrahydroxyaurone (Ciszak et al. 1992) also have a binding affinity for TTR. The potential of flavonoids to interfere with the thyroid system in rats was studied in detail using a synthetic flavonoid 3-methyl-40 ,6-dihydroxy-30 ,50 -dibromoflavone (F21388). The half maximum inhibitory concentration (IC50) of F21388 for [125I]T4 binding to TTR was 2.5 times lower than that of T4 (Ko¨hrle et al. 1988). In vivo, F21388 displaces TH from TTR (Ko¨hrle et al. 1989; Lueprasitsakul et al. 1990; Mendel et al. 1992) and alters TH levels in plasma (Ko¨hrle et al. 1989; Lueprasitsakul et al. 1990; Schro¨der-van der Elst et al. 1997) and TH metabolism (Schro¨der-van der Elst et al. 1991). These findings have been comprehensively reviewed elsewhere (Ko¨hrle et al. 1988; Hamann et al. 2006). Nonsteroidal anti-inflammatory drugs (NSAIDs) also interfere with TH binding to TTR. Munro et al. (1989) investigated the competitive inhibition potency of 26 drugs, most of which were NSAIDs, with [125I]T4 for binding to TTR in vitro. Of those drugs that competed with TH for binding to TTR, the order of the potency was flufenamic acid>mefenamic acid>diflunisal>meclofenamic acid>diclofenac> fenclofenac>>aspirin. In a later study, the binding properties of NSAIDs were directly confirmed by an antibody capture method in combination with highperformance liquid chromatography (HPLC) analysis (Purkey et al. 2001) and mass spectrometry (McCammon et al. 2002). The affinities of NSAIDs for TTR were several orders of magnitude greater than those for TBG (Munro et al. 1989). Salsalate and its metabolite, salicylate, inhibit TH binding to serum transport proteins and decrease TH levels in plasma in vivo. However, these compounds do not bind specifically to TTR, and can displace TH from TBG and albumin too (Wang et al. 1999). Diethylstilbestrol, a synthetic nonsteroidal estrogen, was the most potent competitor, among 17 candidates, of [125I]T3 binding to chicken TTR and had an IC50 37 times less than that for T3 (Ishihara et al. 2003). Resveratrol, a phytoalexin produced naturally by several plants, and N-(m-trifluoromethylphenyl)phenoxazine 4,6-dicarboxylic acid also have binding affinity for TTR (Klabunde et al. 2000; McCammon et al. 2002). As diethylstilbestrol can cause estrogenic effects, the binding of the chemical to TTR could limit its access to target cells, thereby modulating their cellular actions. Polychlorinated biphenyls (PCBs) are industrial chemicals consisting of paired phenyl rings that have various degrees of chlorination. There are 209 possible PCB congeners, depending on the position and number of chlorine atoms. Although their production was banned in the mid-1970s, PCBs are still widespread and persist in the environment, and are concentrated in the food chain. Analysis of the competitive binding of PCBs and [125I]T4 to TTR demonstrates that laterally (3,30 ,5,50 -) substituted PCBs have a higher affinity for TTR than nonlateral (2,20 ,6,60 -) substituted PCBs (Richenbacker et al. 1986). In addition, the PCB congeners that have
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a di-meta substitution in one or both rings, such as 3,30 ,5,50 -tetrachlorobiphenyl (TCB), have the highest binding activity among the PCBs (Chauhan et al. 2000). Hydroxylated PCBs are more potent competitors of [125I]T4 binding to TTR than their parent PCBs (Richenbacker et al. 1986; Lans et al. 1993). Of the hydroxylated PCBs, those that have the hydroxyl group substituted on the meta or para positions, and one or more adjacent chlorine substituents, have the highest competitive binding potency, with Kd’s ranging from 8 to 40 nM (Lans et al. 1993). The hydroxylated derivatives of polychlorinated dibenzo-p-dioxins and dibenzofurans, which have a chlorine substitution adjacent to the hydroxyl group, also have a relatively high binding potency for TTR (Lans et al. 1993). These binding properties were confirmed directly by the antibody capture HPLC method (Purkey et al. 2004). Although there is no direct evidence that supports the hypothesis that the binding of hydroxylated PCBs to TTR lowers total TH levels in plasma by displacing THs bound to TTR, there is a significant inverse relationship between PCB and TH levels in plasma from water birds, polar bears, and humans (Brouwer et al. 1998). Common seals fed on PCB-contaminated fish have significantly lower concentrations of total T4 and T3 and free T4 than those fed on control fish (Brouwer et al. 1989). Of the 65 agricultural and industrial chemicals from 12 chemical groups tested, 21 chemicals from 6 chemical groups, including chlorophenols, phenoxy acids, nitrophenols, chlorobenzenes, dichloro-diphenyl-trichloroethanes (dicofol), and others (bromoxynil), were the most potent competitors for [125I]T4 binding to human TTR in vitro (Van den Berg et al. 1991). In contrast, an in vivo study showed that 2,4dichlorophenoxybutyric acid, dinoseb, bromoxynil, and pentachlorophenol of the 21 chemicals were associated with a decrease in plasma levels of total T4 in rats 6 h after the chemicals were administered (Van den Berg et al. 1991). The competitive binding potency of these chemicals for TTR from various vertebrates differs because of variations in the binding specificity of TTR for TH and in the composition of plasma TH-binding proteins (Ishihara et al. 2003). Structure–activity relationships demonstrate the importance of the hydroxyl group and one or more adjacent halogen substituents for binding to TTR in the chlorinated and brominated derivatives of bisphenol A and nonylphenol (Yamauchi and Ishihara 2006); brominated flame retardants and their related compounds, such as polybrominated diphenyl ethers, pentabromophenol, and tetrabromobisphenol A (Meerts et al. 2000); and chlorinated phenols (Van den Berg 1990). This structural characteristic of these chemicals resembles the structure of THs. However, this structural characteristic does not appear to be essential for chemicals to bind to TTR given that most flavonoids, NSAIDs, and stilbenes have no halogen atoms.
10.3
Chemicals That Affect the Stability of the TTR Tetramer and the Formation of the TTR–RBP Complex
A recent intriguing finding is that ligand binding stabilizes the tetrameric structure of human TTR and prevents TTR-mediated amyloid fibril formation, which is closely linked to the human amyloid diseases, senile systemic amyloidosis, and
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familial amyloid polyneuropathy (Raghu et al. 2004). Most of the compounds that have been found to bind to TTR inhibit TTR-mediated amyloid fibril formation (asterisks in Fig. 10.1), including T4 (Miroy et al. 1996; White and Kelly 2001); flavonoids such as genistein and daidzein (Green et al. 2005); NSAIDs such as flufenanic acid, diclofenac, and diflunisal (Klabunde et al. 2000; Purkey et al. 2001; McCammon et al. 2002; Miller et al. 2004); resveratrol (Klabunde et al. 2000; Purkey et al. 2001; McCammon et al. 2002; Morais-de-Sa´ et al. 2004); diethylstilbestrol (Morais-de-Sa´ et al. 2004); phenoxazine derivatives (Klabunde et al. 2000; McCammon et al. 2002); hydroxylated PCBs (McCammon et al. 2002; Purkey et al. 2004); triiodophenol (Miroy et al. 1996); and 2,4-dinitrophenol (Morais-de-Sa´ et al. 2006). The majority of the chemicals tested by McCammon et al. (2002) exhibit negative cooperativity in binding to TTR, whereas the others exhibit positive cooperativity (e.g., hydroxylated PCB) or noncooperative (e.g., derivatives of Nphenyl phenoxazine) binding modes. A combination of new technical approaches for structure-based drug design, screening, and inhibitory assays facilitated the discovery of a number of novel inhibitors of TTR-mediated amyloid fibril formation, such as derivatives of NSAIDs (Oza et al. 2002; Adamski-Werner et al. 2004; Almeida et al. 2004), bisaryloxime ethers (Johnson et al. 2005), dibenzofurans including dibenzofuran-4,6-dicarboxylic acid (Klabunde et al. 2000; Petrassi et al. 2005), carborane pharmacophores (Julius et al. 2007), and 2-arylbenzoxazoles (Johnson et al. 2008). The structural information of the chemical–TTR complexes will provide a rationale for chemotherapeutic approaches to prevent TTR-associated amyloid diseases. These chemotherapeutic approaches have recently been reviewed in detail (Raghu et al. 2004; see section on TTR Amyloid). When TTR binds RBP, TTR can also transport, indirectly, retinoids and their derivatives, which bind to RBP (Berni et al. 1993). RPB circulates as a 1:1 complex with TTR in blood at a concentration of 1 mM in humans. The TTR–RBP complex prevents filtration of the relatively small RBP molecule through kidney glomeruli (Zanotti and Berni 2004). Retinoid binding to RBP strengthens the TTR–RBP interaction (Fex et al. 1979). When apoRBP is incubated with TTR in vitro, the binding affinity of RBP for TTR was less than that when all-trans retinol or alltrans retinoic acid was added to the reaction mixture (Kd = 1.2 mM vs. 0.2 and 0.8 mM, respectively) (Malpeli et al. 1996). However, the binding affinity of RBP for TTR was decreased with the addition of all-trans retinyl methyl ether (Kd = 6 mM) (Malpeli et al. 1996). In contrast, the synthetic retinoids fenretinide and N-ethylretinamide abolished the binding affinity of RBP for TTR without the apparent loss of binding affinity of RBP for these ligands (Berni and Formelli 1992; Zanotti et al. 1993; Malpeli et al. 1996), indicating that the substitution of the hydroxyl group with an amide group on retinol affects the TTR–RBP interaction. Interestingly, piscine TTR and RBP do not form a complex in plasma (Shidoji and Muto 1977). In support of this finding, the amino acid sequences in piscine TTR and RBP differ from those in human TTR and RBP at positions critical for the interaction between RBP and TTR in higher vertebrates (Zanotti and Berni 2004). Another example demonstrating that a small ligand weakens the TTR-RBP interaction is a metabolite of 3,30 ,4,40 -TCB, but not TCB (Brouwer and Van den
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Berg 1986; Brouwer et al. 1986; Brouwer et al. 1989). Within two days of exposing rats to TCB, plasma levels of RBP, retinol, and T4 in rats had decreased (Brouwer and Van den Berg 1986; Brouwer et al. 1986). A subsequent study identified 4-OH-3,30 ,40 ,5-TCB as the metabolite of TCB that bound to TTR (Brouwer et al. 1990). Given that a number of PCB congeners are enzymatically hydroxylated by members of the cytochrome P-450 family (Bergman et al. 1994), and that several hydroxylated PCBs bind TTR in vitro with high affinity (Rickenbacker et al. 1986; Lans et al. 1993; Cheek et al. 1999; Purkey et al. 2004), other hydroxylated PCBs may have similar effects to those observed for 4-OH-3,30 ,40 ,5-TCB. However, no reports have appeared to date indicating such an action. In contrast, a recent study demonstrated that the in vitro binding of 4,40 diOH-3,30 ,5,50 -TCB to TTR did not inhibit the association of TTR with holoRBP (MaCammon et al. 2002). The binding of holoRBP to TTR, as well as ligand binding to TTR, inhibits TTRmediated amyloid fibril formation (White and Kelly 2001). RBP is secreted from hepatocytes as the holoRBP–TTR complex (Bellovino et al. 1996). Therefore, plasma levels of the natural ligands for TTR and RBP, and of the TTR-RBP complex, would be critical factors for the inhibition of TTR-mediated amyloid fibril formation (White and Kelly 2001). For chemotherapeutical approaches to prevent TTR-mediated amyloid diseases successfully, it would be essential that the binding of chemicals to TTR neither disrupt the thyroid or retinoid system nor exert any adverse effects on humans.
10.4
Application of Competitive TTR Binding Assay to Detect Potential TH-Disrupting Chemicals
The competitive binding assay for TH binding to TTR has been applied to detect TH-disrupting chemicals in various environmental samples, such as airborne particulate matter extracts (Heussen et al. 1993), surface waters (Yamauchi et al. 2003; Murata and Yamauchi 2007), and sediments (Houtman et al. 2004). Extracts from samples of air collected outdoors and of cigarette smoke, which contained 6.6 and 0.23 ng pentachlorophenol per m3 of sample air, respectively, inhibited [125I]T4 binding to TTR in a dose-dependent manner but not [125I]T4 binding to TBG. Plasma levels of total T3 and T4, and free T4, decreased significantly within 6 h after rats were intraperitoneally injected with the extracts from a sample of air collected outdoors (Heussen et al. 1993). Utilizing a similar competitive assay, TH-displacing activity was also detected in effluent from, and downstream of, paper manufacturing plants in Japan (Yamauchi et al. 2003). Contaminants in water samples collected from various locations inhibited [125I]T3 binding to TTR in the following order: effluent>industrial drain>river month>harbor entrance (Fig. 10.2). This result indicated that the causative contaminants are released from the plants and are diluted downstream.
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Fig. 10.2 Detection of potential thyroid hormone-disrupting chemicals in surface water downstream from paper manufacturing plants by TTR competition assay. Open columns, water samples used directly in this assay; shaded columns, contaminant samples extracted from the water samples with dichloromethane. Original data were from Yamauchi et al. (2003)
Although the assay is performed using water samples (open columns in Fig. 10.2) or samples of contaminants that have been extracted from the water samples using solid phase extraction (shaded columns in Fig. 10.2), both samples (at 0.8 times their original concentrations) have similar TH-displacing activity, suggesting that extraction and concentration of contaminants are not necessary at least for surface water samples (Yamauchi et al. 2003; Murata and Yamauchi 2007). There is no direct evidence indicating that the effluent investigated in the aforementioned studies disturbs the thyroid system of the native aquatic inhabitants. This is in contrast to the finding in Finland that juvenile female rainbow trout exposed to diluted pulp mill effluents had increased T4 levels and decreased T3 levels in their plasma (Mattsson et al. 2001). More recently, this competitive binding assay for TH binding to TTR was used to detect contaminants that had TH-displacing activity in sediments from the Rhine Meuse estuary in the Netherlands (Houtman et al. 2004). The Rhine Meuse estuary acts as a sedimentary basin for the river, and contaminants discharged upstream of the estuary tend to accumulate here. There is evidence that indicates that the endocrine system in aquatic wildlife is disturbed following exposure to contaminated sediments in the Rhine Meuse estuary (Vos et al. 2000). Of the samples of sediment collected from 15 locations in the Rhine Meuse estuary, half had significant TH-displacing
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activity that was dose dependent (6–16 pmol T4-equivalents per gram dry weight). In addition to T4 competition binding activity, the authors investigated dioxin responsiveness and estrogen responsiveness, using chemical-activated luciferase gene expression assays, and genotoxicity and nonspecific toxicity in the samples of sediment. However, there were no location-dependent correlations between the T4 competition binding activity and the activities recorded using the other assays. Thus, the sediments potentially contain different endocrine-disrupting activities.
10.5
Perspectives
Chemicals that bind to TTR have the potential to affect TH homeostasis in plasma if they displace TH from TTR. In addition to those chemicals that directly interfere with the thyroid system, a hydroxylated PCB also affects retinoid homeostasis in plasma indirectly by decreasing the TTR–RBP association. Several lines of evidence indicate other physiological roles for TTR and RBP, which are likely to be affected by these chemicals: (1) Chemicals with binding affinity for TTR are also potent inhibitors of TTRmediated amyloid fibril formation (Fig. 10.1). This finding may aid the research of chemotherapeutic approaches that prevent the TTR-associated amyloid diseases, familial amyloid polyneuropathy, and senile systemic amyloidosis. More than 99% of TTR in plasma from eutherians, including that from humans, is not bound by THs. Does this mean that more than 99% of TTR in plasma is apoTTR? It is not known whether endogenous ligands other than THs that bind to TTR, such as all-trans retinoic acid (Kd = 107 M; Smith et al. 1994), inhibit TTR-mediated amyloid fibril formation. (2) Novel roles of TTR within specific cells or cerebrospinal fluid have been suggested in two reports. First, TTR has been found to interact with metalothionein 2 in human liver (Kd = 245 nM), and the complex of the two proteins is found in rat choroid plexus and kidney (Gonc¸alves et al. 2008). Second, in pancreatic b-cells, TTR promotes glucose-induced increases in cytoplasmic free Ca2+ concentration and protects against b-cell apoptosis (Refai et al. 2005). (3) A positive relationship between the level of RBP in plasma and insulin resistance/glucose intolerance in mice and humans who are obese and have type 2 diabetes has been discovered. The synthesis of RBP in, and secretion of RBP from, adipose tissue, but not the liver, resulted in increased levels of RBP in plasma (Yang et al. 2005). This result indicates that RBP may act as an adipocyte-derived signal. Whether these other physiological roles of TTR and RBP are affected by natural and synthetic ligands for TTR or RBP remains to be clarified.
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DeVito M, Biegel L, Brouwer A, Brown S, Brucker-Davis F, Cheek AO, Christensen R, Colborn T, Cooke P, Crissman J, Crofton K, Doerge D, Gray E, Hauser P, Hurley P, Kohn M, Lazar J, McMaster S, McClain M, McConnell E, Meier C, Miller R, Tietge J, Tyl R (1999) Screening methods for thyroid hormone disruptors. Environ Health Perspect 107:407–414 Fex G, Albertsson PA, Hansson B (1979) Interaction between prealbumin and retinol-binding protein studied by affinity chromatography, gel filtration and two-phase partition. Eur J Biochem 99:353–360 Gonc¸alves I, Quintela T, Baltazar G, Almeida MR, Saraiva MJ, Santos CR (2008) Transthyretin interacts with metallothionein 2. Biochemistry 47:2244–2251 Green NS, Foss TR, Kelly JW (2005) Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis. Proc Natl Acad Sci USA 102:14545–14550 Hamann I, Seidlova-Wuttke D, Wuttke W, Ko¨hrle J (2006) Effects of isoflavonoids and other plant-derived compounds on the hypothalamus–pituitary–thyroid hormone axis. Maturitas 55S:S14–S25 Heussen GA, Schefferlie GJ, Talsma MJ, van Til H, Dohmen MJ, Brouwer A, Alink GM (1993) Effects on thyroid hormone metabolism and depletion of lung vitamin A in rats by airborne particulate matter. J Toxicol Environ Health 38:419–434 Houtman CJ, Cenijn PH, Hamers T, Lamoree MH, Legler J, Murk AJ, Brouwer A (2004) Toxicological profiling of sediments using in vitro bioassays, with emphasis on endocrine disruption. Environ Toxicol Chem 23:32–40 Ishihara A, Sawatsubashi S, Yamauchi K (2003) Endocrine disrupting chemicals: interference of thyroid hormone binding to transthyretins and to thyroid hormone receptors. Mol Cell Endocrinol 199:105–117 Johnson SM, Connelly S, Wilson IA, Kelly JW (2008) Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J Med Chem 51:260–270 Johnson SM, Petrassi HM, Palaninathan SK, Mohamedmohaideen NN, Purkey HE, Nichols C, Chiang KP, Walkup T, Sacchettini JC, Sharpless KB, Kelly JW (2005) Bisaryloxime ethers as potent inhibitors of transthyretin amyloid fibril formation. J Med Chem 48:1576–1587 Julius RL, Farha OK, Chiang J, Perry LJ, Hawthorne MF (2007) Synthesis and evaluation of transthyretin amyloidosis inhibitors containing carborane pharmacophores. Proc Natl Acad Sci USA 104:4808–4813 Klabunde T, Petrassi HM, Oza VB, Raman P, Kelly JW, Sacchettini JC (2000) Rational design of potent human transthyretin amyloid disease inhibitors. Nat Struct Biol 7:312–321 Ko¨hrle J, Fang SL, Yang Y, Irmscher K, Hesch RD, Pino S, Alex S, Braverman LE (1989) Rapid effects of the flavonoid EMD 21388 on serum thyroid hormone binding and thyrotropin regulation in the rat. Endocrinology 125:532–537 Ko¨hrle J, Spanka M, Irmscher K, Hesch RD (1988) Flavonoid effects on transport, metabolism and action of thyroid hormones. In: Cody V, Middleton E, Harborne JB, Beretz A (eds), Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular, and Medical Properties, Alan R Liss, New York, pp 323–340 Lans MC, Klasson-Wehler E, Willemsen M, Meussen E, Safe S, Brouwer A (1993) Structuredependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin. Chem Biol Interact 88:7–21 Lueprasitsakul W, Alex S, Fang SL, Pino S, Irmscher K, Ko¨hrle J, Braverman LE (1990) Flavonoid administration immediately displaces thyroxine (T4) from serum transthyretin, increases serum free T4, and decreases serum thyrotropin in the rat. Endocrinology 126:2890–2895 Malpeli G, Folli C, Berni R (1996) Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin. Biochim Biophys Acta 1294:48–54 Mattsson K, Lehtinen KJ, Tana J, Ha¨rdig J, Kukkonen J, Nakari T, Engstro¨m C (2001) Effects of pulp mill effluents and restricted diet on growth and physiology of rainbow trout (Oncorhynchus mykiss). Ecotoxicol Environ Saf 49:144–154
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McCammon MG, Scott DJ, Keetch CA, Greene LH, Purkey HE, Petrassi HM, Kelly JW, Robinson CV (2002) Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein, multiligand complexes by mass spectrometry. Structure 10:851–863 McKinney JD, Chae K, Oatley SJ, Blake CC (1985) Molecular interactions of toxic chlorinated dibenzo-p-dioxins and dibenzofurans with thyroxine binding prealbumin. J Med Chem 28:375–381 Meerts IATM, van Zanden JJ, Luijks EAC, van Leeuwen-Bol I, Marsh G, Jakobsson E, Bergman ˚ , Brouwer A (2000) Potent competitive interactions of some brominated flame retardants and A related compounds with human transthyretin in vitro. Toxicol Sci 56:95–104 Mendel CM, Cavalieri RR, Ko¨hrle J (1992) Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin. Endocrinology 130:1525–1532 Miller SR, Sekijima Y, Kelly JW (2004) Native state stabilization by NSAIDs inhibits transthyretin amyloidogenesis from the most common familial disease variants. Lab Invest 84:545–552 Miroy GJ, Lai Z, Lashuel HA, Peterson SA, Strang C, Kelly JW (1996) Inhibiting transthyretin amyloid fibril formation via protein stabilization. Proc Natl Acad Sci USA 93:15051–15056 Morais-de-Sa´ E, Neto-Silva RM, Pereira PJ, Saraiva MJ, Damas AM (2006) The binding of 2,4dinitrophenol to wild-type and amyloidogenic transthyretin. Acta Crystallogr D Biol Crystallogr 62:512–519 Morais-de-Sa´ E, Pereira PJ, Saraiva MJ, Damas AM (2004) The crystal structure of transthyretin in complex with diethylstilbestrol: a promising template for the design of amyloid inhibitors. J Biol Chem 279:53483–53490 Munro SL, Lim CF, Hall JG, Barlow JW, Craik DJ, Topliss DJ, Stockigt JR (1989) Drug competition for thyroxine binding to transthyretin (prealbumin): comparison with effects on thyroxine-binding globulin. J Clin Endocrinol Metab 68:1141–1147 Murata T, Yamauchi K (2007) 3,30 ,5-Triiodo-l-thyronine-like activity in effluents from domestic sewage treatment plants detected by in vitro and in vivo bioassays. Toxicol Appl Pharmacol 226:309–317 Oza VB, Smith C, Raman P, Koepf EK, Lashuel HA, Petrassi HM, Chiang KP, Powers ET, Sachettinni J, Kelly JW (2002) Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors. J Med Chem 45:321–332 Peterson SA, Klabunde T, Lashuel HA, Purkey H, Sacchettini JC, Kelly JW (1998) Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc Natl Acad Sci USA 95:12956–12960 Petrassi HM, Johnson SM, Purkey HE, Chiang KP, Walkup T, Jiang X, Powers ET, Kelly JW (2005) Potent and selective structure-based dibenzofuran inhibitors of transthyretin amyloidogenesis: kinetic stabilization of the native state. J Am Chem Soc 127:6662–6671 Purkey HE, Dorrell MI, Kelly JW (2001) Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma. Proc Natl Acad Sci USA 98:5566–5571 Purkey HE, Palaninathan SK, Kent KC, Smith C, Safe SH, Sacchettini JC, Kelly JW (2004) Hydroxylated polychlorinated biphenyls selectively bind transthyretin in blood and inhibit amyloidogenesis: rationalizing rodent PCB toxicity. Chem Biol 11:1719–1728 Raghu P, Reddy GB, Sivakumar B (2004) Inhibition of transthyretin amyloid fibril formation by 2,4-dinitrophenol through tetramer stabilization. Arch Biochem Biophys 400:43–47 Refai E, Dekki N, Yang SN, Imreh G, Cabrera O, Yu L, Yang G, Norgren S, Ro¨ssner SM, Inverardi L, Ricordi C, Olivecrona G, Andersson M, Jo¨rnvall H, Berggren PO, Juntti-Berggren L (2005) Transthyretin constitutes a functional component in pancreatic b-cell stimulussecretion coupling. Proc Natl Acad Sci USA 102:17020–17025 Rickenbacher U, McKinney JD, Oatley SJ, Blake CC (1986) Structurally specific binding of halogenated biphenyls to thyroxine transport protein. J Med Chem 29:641–648
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Robbins J (1996) Thyroid hormone transport proteins and the physiology of hormone binding. In: Braverman LE, Utiger RD (eds) Weiner and Ingbar’s The Thyroid, 7th edn. Lippincott-Raven, Philadelphia, pp 96–110 Schmutzler C, Gotthardt I, Hofmann PJ, Radovic B, Kovacs G, Stemmler L, Nobis I, Bacinski A, Mentrup B, Ambrugger P, Gru¨ters A, Malendowicz LK, Christoffel J, Jarry H, Seidlova`Wuttke D, Wuttke W, Ko¨hrle J (2007) Endocrine disruptors and the thyroid gland-a combined in vitro and in vivo analysis of potential new biomarkers. Environ Health Perspect 115 (Suppl 1):77–83 Schro¨der-van der Elst JP, Smit JW, Romijn HA, van der Heide D (2003) Dietary flavonoids and iodine metabolism. Biofactors 19:171–176 Schro¨der-van der Elst JP, van der Heide D, Ko¨hrle J (1991) In vivo effects of flavonoid EMD 21388 on thyroid hormone secretion and metabolism in rats. Am J Physiol 261:E227–E232 Schro¨der-van der Elst JP, van der Heide D, Rokos H, Ko¨hrle J, Morreale de Escobar G (1997) Different tissue distribution, elimination, and kinetics of thyroxine and its conformational analog, the synthetic flavonoid EMD 49209 in the rat. Endocrinology 138:79–84 Schreiber G, Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B Biochem Mol Biol 116:137–160 Shidoji Y, Muto Y (1977) Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein. J Lipid Res 18:679–691 Smith TJ, Davis FB, Deziel MR, Davis PJ, Ramsden DB, Schoenl M (1994) Retinoic acid inhibition of thyroxine binding to human transthyretin. Biochim Biophys Acta 1199:76–80 Van den Berg KJ (1990) Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin. Chem Biol Interact 76:63–75 Van den Berg KJ, van Raaij JA, Bragt PC, Notten WR (1991) Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo. Arch Toxicol 65:15–19 van der Heide D, Kastelijn J, Schro¨der-van der Elst JP (2003) Flavonoids and thyroid disease. Biofactors 19:113–119 Vos JG, Dybing E, Greim HA, Ladefoged O, Lambre´ C, Tarazona JV, Brandt I, Vethaak AD (2000) Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit Rev Toxicol 30:71–133 Wang R, Nelson JC, Wilcox RB (1999) Salsalate and salicylate binding to and their displacement of thyroxine from thyroxine-binding globulin, transthyretin, and albumin. Thyroid 9:359–364 White JT, Kelly JW (2001) Support for the multigenic hypothesis of amyloidosis: the binding stoichiometry of retinol-binding protein, vitamin A, and thyroid hormone influences transthyretin amyloidogenicity in vitro. Proc Natl Acad Sci USA 98:13019–13024 Yamauchi K, Ishihara A (2006) Thyroid system-disrupting chemicals: interference with thyroid hormone binding to plasma proteins and the cellular thyroid hormone signaling pathway. Rev Environ Health 21:229–251 Yamauchi K, Ishihara A, Fukazawa H, Terao Y (2003) Competitive interactions of chlorinated phenol compounds with 3,30 ,5-triiodothyronine binding to transthyretin: detection of possible thyroid-disrupting chemicals in environmental waste water. Toxicol Appl Pharmacol 187: 110–117 Yang Q, Graham TE, Mody N, Preitner F, Peroni OD, Zabolotny JM, Kotani K, Quadro L, Kahn BB (2005) Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 436:356–362 Zanotti G, Malpeli G, Berni R (1993) The interaction of N-ethyl retinamide with plasma retinol˚ resolubinding protein (RBP) and the crystal structure of the retinoid-RBP complex at 1.9-A tion. J Biol Chem 268:24873–24879 Zanotti G, Berni R (2004) Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin. Vitam Horm 69:271–295
Chapter 11
Genetics: Clinical Implications of TTR Amyloidosis Merrill D Benson
Abstract Transthyretin (TTR) is a fascinating protein. Its transport properties for thyroxin and Vitamin A, its classic b-barrel structure, stable homo-tetrameric structure in solution and its phylogenetic evolution have all been of great interest to scientific study. Even the lack of pathology in its absence, in TTR gene knockout mice, has been of great interest. Its entire notoriety in the area of human pathology, however, is that it readily forms amyloid fibrils, usually due to single amino acid mutations in its primary structure but in some cases even when there is no gene or amino acid structural alteration. TTR amyloidosis is strictly a human disease and more than 100 TTR mutations having been associated with the disease. A few of the mutations are found in large kindreds worldwide while many mutations have been found in only single individuals or single families. In depth analysis of this autosomal dominant disease with an appreciation of the variations in phenotypes suggests clues which may help rationalize basic physical and chemical studies of TTR with biochemical processes which must be involved in the generation of clinical disease. This chapter discusses the clinical aspects of the disease and tries to use these observations to contemplate the intricacies of pathogenesis. Keywords Amyloid, Amyloidosis, Cardiomyopthy, Neuropathy
11.1
Introduction
Transthyretin (TTR) amyloidosis is the most common type of hereditary amyloidosis. It may also occur as a sporadic disease of the elderly as senile systemic amyloidosis (Benson 2003; Cornwell et al. 1981; Pitka¨nen et al. 1984). The hereditary M.D. Benson (*) Indiana University School of Medicine, 635 Barnhill Drive, MS-128, Indianapolis, IN 462025126 USA e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_11, # Springer‐Verlag Berlin Heidelberg 2009
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form is associated with mutations in TTR of which over a 100 have been identified (Connors et al. 2003). In senile systemic amyloidosis (SSA), also known as senile cardiac amyloidosis (SCA), amyloid deposits are derived from normal TTR and, thus far, no genetic predisposition has been identified (Westermark et al. 1990). This occurrence suggests that the highly b-sheet structured TTR protein is innately prone to form b-pleated sheet fibrils and alterations in its primary structure are likely to encourage this fate. However, the story is not that simple. Variations in the age of the disease onset, penetrance, organ involvement by amyloid deposits, and male versus female disease presentation indicate that there are factors, yet unknown, that must be involved in the disease process that is called TTR amyloidosis. Analysis of the data on TTR amyloidosis that have been accumulated since Andrade’s description of ‘‘A peculiar form of peripheral neuropathy. . .,’’ later called Familial Amyloidotic Polyneuropathy (FAP), requires weighing the importance of each observation of the disease and integration of these observations into an overall picture which may aid in understanding the pathogenesis of the disease (Andrade 1952). The key word is ‘‘observation’’ for we have little to rely on in the realm of experimentation. TTR amyloidosis is a disease of humans. No other species has been found to have a similar disease, and attempts to create an animal model of the disease have limited success in revealing the pathogenic mechanisms that are involved in the transition of a soluble plasma protein to the tissue deposits of inert b-pleated fibrils which we call amyloid. Acknowledging that we must start with predominantly descriptive research we will try to piece together the puzzle of clinical TTR amyloidosis with the hope that it will eventually be validated by experimental reasoning and research. To start with, we will consider the clinical presentation of TTR amyloidosis and the ‘‘variations on a theme’’ that may add to the overall picture. This requires a composite view of the disease plus a consideration of the clinical disease in the larger kindreds with specific TTR mutations. Then it is important to ask what might be learned by the analysis of clinical variations which are seen in families or single individuals where rare, uncommon mutations have been found. If we are able to identify from our clinical observations those factors which are most pertinent to the evolution of the clinical disease, we can attempt to reconcile them with the sparse experimental data we have on TTR expression and metabolism to formulate hypotheses on pathogenesis.
11.2
Clinical Picture of TTR Amyloidosis
The original description of FAP by Andrade emphasized the peripheral neuropathy which – while a major feature of most of the syndromes associated with TTR mutations – is now known to be only one part of the picture (Andrade 1952). Andrade also noted extensive renal amyloid deposition in some cases, but we now know that this is relatively uncommon, especially when compared with the incidence of amyloid cardiomyopathy (Dubrey et al. 1997).
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The typical clinical presentation in Portuguese patients is with loss of small fiber function in the lower extremities. Loss of temperature perception followed by loss of touch and pain modality is the rule with progression in a distal to proximal pattern (Fig. 11.1) (Benson and Kincaid 2007). Parasthesiae and painful dysasthesias may occur mainly in the lower extremities. Motor impairment occurs with foot drop and muscle atrophy. The need for walking aids and, finally, the use of a wheelchair is not uncommon. At the same time bowel dysfunction with constipation but more frequently persistent diarrhea and early satiety results in varying degrees of weight loss and malnutrition. It may be difficult to judge if the degree of physical impairment is due to neuropathy or to generalized inanition. Sexual impotence tends to occur in males and occasionally may be the first manifestation of the clinical disease. Features noted by Andrade included trophic skin changes and nonhealing ulcers of the feet, hand muscle atrophy which was probably the result of median nerve compression (carpal tunnel syndrome) and, rarely, irregular pupil conformation (Fig. 11.2; Lessell et al. 1975). In Portugal, the age of onset of the clinical disease was originally noted as the late twenties to early thirties and this has remained consistent over the past 50 years. Some individuals in Portugal have been found to have late onset of the clinical disease (after age 60) but this is a small percentage of the whole (Sequeiros and Saraiva 1987). Life expectancy was originally reported as less than ten years from disease onset and modern medicine has not changed this figure dramatically. Early descriptions of TTR amyloidosis in Japanese patients were similar to those of the Portuguese disease with an emphasis on neuropathy and muscle wasting (Araki et al. 1968). Disease onset was similar (20–30 years) and with progression of peripheral neuropathy, diarrhea, and generalized debilitation. More recently we have realized the importance of amyloid cardiomyopathy with congestive heart
Fig. 11.1 Distribution of typical nerve function loss in TTR amyloidosis
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Fig. 11.2 Scalloped pupil deformity due to amyloid involvement of ciliary nerves in a patient with TTR Val30Met
failure and arrhythmias in overall survival. It is generally considered that the larger kindreds with FAP in Japan represent introduction of the TTR Val30Met mutation by Portuguese colonization. This is supported by DNA studies showing a common TTR allele haplotype for Portuguese and most Japanese patients (Yoshioka et al. 1980). TTR amyloidosis was also recognized as a hereditary disease at an early date in Northern Sweden. As early as 1968 Andersen reported amyloidosis in association with vitreous opacities and in 1970 emphasized the hereditary nature of this peripheral neuropathy (Andersson 1970, 1968). While we now know that the Swedish patients have the same TTR mutation (Val30Met) as the Portuguese and the Japanese, the clinical disease is quite different. Age of disease onset is in the late fifties and survival after diagnosis is often 10–20 years (Holmgren et al. 1988). Cardiomyopathy and vitreous opacities are common and peripheral neuropathy, while a significant part of the syndrome, tends to be less rapid in progression and, therefore, not as debilitating (Sandgren 1995). Diarrhea, however, can be problematic with resultant weight loss and physical disability (Shur et al. 1994).
11.3
Larger Kindreds
Over 100 TTR mutations have been identified which are associated with amyloidosis. Most mutations have been found in only one family and some in only a single individual. While many of the mutations have not been linked to an inheritance
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pattern, only two have been shown to represent new mutation events (Murakami et al.1992; Yazaki et al. 2002b). Many of these uncommon mutations have been found in individuals who presented with vitreous opacities or cardiomyopathy which have brought the patients to medical attention (Yazaki et al. 2002a). On the other hand, often a family has historically been afflicted with peripheral neuropathy or heart disease and several generations have passed before the causative amyloid has been recognized (Benson and Cohen 1977). This has been the case with some of the TTR mutations that are well represented by extensive kindreds. Included in this group are: Val30Met, Leu58His, Thr60Ala, Ser77Tyr, Ile84Ser, and Val122Ile. To appreciate the phenotypes of clinical disease special consideration of the more common TTR mutations is in order to see variations between kindreds and within kindreds and families (Table 11.1).
11.4
Clinical Aspects of Common TTR Mutations
Val30Met amyloidosis characteristically presents as sensorimotor polyneuropathy with varying degrees of cardiomyopathy. Bowel dysfunction is common with constipation but more often persistent diarrhea is a major manifestation of the disease. Vitreous opacities may or may not occur and this varies within families. Renal amyloid may be present but is less common than cardiac amyloid. The wide variation in mean age of the onset of the disease that is seen for Portuguese and Japanese patients (25–40 years) compared with Swedish patients (55–60 years) tells us that there must be factors, most likely related to metabolism and aging, that modify the disease. The high incidence of vitreous amyloid in older Swedish patients is another observation that suggests age related modifiers (Sandgren 1995).
Table 11.1 Clinical features of the most frequent TTR amyloidoses Mutation Typical age Neuropathy Cardiac Intestinal Eye of onset/ 0–4+ 0–4+ 0–4+ vitreous years yes = +/ no = Val30Met
Leu58His Thr60Ala Ser77Tyr Ile84Ser Val122Ile
30–35 (Portugal, Japan) 55–60 (Sweden) 50–60 60–65 50–60 45–55 60–65
Leptomeninges yes = +/ no =
Renal yes = +/ no =
3–4
2–3
2–4
Rare
2–3 1–2 3–4 2–3 1–2
3–4 4 3–4 4 4
– 0–4 2–3 2–3 1–2
– – – + –
– – – – –
– – – –
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In addition, DNA studies have identified at least three haplotypes that segregate with the Val30Met mutation (Yoshioka et al. 1980). Portuguese, Swedish, and most Japanese patients share TTR haplotype I. Many English patients have TTR haplotype III and show later onset of disease, less extensive neuropathy and more prominent cardiomyopathy (Kincaid et al. 1989). Haplotype II was described only in the original report of this genetic analysis. Leu58His amyloidosis was described in 1969 but the TTR mutation was not identified until 1989 (Mahloudji et al. 1969; Nichols et al. 1991). The disease presented in Americans of German heritage and the mutation has subsequently been found in families in the Rhine Valley of Germany (Goebel et al. 1997). The clinical phenotype is notable for the early onset of the carpal tunnel syndrome which is followed much later by polyneuropathy and cardiomyopathy. Bowel dysfunction is less common and vitreous opacities have not been reported. Thr60Ala was discovered in members of a large kindred in the Appalachian region of the United States (Wallace et al. 1986; Benson et al. 1987). The clinical picture is characterized by cardiomyopathy presenting around age 60 but older onset and decreased penetrance has obscured the genetic nature of the disease in some families. Polyneuropathy tends to be slowly progressive over 10–20 years but bowel dysfunction with diarrhea can be very debilitating. Vitreous amyloid has not been found with this mutation. Ser77Tyr amyloidosis was discovered in a Midwest kindred but has subsequently been found in other American and many European families (Wallace et al. 1988; Libbey et al. 1984; Satier et al. 1990; Zhao et al. 1994). Both cardiac and renal amyloid may occur in addition to the characteristic polyneuropathy starting around age 50 years. Vitreous opacities are not a part of this syndrome. Ile84Ser amyloidosis was first noted in an Indiana family because of vitreous amyloidosis (Falls et al. 1955). Essentially all affected individuals have eye involvement and this often occurs (age 40–50) before systemic manifestations (Rukavina et al. 1956). Carpal tunnel syndrome occurs early as seen with Leu58His. Death is usually from cardiomyopathy. A feature of this disease is the essentially 100% penetrance of the trait. The Indiana kindred is of Swiss origin and affected individuals in Switzerland have been identified but not been the subject of scientific publication (Dwulet and Benson 1986). A family in Hungary carrying the Ile84Ser mutation, with the same clinical phenotype has the same TTR haplotype but has not been linked by genealogy (Zo´lyomi et al. 1998). Val122Ile amyloidosis is mainly a disease of African Americans. The disease allele most likely originated in the West Coast of Africa (Sierra Leone, Gold Coast, etc.) but was first identified in African Americans with onset of cardiomyopathy after age of 60 (Gorevic et al. 1989; Nichols et al. 1991). Approximately 3–4% of African Americans carry this trait but the late age of onset for the clinical disease and the predominance of cardiomyopathy rather than neuropathy has obscured the true incidence of the disease (Jacobson et al. 1997; Yamashita et al. 2005). The degree of penetrance is still to be ascertained.
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Clinical Syndromes of FAP
11.5.1 Neuropathy Two forms of neuropathy are common in TTR amyloidosis: 1) Carpal tunnel syndrome – a compression neuropathy at the wrist, and; 2) Polyneuropathy affecting the longest fibers first (Benson and Kincaid 2007). The carpal tunnel syndrome may occur as the first sign of TTR amyloidosis or it may occur later, after bowel, cardiac, or peripheral nerve symptoms are present. Clinically it causes numbness and tingling in the distribution of the median nerve which is compressed in the carpal tunnel. It often disrupts sleep and patients report waking to shake their hands to relieve symptoms. Later it may cause sensory loss, atrophy of the thenar muscles with loss of hand grip and dexterity. Nerve conduction studies of the median nerve typically show delayed latency across the wrist. Nerve conduction may improve after surgical decompression of the median nerve but usually remains in the abnormal range. The peripheral neuropathy of TTR amyloidosis is axonal. Nerve conduction velocities are relatively maintained but response amplitude is decreased. The polyneuropathy is progressive with loss of response in longer sensory nerves (e.g., sural) occurring early. Autonomic neuropathy is often a significant aspect of TTR amyloidosis. Impotence is common in males and bladder retention may occur. Rectal sphincter dysfunction can be very debilitating when coupled with persistent diarrhea. Orthostatic hypotension is common and, when present along with restrictive cardiomyopathy, may severely limit physical activity. Autonomic neuropathy may be implicated in delayed gastric emptying but much of the intestinal dysfunction may be related to amyloid disruption of intrinsic bowel motility.
11.5.2 Cardiomyopathy A majority of the TTR mutations cause amyloidosis with cardiac involvement. This is also the case with SSA (SCA) in which cardiac amyloid deposition is the most common and usually the only clinically significant feature. The cardiomyopathy produces restrictive hemodynamics due to poor compliance of the ventricular walls. Left ventricular filling is impeded and stroke volume is lowered. The result is decreased cardiac output, rise in pressures of the pulmonary bed and subsequent features of congestive heart failure with generalized weakness, fatigue and fluid retention. Decreased cardiac output may commonly lead to hypotension and orthostatic symptoms which limit physical activity. In terminal stages, cardiac function may fall below that needed for tissue perfusion, especially the kidneys, and death may be the result of renal failure. Left atrial enlargement, the result of left ventricular diastolic dysfunction, often leads to atrial
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fibrillation which impairs the left ventricular filling with resultant further reduction in cardiac output. Amyloid deposition also may impair the cardiac conduction leading to AV block, atrial arrhythmias or sinus exit block. Ventricular tachycardia leading to ventricular fibrillation may be a terminal event.
11.5.3 Gastroenteropathy Bowel motility is often a major problem for patients with TTR amyloidosis. Clinically, the gastrointestinal (GI) problems associated with TTR amyloidosis occur in two anatomic areas: the upper GI track and the lower GI track. Upper GI dysfunction is mainly a result of gastroparesis. The stomach does not empty in the appropriate fashion after ingestion of a meal and the patient reports early satiety. Nausea and vomiting are not significant problems. The patient just feels ‘‘full’’ after taking a small portion of nourishment. The problem is one of decreased gastric motility which may result from a combination of factors. Autonomic neuropathy affecting the vagus nerve may lead to gastric motility deficit. In addition, infiltration of the submucosa and muscular layers of the stomach may lead to loss of interstitial cells of Cajal with a decline in intrinsic gastric motility. Lower bowel dysfunction may be a major manifestation of TTR amyloidosis and most frequently is expressed as chronic and unremitting diarrhea. A decrease in transit time of the liquefied intestinal contents seems to be provoked by any attempt at oral intake. The rapid gastrointestinal transit time, coupled with the psychological impact of eating followed by diarrhea, probably are the major factors in poor nutritional status and progressive body weight loss. Neurologically the lower intestinal dysfunction is probably related to the loss of neural inhibitory signals in the lower GI track. Normally, motility of the colon is inhibited by the autonomic nervous system and this allows increased time for reabsorbtion of water resulting in the formation of stools with a normal soft consistency. This process allows for storage in the rectosigmoid part of the colon and with normal function of the intrinsic bowel mechanisms along with higher Central Nervous System control allows for normal defecation.
11.5.4 Leptomeningeal Amyloidosis Leptomeningeal amyloidosis is a rare manifestation of TTR amyloidosis. It has been associated with 12 different mutations of TTR, and with these mutations it may be the most clinically significant and life threatening aspect of the disease. TTR mutations which have been associated with this phenotype range from the Val30Met mutation where leptomeningeal involvement is rarely of clinical significance to other mutations including Val30Gly and Thr114Cys, where most individuals
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in the kindreds have this manifestation (Goren et al. 1980; Petersen et al. 1998; Ueno et al. 1990). Clinically, leptomeningeal amyloidosis is characterized by repeated intra-cerebral hemorrhages which may manifest as seizures or obtundation. Occasionally episodes of obtundation without radiologic evidence of cerebral hemorrhage or infarction may occur. Some affected individuals develop hydrocephalus with increased cerebral spinal fluid (CSF) pressure and relatively high CSF protein levels. Leptomeningeal amyloidosis may or may not be associated with vitreous opacities but when this occurs, such as in the Val30Gly kindred, it is typically termed oculoleptomeningeal amyloidosis. This is a frequent finding with the Thr114Cys patients. Vitreous opacities have been described for approximately 25% of the TTR mutations and may or may not be associated with leptomeningeal manifestations. Pathologically dense amyloid deposition is found in cerebral vessels including those perforating the cortex. The leptomeninges are thickened by deposition of amyloid and this can be appreciated by MRI (Fig. 11.3) (Dowell et al. 2007). Leptomeningeal involvement extends down the spinal column and can give symptoms related to spinal cord or spinal nerve compression. Death is very often due to massive cerebral hemorrhage. Many of the TTR mutations associated with leptomeningeal amyloidosis have been described in only single families, but as with the larger kindreds with this phenotype, affected individuals tend to be relatively young (i.e., fourth or fifth decade of life). Survival is governed by the degree of leptomeningeal involvement. Most patients have little, if any, systemic deposition of amyloid. It is generally considered that leptomeningeal amyloid is the result of TTR produced by the choroid plexus and is not of hepatic origin as is systemically deposited amyloid. In favor of this hypothesis is the fact that in individuals heterozygous for a TTR mutation, the leptomeningeal amyloid is, for all practical purposes, the product of only the variant protein. This is unlike systemic amyloid isolated from the heart or the kidney where typically 65–70% of the amyloid is from a variant TTR and the remainder is from normal TTR. Similar pathogenic mechanisms may be operative in the eye where typically 85–90% of the vitreous amyloid fibrils are derived from a variant TTR (Liepnieks et al. 2006).
Fig. 11.3 Brain MRI of a patient with TTR Val30Gly amyloidosis. (a) Sagital T1 post gadolinium view. Arrows indicate thickened leptomeninges. (b) Sagital view of cervical spine. Arrow indicates leptomeningeal thickening extending down the spinal column
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Pathogenesis of TTR Amyloidosis From a Clinical Aspect
The pathogenesis of TTR amyloidosis has been the subject of considerable experimental study. Since the predisposition to TTR amyloidosis is associated with inheritance of mutations in the TTR protein, it is logical that alterations in protein structure should be a determining factor in the development of amyloid fibril formation. Tertiary structures of a number of amyloid associated TTR variant proteins have been determined by X-ray crystallography using either native variant plasma TTR or variant TTR produced by recombinant means (Hamilton and Benson 2001). These studies have shown definite alterations in tertiary structure; however, no unifying structural variation has been identified that would aid in predicting amyloid fibril formation. Thyroxin binding by a number of amyloid associated TTR variants has been studied showing considerable variation in hormone affinity (Rosen et al. 1993). This finding has no obvious correlation with the amyloid forming potential of TTR variants that were studied. Subsequent studies have shown that thyroxin binding to TTR thermodynamically stabilizes the tetramer in vitro (Peterson et al. 1998). This is the basis for the hypothesis that tetramer stability is an important factor in the formation of amyloid fibrils. Several small organic molecules have been identified which have high affinity for the TTR thyroxin binding pocket and, thereby, cause increased thermodynamic stability of the TTR tetramer (Baures et al. 1999; Hammarstro¨m et al. 2003). Considerable data have been generated from studying the primary structure of TTR peptides isolated from amyloid deposits. A most consistent finding has been the fragmentation of TTR with cleavage at or around amino residue 49; although some amount of full length TTR protein is always present in the tissue extracts (Liepnieks and Benson 2007). Studies in Westermark’s lab have shown experimentally that the carboxyl terminal portion of TTR is more prone to form fibril structures in vitro (Gustavsson et al. 1994). Several studies have shown that subjects heterozygous for an amyloid associated TTR mutant have lower plasma levels of TTR than normal (Benson and Dwulet 1983; Skinner et al. 1985; Westermark et al. 1985). This would not appear to be due to altered nutrition since it has been observed in young healthy individuals long before any evidence of amyloid has occurred (Waits et al. 1995). Low plasma levels of TTR may be a result of increased metabolic turnover associated with variant TTR proteins and this could be a significant factor in pathogenesis. Studies by Vasquez et al., more than 50 years ago, showed that the plasma half-life of TTR (prealbumin) was only one or two days, and subsequent work has shown increased plasma clearance of Val30Met TTR in both affected and nonaffected subjects (Benson et al. 1996; Vahlquist et al. 1973). Transgenic animal models of TTR amyloidosis have been studied but have not, in general, shed much light on the pathogenic mechanisms of the disease. From a clinical perspective a number of factors speak to the pathogenesis of TTR amyloidosis. These are all observational; none is the result of experimental design. The disease displays autosomal dominant inheritance. Most patients are heterozygous
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for one of the 100 plus TTR mutations. A few patients are homozygous for Val30Met or Val122Ile and have been reported but do not have earlier onset or more aggressive disease than their heterozygous kin (Holmgren et al. 1988; Nichols et al. 1991). While patients homozygous for Leu58His have been reported to have more severe disease, the general lack of clinical difference between homozygous and heterozygous subjects is against significant importance either of TTR variant concentration or tetramer stability from hormone binding. Not all TTR variant gene carriers develop amyloidosis, indicating that other factors must be involved in expressing the clinical disease. TTR amyloidosis is an adult onset disease and, while a few individuals have been reported affected in their teenage years (Leu55Pro, Trp41Leu), most do not develop symptoms of the disease until the fourth or fifth decade of life. It is obvious that the clinical disease is significantly affected by age. Organ impairment from amyloid deposition is, in general, faster in early onset disease patients as seen in the Portuguese and Japanese Val30Met kindreds. Progression is slower in the Swedish and English Val30Met patients who have later onset of the clinical disease. Senile systemic TTR (senile cardiac) amyloidosis occurs in patients over the age of 60 and is usually not of clinical significance until after the age of 70 years. The disease occurs in the absence of any TTR mutation and no other inherited factor has been identified except gender. Essentially all patients with SSA (SCA) are male. This suggests that the male sex is a significant disease modifier and this is consistent with the approximately 60:40 male to female ratio of patients that are diagnosed with TTR amyloidosis. This, as yet unidentified factor, may be a general metabolic variable since the 60:40 ratio of affected individuals is also observed with AL (immunoglobulin light chain) amyloidosis in most published series. Advanced age onset of amyloid deposition from normal protein is also seen in Alzheimer disease (AD). In both TTR and AD amyloidoses the presence of a mutation in the amyloid precursor protein appears to cause an earlier onset of amyloid formation. In the Alzheimer disease, in the absence of a mutation, the disease is affected by inheritance of the apolipoprotein E haplotype. In TTR amyloidosis no similar modifier has been identified (Saunders et al. 1993). Organ system involvement by TTR amyloidosis is quite variable but does suggest underlying modifiers and, therefore, possible clues for pathogenesis. The nervous system (both peripheral and autonomic), the heart, kidneys, bowel, eyes, skin and leptomeningeal tissue may all be sites of amyloid deposition. The apparent syndromic picture associated with certain TTR mutations might suggest dominant control of phenotype by certain specific mutations. However, many of the mutations are in limited kindreds where shared genetic background may not allow diversity. The Val30Met mutation, which is the most widespread worldwide, shows the greatest clinical diversity and, while usually characterized principally by peripheral neuropathy, many patients have cardiomyopathy, vitreous opacities, nephropathy, and occasionally leptomeningeal amyloid deposition. The Thr60Ala mutation, however, has been widely dispersed but, so far, has not been found to be associated with renal, vitreous or leptomeningeal amyloid. This would suggest a greater influence on pathogenesis by this specific TTR mutation.
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So even when combining experimental data on TTR structure and function with clinical observations of TTR amyloidosis we are far from understanding the pathogenesis of the disease. We can, however, consider specific studies that might pertain to pathogenesis and/or search for effective therapies. More information on TTR metabolism including cellular uptake and catabolic processing can be undertaken in animal studies. Specific information on amyloid formation will have to result from human experimentation until an animal model that faithfully represents the human disease is available.
11.7
Therapy
Nonspecific therapy aimed at alternating the ravages of organ system destruction has been, and remains, the most important aspect of treatment for the patient with TTR amyloidosis. Treatments for the neuropathic and psychological consequences of TTR amyloidosis are addressed in other chapters of this book. Analgesic, antiepileptic and psychotropic drugs are a mainstay of treatment for TTR neuropathic symptoms. Surgical decompression for the carpal tunnel syndrome is very effective when done early in the course of disease. Laminectomy has been shown to be effective in a few cases of spinal claudication due to epidural amyloid deposition (Harats et al. 1989). A few notes on treatment of cardiomyopathy are in order since this is a major manifestation in many patients with TTR amyloidosis. Amyloid deposition in the heart results in restrictive hemodynamics. Cardiac walls are thickened and noncompliant (Figs. 11.4a, b). The amyloid causes impairment of ventricular filling during diastole and, therefore, decreased cardiac output. This effect is amplified by any increase in heart rate since the time for diastolic filling is shortened. Thus an increase in heart rate from sudden exercise will decrease ventricular filling and may cause syncope due to decreased cerebral blood flow. Sustained impairment of
Fig. 11.4 Echocardiographic and gross anatomical findings in TTR cardiomyopathy. (a) Echocardiographic image in a patient with TTR Thr60Ala showing thickened left ventricular walls and a dilated left atrium. (b) Transverse section of a heart showing thickened ventricular walls and a small left ventricular cavity
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ventricular filling will lead to congestive heart failure with weakness, fatigue and fluid retention. This often occurs during prolonged tachyarrhymias such as atrial fibrillation, a result of atrial enlargement from restrictive hemodynamics. Therapeutically, it is important to maintain normal sinus heart rhythm for as long as possible. Arrhythmias are bad for cardiac function and may be prevented with use of modest doses of antiarrhythmics such as Beta adrenergic blocking agents. Calcium channel blocking agents should be avoided (Pollak and Falk 1993). They have major negative inotropic properties and may exacerbate congestive heart failure. Diuretics are a major therapeutic modality for treatment of congestive heart failure due to amyloid cardiomyopathy. It is important, however, to adjust their use so that systolic blood pressure is maintained well enough for adequate renal perfusion. Some patients develop cardiac conduction disorders such as sinus exit, atrial, or atrioventricular blocks and then require cardiac pacemaking. Cardiac defibrillators (ICD’s) are of questionable value in amyloid cardiomyopathy. Cardiac transplantation with or without concomitant liver transplant has proven effective for a number of patients with TTR amyloidosis. In patients who have had a heart transplant in conjunction with orthotopic liver transplant, none has developed amyloid in the heart graft even though some have died from amyloid progression in other organs (lung, nerves and bowel). Limited clinical experience indicates that amyloid deposition in cardiac grafts without concomitant liver transplant may be delayed for several years or may not become manifest in the lifetime of the recipient. The only treatment for TTR amyloidosis that has been proven to be effective is orthotopic liver transplantation (OLT) (Holmgren et al. 1991). As noted elsewhere in this book, many patients have benefited from liver transplantation. Even so, some patients have had progression of disease after OLT and this has been shown to be due to continued amyloid formation from normal TTR (Dubrey et al. 1997; Liepnieks and Benson 2007). This emphasizes the importance of developing new therapies. To date, trials are ongoing to determine the effect of small organic molecules to stabilize the TTR tetramer and delay fibril formation. Studies designed to restrict production of TTR fibril precursor synthesis with antisense oligonucleotide or siRNA technology are in progress but are not yet ready for human trials (Benson et al. 2006). Clinical studies resulting from advances in medical diagnostic technology are showing that TTR amyloidosis is not such a rare disease after all. New therapies are sorely needed.
11.8
Conclusion
So here we are. We have amassed a tremendous amount of knowledge about TTR and its single human disease, amyloidosis. This type of amyloidosis has so much phenotypic variation that it could be argued that we are really dealing with many diseases. Each, however, is characterized by the formation and deposition of b-structured protein fibrils with resultant disruption of organ function. As with most types of
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amyloidosis, the major question remains: how does a physiological protein transit from its normal soluble state to one of insoluble proteolytically resistant fibrils? Hopefully, the answer to this question will lead to the development of means to disrupt this pathological process.
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Goebel HH, Seddigh S, Hopf HC, Uemichi T, Benson MD, McKusick VA (1997) A European family with histidine 58 transthyretin mutation in familial amyloid polyneuropathy. Neuromuscular Disorders 7:229–230. Gorevic PD, Prelli FC, Wright J, Pras M, Frangione B (1989) Systemic senile amyloidosis. Identification of a newprealbumin (transthyretin) variant in cardiac tissue: immunologic and biochemical similarity to one form of familial amyloidotic polyneuropathy. Clin Invest 83: 836–843. Goren H, Steinberg MC, Farboody GH (1980) Familial oculoleptomeningeal amyloidosis. Brain 103:473–495. ˚ , Engstro¨m U, Westermark P (1994) Mechanisms of transthyretin amyloidogenesis. Gustavsson A Antigenic mapping of transthyretin purified from plasma and amyloid fibrils and within in situ tissue localizations. Am J Pathol 144:1301–1311. Hamilton JA, Benson MD (2001) Transthyretin: A review from a structural perspective. Cell Mol Life Sci 58:1491–1521. Hammarstro¨m P, Wiseman RL, Powers ET, Kelly JW (2003) Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science 299:713–716. Harats N, Worth R, Benson MD (1989) Spinal claudication in systemic amyloidosis. J Rheum 16:1003–1006. Holmgren G, Haettner E, Nordenson I, Sandgren O, Steen L, Lundgren E (1988) Homozygosity for the transthyretin-Met30-gene in two Swedish sibs with familial amyloidotic polyneuropathy. Clin Genet 34:333–338. Holmgren G, Holmberg E, Lindstrom A, Lindstrom E, Nordenson I, Sandgren O, Steen L, Svensson B, Lndgren E, von Gabain A (1988) Diagnosis of familial amyloidotic polyneuropathy in Sweden by RFLP analysis. Clin Genet 33:176–180. Holmgren G, Steen L, Ekstedt J, Groth CG, Ericzon BG, Eriksson S, Andersen O, Karlberg I, Norden G, Nakazato M, Hawkins P, Richardson S, Pepys M (1991) Biochemical effect of liver transplantation in two Swedish patients with familial amyloidotic polyneuropathy (FAPmet30). Clin Genet 40:242–246. Jacobson DR, Pastore RD, Yaghoubian R, Kane I, Gallo G, Buck FS, Buxbaum JN (1997) Variantsequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans. N Engl J Med 336:466–473. Kincaid JC, Wallace RW, Benson MD (1989) Late onset familial amyloid polyneuropathy in an American family of English origin. Neurology 39:861–863. Lessell S, Wolf PA, Benson MD, Cohen AS (1975) Scalloped pupils in familial amyloidosis. N Engl J Med 293:914–915. Libbey CA, Rubinow A, Shirahama T, Deal C, Cohen AS (1984) Familial amyloid polyneuropathy. Demonstration of prealbumin in a kinship of German/English ancestry with onset in the seventh decade. Am J Med 76:18–24. Liepnieks JJ, Benson MD (2007) Progression of cardiac amyloid deposition in hereditary transthyretin amyloidosis patients after liver transplantation. Amyloid 14:277–282. Liepnieks JJ, Wilson DL, Benson MD (2006) Biochemical characterization of vitreous and cardiac amyloid in Ile84Ser transthyretin amyloidosis. Amyloid 13:170–177. Mahloudji M, Teasdall RD, Adamkiewicz JJ, Hartmann WH, Lambird PA, McKusick VA (1969) The genetic amyloidoses. With particular reference to hereditary neuropathic amyloidosis, type II (Indiana or Rukavina type). Medicine 48:1–37. Murakami T, Maeda S, Yi S, Ikegawa S, Kawashima E, Onodera S, Shimada K, Araki S (1992) A novel transthyretin mutation associated with familial amyloidotic polyneuropoathy. Biochem Biophys Res Commun 182:520–526. Nichols WC, Liepnieks JJ, McKusick VA, Benson MD (1989) Direct sequencing of the gene for Maryland/German familial amyloidotic polyneuropathy type II and genotyping by allelespecific enzymatic amplification. Genomics 5:535–540. Nichols WC, Liepnieks JJ, Snyder EL, Benson MD (1991). Senile cardiac amyloidosis associated with homozygosity for a transthyretin variant (Ile-122). J Lab Clin Med 117:175–180.
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Petersen RB, Goren H, Cohen M, Richardson SL, Tresser N, Lynn A, Gali M, Estes M, Gambetti P. (1997) Transthyretin amyloidosis: a new mutation associated with dementia. Ann Neurol 41: 307–313. Peterson SA, Klabunde T, Lashuel HA, Purkey H, Sacchettini JC, Kelly JW (1998) Inhibiting transthyretin conformational changes that lead to amyloid fibril formation. Proc Natl Acad Sci USA 95:12957–12960. Pitka¨nen P, Westermark P, Cornwell GG (1984) Senile systemic amyloidosis. Am J Pathol 117: 391–399. Pollak A, Falk RH (1993) Left ventricular systeolic dysfunction precipitated by verapamil in cardiac amyloidosis. Chest 104:618–620. Rosen HN, Moses AC, Murrell JR, Liepnieks JJ, Holmgren G, Sandgren O, Steen L, Benson MD (1993) Thyroxine interactions with transthyretin: a comparison of 10 different naturallyoccurring transthyretin variants. J Clin Endo Metab 77:370–374 Rukavina JG, Block WD, Jackson CE, Falls HF, Carey JH, Curtis AC (1956) Primary systemic amyloidosis: a review and an experimental, genetic, and clinical study of 29 cases with particular emphasis on the familial form. Medicine 35:239–334. Saunders AM, Schmader K, Breitner JC, Benson MD, Brown WT, Goldfard L, Goldgaber D, Manwaring MG, Szymanski MH, McCrown N, Dole KC, Schmechel DE, Strittmatter WJ, Pericak-Vance MA, Roses AD (1993) Apolipoprotein E 4 allele distributions in late-onset Alzheimer’s disease and in other amyloid-forming diseases. Lancet 342:710–711. Sandgren O (1995) Ocular amyloidosis, with special reference to the hereditary forms with vitreous involvement. Surv Ophthalmol 40:173–196. Satier F, Nichols WC, Benson MD (1990) Diagnosis of familial amyloidotic polyneuropathy in France. Clin Genet 38:469–473. Sequeiros J, Saraiva MJ (1987) Onset in the seventh decade and lack of symptoms in heterozygotes for the TTRMet30 mutation in hereditary amyloid neuropathy type I (Portuguese, Andrade). Am J Med Genet 27:345–357. Shur O, Danielsson A, Holmgren G, Steen L (1994) Malnutrition and gastrointestinal dysfunction as prognostic factors for survival in familial amyloidotic polyneuropathy. J Int Med 479-485. Skinner M, Connors LH, Rubinow A, Libbey C, Sipe JD, Cohen AS (1985) Lowered prealbumin levels in patients with familial amyloid polyneuropathy (FAP) and their non-affected but at risk relatives. Am J Med Sci 289:17–21. Ueno S, Uemichi T, Yorifuji S, Tarui S (1990) A novel variant of transthyretin (Tyr114 to Cys) deduced from the nucleotide sequences of gene fragments from familial amyloidotic polyneuropathy in Japanese sibling cases. Biochem Biophys Res Commun 169:143–147. Vahlquist A, Peterson PA, Wibell L (1973) Metabolism of the vitamin A transporting protein complex. 1. Turnover studies in normal persons and in patients with chronic renal failure. Eur J Clin Invest 3:352–362. Waits RP, Yamada T, Uemichi T, Benson MD (1995) Low plasma concentrations of retinol-binding protein in individuals with mutations affecting position 84 of the transthyretin molecule. Clin Chem 41:1288–1291. Wallace MR, Dwulet FE, Conneally PM, Benson MD (1986) Biochemical and molecular genetic characteristic of a new variant prealbumin associated with hereditary amyloidosis. J Clin Invest 78:6–12. Wallace MR, Dwulet FE, Williams EC, Conneally PM, Benson MD (1988) Identification of a new hereditary amyloidosis prealbumin variant, tyr77, and detection of the gene by DNA analysis. J Clin Invest 81:189–193. Westermark P, Pitkanen P, Benson L, Vahlquist A, Olofsson BO, Cornwell GG III (1985) Serum prealbumin and retinol-binding protein in the prealbumin-related senile and familial forms of systemic amyloidosis. Lab Invest 52:314–318. Westermark P, Sletten K, Johansson B, Cornwell GG (1990) Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc Natl Acad Sci USA 87:2843–2845.
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Yamashita T, Hamidi Asl K, Yazaki M, Benson MD (2005) A prospective evaluation of the transthyretin Ile122 allele frequency in an African-American population. Amyloid J Protein Folding Disord 12:127–130. Yazaki M, Connors LH, Eagle Jr, RC, Leff SR, Skinner M, Benson MD (2002) Transthyretin amyloidosis associated with a novel variant (Trp41Leu) presenting with vitreous opacities. Amyloid J Protein Folding Disord 9:263–267. Yazaki M, Yamashita T, Kincaid J, Scott J, Auger R, Dyck P, Benson MD (2002) Rapidly progressive amyloid polyneuropathy associated with a novel variant transthyretin Ser 25. Muscle Nerve 25:244–250. Yoshioka K, Furuya H, Sasaki H, Saraiva MJM, Costa PP, Sakaki Y (1980) Haplotype analysis of familial amyloidotic polyneuropathy. Hum Genet 82:9–13. Zhao N, Aoyama N, Benson MD, Skinner M, Satier F, Sakaki Y (1994) Haplotype analysis of His58, Ala60 and Tyr77 types of familial amyloidotic polyneuropathy. Amyloid Int J Exp Clin Invest 1:75–79. Zo´lyomi Z, Benson MD, Hala´sz K, Uemichi T, Fekete G (1998) Transthyretin mutation (Serine 84) associated with familial amyloid polyneuropathy in a Hungarian family. Amyloid Int J Exp Clin Invest 5:30–34.
Chapter 12
Molecular Pathogenesis Associated with Familial Amyloidotic Polyneuropathy Maria Joa˜o Saraiva
Abstract Human plasma transthyretin (TTR) can undergo conformational changes and form amyloid fibrils, in both acquired and hereditary forms of systemic amyloidosis. More than 100 TTR mutations have been associated with autosomal dominant amyloidosis, usually presenting with peripheral and autonomic neuropathy (Familial amyloidotic polyneuropathy (FAP)) and/or cardiomyopathy (Familial amyloidotic cardiomyopathy (FAC)). The mechanisms underlying cell death in TTR-related amyloidoses need to be addressed for the development of future therapies in FAP. These issues are the subject of this review. Keywords Amyloid, Aggregation, Stress response
12.1
Introduction
Hereditary transthyretin (TTR) amyloidosis is a genetically transmitted disease that results from a mutation in the gene encoding the plasma TTR protein. TTR is a transport protein for thyroid hormones and vitamin A and is predominantly synthesized in the liver. Most known TTR mutations increase the potential for the protein to destabilize and aggregate as amyloid fibrils extracellularly in different organs and tissues, with predominance in the peripheral nervous system (PNS). Brain and liver are spared from deposition. The most common TTR mutation associated with peripheral neuropathy in familial amyloidotic polyneuropathy (FAP) is TTR Val30Met described in the Portuguese population (Saraiva et al. 1984; for mutations M.J. Saraiva Molecular Neurobiology Group, IBMC – Instituto de Biologia Molecular e Celular and ICBAS – Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, 4150 Porto Portugal e-mail:
[email protected]
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listing see http://www.ibmc.up.pt/mjsaraiva/ttrmut.html). The first symptoms relate to a sensory neuropathy beginning at the extremities of the lower limbs, with paresthesias, dysaesthesias and loss of thermal and pain sensations. The disease advances proximally and the upper extremities are affected later, when the loss of sensation has reached the level of the knee. Usually 2 or 3 years after the first sensory manifestations, motor disturbances are noticeable, with atrophy and muscular weakness, beginning also by the lower extremities. In the more advanced stages, patients are confined to a wheel-chair (Andrade 1952). Autonomic neuropathy is particularly severe in Portuguese FAP patients and can constitute the first manifestation in many patients. Autonomic involvement manifests as gastrointestinal disturbances, with alternating periods of constipation and diarrhea, gastric stasis, nausea and vomiting. Loss of weight and asthenia frequently precede gastrointestinal disturbances. Sphincter dysfunction is responsible for both urinary and fecal incontinence as well as impotence. Electrocardiographic abnormalities are common in Portuguese FAP patients and are expressed as conduction disturbances leading to branch blocks which demands pacemaker implantation in some instances. The progression of the disease is slow and relentless, leading to cachexia and death in 10–15 years from its onset. At present, studies on cellular toxicity and neurodegeneration in FAP are emerging. They encompass analyses both on clinical samples and animal models and will guide potential forms of treatment aimed at counteracting toxic cascades generated by deposition of aggregated TTR. We will next describe molecular pathways that have been reported in FAP and how they can guide us in treatments.
12.2
Pathological Features in the Peripheral Nervous System in FAP
TTR amyloid deposits can be found in any part of the peripheral nervous system, including the nerve trunks, plexuses and sensory and autonomic ganglia. In peripheral nerves, deposition occurs extracellularly, particularly in the endoneurium close to Schwann cells (SC) and to collagen fibrils; in the endoneurial space it usually takes the form of globoid deposits (Fig. 12.1a) near the perineurial envelope or near the endoneurial capillaries, or surrounding the capillary wall. Amyloid is also present in the adventicia of the vessels or as small deposits in the epinerve. In severely affected nerves, almost the whole endoneurial contents may be replaced by amyloid and very few nerve fibers survive. FAP is characterized by initial axonal loss affecting unmyelinated and small myelinated fibres and later affecting the larger fibres (Coimbra and Andrade 1971). In the autonomic and sensory nervous system, advanced neuronal degeneration which might cause progressive ascending neuropathy (dying back type) is evident in addition to axonopathy. At the ultrastructural level, the fibrils of amyloid deposits have the usual aspect, sometimes forming bundles that participate in the constitution of globoid deposits, involving and being surrounded by collagen fibers (Fig. 12.1b). In suitable fixed
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Fig. 12.1 Amyloid in the nerve. (a) Anti-TTR immunohistochemistry (scale bar = 0.5 mm); (b) Electron microscopy (Scale bar = 100 nm)
nerve trunks, without shrinkage artifacts, the fibrils are present near the SC basal membrane, but without penetrating the cell membrane or cytoplasm (the same is true for the smooth muscle cells or endothelial cells of blood vessels). The dorsal root ganglia and the sympathetic and parasympathetic intramural ganglia also present progressive loss of neurons and deposition of amyloid in their stroma. There is loss of ganglionic neuronal cells, some of them presenting central chromatolysis and some empty spaces surrounded by satellite cells.
12.3
Early Deposition of TTR in an Asymptomatic Phase in a Nonfibrillar Fashion
12.3.1 Ex Vivo Analyses in Clinical Samples TTR deposition was assessed in nerves from asymptomatic TTR Val30Met carriers and FAP patients in different stages of disease progression. The scoring system of patients’ material was performed by morphometric measurements of myelinated (MF) and unmyelinated (UF) fibers: FAP 0- the least affected nerves (no nerve degeneration, no amyloid deposited) of all individuals were asymptomatic. From stage 1–3, individuals were all presenting symptoms as follows: FAP 1- discrete reduction in the number of nerve fibres and modest fibrillar deposits; FAP 2evident reduction of nerve fibres and amyloid throughout the nerve; and FAP 3severe reduction of nerve fibres and extensive amyloid deposition. It was observed that in the stage prior to loss of UF and MF and major nerve fiber degeneration (FAP 0), despite the absence of Congo red birefringence (the hallmark of amyloid) TTR was present, as revealed by immunohistochemistry with an antiTTR antibody. Thus, TTR deposits in a nonfibrillar, or prefibrillar form in the early stages of FAP, before assembling into mature amyloid fibrils, that give the characteristic green-birefringence by Congo red staining. Furthermore, immunocytolabeling of TTR was observed extracellularly in the proximity of SC, in a nonfibrillar
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form; and very small contiguous fibrillar-like assemblies were noticed, but were most likely too small to give bi-refringence with Congo-red staining on histochemistry. It was concluded that TTR deposits early in the asymptomatic phase in a nonfibrillar fashion (Sousa et al. 2001a).
12.3.2 Ex Vivo Analyses in Animal Models In order to understand the mechanisms underlying fibril formation, deposition and cytotoxic effects caused by aggregates, many questions remain to be investigated. In an attempt to gain insights into the pathogenesis of FAP, several groups generated transgenic mice carrying the human TTR Val30Met gene. Using the human homologous TTR promoter sequences to generate TTR Val30Met transgenics, amyloid was observed starting at 6 months. At the age of 24 months, the pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems (Yi et al. 1991). The same pattern of amyloid deposition was reported when these transgenics were backcrossed to a TTR-null background (Kohno et al. 1997). Animals presented widespread TTR staining as assessed by immunohistochemistry, with particular involvement of the gastrointestinal tract, starting as early as 3 months. At this stage no fibrillar material was detected both by Congo red staining and by immunocytolabeling. Congo red positive deposition was only observed at older ages affecting all mice after 21 months of age and starting at 9 months (Sousa et al. 2002).
12.4
Inflammation and Oxidative Stress Pathways in FAP and Involvement of the Receptor for Advanced Glycation-End Products (RAGE)
The toxicity of synthetic TTR fibrils formed in vitro at physiological pH was studied on a Schwannoma cell line by caspase-3 activation assays and showed that early aggregates, but not mature fibrils activate caspases, indicative of cytotoxicity by nonfibrillar deposits occurring in early stages of FAP (Sousa et al. 2001b). Increased expression of the receptor for glycation end products (RAGE) has been observed in TTR amyloid - laden tissues. Binding of RAGE by fibrillar TTR triggers activation of the transcription factor NF-kB. RAGE is a member of the immunoglobulin superfamily with a broad repertoire of ligands in addition to amyloid-associated macromolecules, including products of nonenzymatic glyeation (advanced glycation endproducts, AGEs), proinflammatory mediators (S100/ calgranulins) and amphoterin (Schmidt et al. 2000). In each case, the receptor recruits signal-transduction mechanisms, often resulting in a sustained and pathogenic
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inflammatory and stress response. This response might underlie peripheral nerve dysfunction. Analyses of nerve biopsy samples from Portuguese FAP patients at different stages of the disease (including the presymptomatic stage), were compared with age-matched controls by semiquantitative immunohistochemistry and in situ hybridization and showed upregulation of proinflammatory cytokines (tumor necrosis factor and interleukin 1), and the inducible form of nitric oxide synthase (iNOS), as well as increased tyrosine nitration and activated caspase-3 (Sousa et al. 2001b) in axons. Recent work showed that extracellular signalregulated kinases 1/2 (ERK1/2) displayed increased activation in FAP tissues and in TTR transgenic mice. Treatment of a rat Schwannoma cell line with TTR aggregates stimulated ERK1/2 activation, which was partially mediated by RAGE and abrogated by specific ERK1/2 inhibitors. These data suggested that abnormally sustained activation of ERK in FAP may represent an early signaling cascade leading to neurodegeneration (Monteiro et al. 2006).
12.5
The Ubiquitin–Proteasome System in FAP
The ubiquitin-proteasome system (UPS) has been associated with neurodegenerative disorders of intracellular protein aggregation. Studies on the UPS in FAP in TTR synthesizing and nonsynthesizing tissues from affected individuals and in the transgenic Val30Met mice model showed that ubiquitin protein conjugates were upregulated, the proteasome levels were decreased and parkin, and alpha-synuclein expression were both decreased (Santos et al. 2007). On the other hand, the liver, that normally synthesizes a variant of TTR Val30Met, did not show this response. Furthermore, transgenic mice immunized to decrease TTR deposition (Terazaki et al. 2006) showed a significant reduction in ubiquitin levels and an increase in parkin and alpha-synuclein levels in comparison to control mice. When neuronal or Schwannoma cell lines were cultured with TTR aggregates, an increase in ubiquitin and decrease in parkin levels were demonstrated. The overall results indicate that TTR extracellular deposition is an external stimulus to an intracellular UPS response in FAP.
12.6
The Unfolded Protein Response in FAP
12.6.1 Activation of the Heat Shock Response The heat shock proteins (hsp) have been implicated in a variety of neurodegenerative diseases in which the underlying pathology is protein aggregation. The heat shock response was investigated in FAP. Upregulation of hsp27 and hsp70 expression related to the presence of extracellular TTR aggregates in human FAP biopsies, as compared to normal controls, was documented. TTR aggregates did not
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colocalize with hsps, suggesting that extracellular TTR tissue deposits are able to induce an intracellular stress response. Moreover, the heat shock transcription factor 1 (HSF1) was upregulated and localized to the nucleus. Transgenic mice models expressing the Val30Met mutant form of TTR showed a similar response: the presence of TTR deposits induced a stress response with the activation of HSF1 and increased synthesis of hsps. In cell culture, the upregulation of hsp70 and hsp27 in the presence of toxic TTR aggregates was found (Santos et al. 2008a). Taken together, these findings prompt new avenues for the research on pathogenic mechanisms in FAP and the use of the heat shock response as a pharmacological target to treat FAP and other neurodegenerative disorders.
12.6.2 A Compromised Heat Shock Response Increases Systemic Extracellular Transthyretin Deposition and Affects the Peripheral and Autonomic Nervous Systems: Lessons from an Animal Model Protein misfolding diseases result to a certain extent from the incapacity of the cell to stay below the dangerous limits of intra and/or extracellular accumulated aggregated proteins as in Alzheimer’s disease, Parkinson’s disease or amyotrophic lateral sclerosis. The proteins involved are predisposed to aggregate, forming protofilaments and eventually amyloid fibrils. In the case of FAP, extracellular accumulation of aggregated TTR affects mainly the PNS, leading to inflammatory and oxidative stress and ultimately neurodegeneration. It was hypothesized that HSF1 could be involved in FAP pathogenesis as a cellular defense mechanism against the presence of TTR extracellular deposits and that disruption of the heat shock response would aggravate TTR deposition. A mouse model expressing the human TTR- Val30Met in a HSF1 null background was characterized. The lack of HSF1 expression lead to extensive and earlier nonfibrillar TTR, evolving into fibrillar material in distinct organs including the peripheral nervous system. As in the human disease, liver, brain and spinal cord did not present deposition. Furthermore, inflammatory stress and a reduction in unmyielinated nerve fibres were observed, as in human patients, indicating that HSF1 regulated genes are involved in FAP, modulating TTR tissue deposition (Santos et al. 2008b). The TTR/HSF1 model allows different studies, not possible with previous models. Sural nerve biopsies, on which most of the pathological analysis in FAP has been performed, represent a restricted portion of the PNS, and it is clearly possible that TTR deposits in ganglia, or more proximally in nerve trunks, which could be responsible for distal nerve fiber loss. The possibility to analyze dorsal root ganglia (DRG) with deposition in the TTR/HSF1 mice model is invaluable, and permits a close evaluation of events occurring in sensory and autonomic neurons responsible for neurodegeneration.
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The ER Response
The availability of a transgenic mouse model with extracellular TTR aggregates in DRG is a unique tool to study molecular mechanisms underlying pathogenesis in FAP. One of the first pathways investigated was ER stress. In mouse DRG, BiP expression was only visible in neurons of the ganglia in close contact with TTR extracellular deposits, suggesting a cause–effect relation, first suggesting a connexion between the ER stress response and FAP (Teixeira et al. 2006). Increased levels of the ER-resident chaperone BiP were found, in a consistent way, in human salivary gland biopsies of FAP patients, in TTR transgenic mouse DRG in and cell line models. These results were further confirmed in human salivary gland biopsies, by western blot and immunohistochemistry; increase in BiP level was visible in areas where mutant TTR was deposited. Using an ND7/23 cell line, one of the few cell lines of peripheral neurons, and a human neuroblastoma cell line it was found that extracellular TTR oligomers can induce BiP expression and activation of eIF2a in a way similar to the chemical inducer thapsigargin. This increase in BiP levels involved the mobilization of the secondary messenger Ca2+ from the ER, as it was blocked by Ca2+-channel inhibitors. These inhibitors lowered caspase-3 activation as response to TTR oligomers added to cultures, re-enforcing the notion of ER stress by extracellular TTR deposition as a toxic event in this system.
12.8
Activation of Extracellular Matrix Remodeling Genes
Although inflammation and oxidative stress are already triggered in presymptomatic individuals, clinical disease appears only after amyloid deposition. Gene arrays on clinical material (salivary glands) and control tissues allowed analyses of differential gene expression between pathogenic and healthy tissues. Among the differentially expressed genes, upregulation of genes related to extracellular matrix (ECM) remodeling was evident in patients’ tissues. Matrix metaloproteinase 9 (MMP9) is overexpressed only in amyloid laden tissues, but not in tissues from presymptomatic individuals (Sousa et al. 2005), suggesting that fibrils trigger specific signaling pathways leading to ECM remodeling and that this process causes tissue damage with pathological consequences. In the field of therapeutics it is important to define disease markers, not only to characterize the pathology but also to follow-up and evaluate therapeutic protocols. MMP9 apparently is a good biomarker for the presence of TTR amyloid as suggested by the above described array data but also from studies on TTR transgenic mice. These mice recapitulate the human situation as only mice with amyloid deposits displayed increased levels and activity of MMP-9 as compared to younger animals with nonfibrillar deposits. When treated with doxycycline, a TTR disrupter (Cardoso et al. 2003; Cardoso and Saraiva 2006), tissue MMP-9 expression and activity were decreased to levels comparable to those found in animals
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without TTR deposition. Other amyloid-related components, namely (Serum amyloid protein) SAP, were also decreased, indicating fibril removal.
12.9
Antiapoptotic Treatments
Tauroursodeoxycholic acid (TUDCA) is a unique natural compound that acts as a potent antiapoptotic and antioxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with TTR deposition in FAP, the possible TUDCA therapeutical application in this disease has been investigated. It was shown by semi-quantitative immunohistochemistry that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER chaperone BiP, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most importantly, TUDCA treatment significantly reduced TTR toxic aggregates by as much as 75% (Macedo et al. 2008). Since TUDCA has no effect on TTR aggregation ‘‘in vitro’’, this finding pointed for the ‘‘in vivo’’ modulation of TTR aggregation by oxidative stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutical measures in the FAP population.
Intermediate species
Amyloid
?
ER stress UPR Inflammation Oxidative Stress Matrix remodeling
Fig. 12.2 Identified signaling pathways related to extracellular TTR deposition: a possible contribution to amyloid formation; for explanation, see text
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Concluding Remarks
Protein misfolding occurring in the PNS triggers inflammatory and oxidative stress, matrix remodeling, UPR, and ER pathways that resemble in many aspects (including common molecular players and scenarios) those described in CNS for misfolding disorders, such as Alzheimer Disease. Thus, similarities and dissimilarities between the two systems are very useful to pinpoint and guide us in the treatment of age related neurodegenerative disorders. At this point, the various studies on the molecular pathology associated with FAP suggest that signaling pathways in FAP can contribute to extracellular polymerization of TTR in tissues, as depicted in Fig. 12.2. This avenue of research should be further explored.
References Andrade C (1952) A peculiar form of peripheral neuropathy. Familial atypical generalized amyloidosis with special involvement of the peripheral nerves. Brain 175:408–427. Cardoso I, Merlini G, Saraiva MJ (2003) 40 -iodo-40 -Deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producing non-cytotoxic species. Screening for TTR fibril disrupters. FASEB J 17:803–809. Cardoso I, Saraiva MJ (2006) Doxycycline disrupts transthyretin amyloid: evidence from studies in a FAP transgenic mice model. FASEB J 20:234–239. Coimbra A, Andrade C (1971) Familial amyloid polyneuropathy: and electron microscope study of peripheral nerve in five cases. I. Interstitial changes. Brain 94:199–206. Kohno K, Palha JA, Miyakawa K, Saraiva MJ, Ito S, Mabuchu T, Blaner WS, Iijima H, Tsukahara S, Episkopou V, Gottesman ME, Shimada K, Takahashi K, Yamamura K, Maeda S (1997) Analysis of amyloid deposition in a transgenic mouse model of homozygous familial amyloidotic polyneuropathy. Am J Pathol 150:1497–1508. Macedo B, Batista AR, Ferreira N, Almeida MR, Saraiva MJ (2008) Anti-apoptotic treatment reduces transthyretin deposition in a transgenic mouse model of familial amyloidotic polyneuropathy. Biochem Biophys Acta 1782:517–522. Monteiro F, Sousa MM, Cardoso I, Barbas do Amaral J, Guimara˜es A, Saraiva MJ (2006) Activation of ERK1/2 MAP kinases in familial amyloidotic polyneuropathy. J Neurochemistry 97:151–161. Santos SD, Cardoso I, Magalha˜es J, Saraiva MJ (2007) Impairment of the ubiquitin-proteasome system associated with extracellular transthyretin aggregates in familial amyloidotic polyneuropathy. J Pathology 213:200–209. Santos SD, Magalha˜es J, Saraiva MJ (2008a) Activation of the heat shock response in familial amyloidotic polyneuropathy. J Neuropath Exp Neurol 67:449–455. Santos SD, Fernandes R, Saraiva MJ (2008b) The heat shock response modulates transthyretin deposition in the peripheral and autonomic nervous systems. Neurobiol Aging, May 14 [Epub ahead of print]. Saraiva MJM, Birken S, Costa PP, Goodman DS (1984) Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of a molecular abnormality in transthyretin (prealbumin). J Clin Invest 74:104–119. Schmidt AM, Yan SD, Yan SF, Stern DM (2000) The biology of the receptor for advanced glycation end products and its ligands. Biochim Biophys Acta 1498:99–111. Sousa MM, Cardoso I, Fernandes R, Guimara˜es A, Saraiva MJ (2001a) Deposition of transthyretin in early stages of familial amyloidotic polyneuropathy: evidence for toxicity of non-fibrillar aggregates. Am J Pathol 159:1993–2000.
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Sousa MM, Yan SD, Fernandes R, Guimara˜es A, Stern D, Saraiva MJM (2001b) Familial amyloid polyneuropathy: RAGE-dependent triggering of neuronal inflammatory and apoptotic pathways. J Neuroscience 21:7576–7586. Sousa MM, Fernandes R, Palha JA, Taboada A, Vieira P, Saraiva MJ (2002) Evidence for early cytotoxic aggregates in transgenic mice for human transthyretin Leu55Pro. Am J Pathol 161: 1935–1948. Sousa MM, Barbas do Amaral J, Guimara˜es A, Saraiva MJ (2005) Upregulation of the extracellular matrix remodeling genes, biglycan, neutrophil gelatinase-associated lipocalin and matrix metalloproteinase-9 in familial amyloid polyneuropathy. FASEB J 19:124–126. Teixeira P, Cerca F, Santos SD, Saraiva MJ (2006) Endoplasmic reticulum stress associated with extracellular aggregates: evidence from transthyretin deposition in familial amyloid polyneuropathy. J Biol Chem 281:21998–22003. Terazaki H, Ando Y, Fernandes R, Yamamura K, Maeda S, Saraiva MJ (2006). Immunization in familial amyloidotic polyneuropathy: Counteracting deposition by immunization with a Y78F TTR mutant. Lab Invest 86:23–31. Yi S, Takahashi K, Naito M, Tashiro F, Wakasugi S, Maeda S, Shimada K, Yamamura K, Araki S (1991). Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met30) gene. Pathologic similarity to human familial amyloidotic polyneuropathy, type I. Am J Pathol 138:403–412.
Chapter 13
Histidine 31: The Achilles’ Heel of Human Transthyretin. Microheterogeneity is Not Enough to Understand the Molecular Causes of Amyloidogenicity Klaus Altland and Samantha J. Richardson
Abstract The microheterogeneity of human transthyretin (TTR) is mainly one of ligand and amino acid substitutions. These substitutions modify the conformational stability of monomers, dimers, and tetramers and may eventually result in unfolding– refolding transitions with the endpoint of amyloidosis. In this chapter we focus on a structural peculiarity of human TTR, i.e., a hydrogen bridge between His31 (b-strand B) and Ser46 (b-strand C), which appears to be the vulnerable site for changes of pH within a range (pH 7.4–6.5) observed under conditions of interstitial acidosis. We present arguments in favor of a cooperative interaction of all sites in the TTR monomer in modifying its conformational stability and reversible unfolding-refolding transitions which also affect the dimer and tetramer. We postulate that the unfolded monomer is the pool from which amyloidogenic aggregates are generated. Keywords Cooperative interaction, Histidines, Inflammation, Interstitial acidosis, Unfolding-refolding transition, Wallaby
Abbreviations ATTR CA DTT FAP
Amyloidogenic transthyretin Carrier ampholytes Dithiothreitol Familial amyloidotic polyneuropathy
K. Altland and S.J. Richardson Justus-Liebig-University, Institute of Human Genetics, Schlangenzahl 14, D-35392 Giessen, Germany e-mail:
[email protected] School of Medical Sciences (building 223, level 2, rootm 51), RMIT University, PO Box 71, Bundoora 3083, Victoria Australia e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_13, # Springer‐Verlag Berlin Heidelberg 2009
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HEPES IEF MOPS PAGE PIPES RBP SDS TTR
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2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid Isoelectric focusing 3-(N-Morpholino)-propanesulfonic acid Polyacrylamide gel electrophoresis 1,4-Piperazinediethane sulfonic acid Retinol-binding protein Sodium dodecylsulfate Transthyretin
Introduction
More than 100 variants of human transthyretin (TTR) have been identified worldwide and the great majority of these variants have been found to be associated with the inherited forms of TTR amyloidosis (Connors et al. 2003; Benson 2003). No variants have been detected in patients with senile systemic TTR amyloidosis at age >60 years (Cornwell et al. 1983; Westermark et al. 1990, 2003; Kyle et al. 1996). With one exception (Jacobson et al. 1992), inherited TTR amyloidosis is not observed within the first two decades of life. Among carriers of the most frequent amyloidogenic variant ATTR-V30M, the age of onset of the disease significantly varies with the origin of the population (Plante´-Bordeneuve et al. 2003) and even among identical twins (Holmgren et al. 1997). Amyloid has been identified in biopsy specimens from patients with carpal tunnel syndrome (CTS) who are not carriers of a TTR variant (Kyle et al. 1992). CTS is frequently observed in carriers of an amyloidogenic variant years before the onset of systemic amyloidosis. Similarly, TTR amyloid has been detected in tumors from otherwise healthy carriers of an amyloidogenic TTR variant gene (Altland, personal observations). Obviously, these observations require the presence of several causal factors to understand the development of TTR amyloidosis, which include the normal TTR molecule, its inherited heterogeneity, and the affected individuals with their specific developmental, environmental and genetic background. In this chapter we focus on some properties of the human TTR monomer that might support our understanding of the pathway from native TTR to its unfolded conformational state as the source for the development of the disease.
13.2
Microheterogeneity of Human TTR by Ligands and Amino Acid Substitutions
The TTR tetramer has binding sites for three different types of ligands. The channel inside the TTR tetramer contains two binding sites for thyroxine and structurally related drugs, described elsewhere in this book. The outer surface of the TTR
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tetramer has four binding sites for complexes of retinol and retinol-binding protein (RBP). Each of these binding sites covers areas of three TTR monomers. Because of steric hindrance only a maximum of two RBP molecules can bind to a TTR tetramer at a time (Monaco et al. 1995). TTR monomers have one cysteine (Cys10), and its thiol is exposed to the solvent and can be reversibly bound to other sulfhydryls or sulfites from the aqueous environment or may be irreversibly alkylated or oxidized (The´berge et al. 1999; Kishikawa et al. 1999; Altland et al. 1999). The relative quantities of the complexes between monomers and substituents at Cys10 are expected to vary with the affinities, concentrations, and redox states of the free substituents (e.g., sulfhydryls, sulfite) in the solvent under normal and pathological conditions (Mansoor et al. 1992; Nolin et al. 2007; Hack et al. 1998; Mills et al. 2000; Moriarty-Craige et al. 2005; Jones 2006). The quantification of in vivo plasma concentrations of free and protein-bound thiols requires special care to avoid significant deviations by oxidation. Inadequate storage of biological fluids (i.e., plasma, cerebrospinal fluid) appears to be associated with changes of the electrophoretic patterns of human TTR mainly due to a loss of cysteinylated and a gain of sulfonated Cys10 (Feldin and Fex 1984, Altland, personal observation). The genetic microheterogeneity of human TTR mostly consists of amino acid substitutions, but deletions of single amino acids have also been observed (Connors et al. 2003). The great majority of TTR variants were detected by amyloidosis of amyloidogenic transthyretin (ATTR) gene carriers, while non-amyloidogenic variants were identified by electrophoretic screening procedures (Altland et al. 1982; Jenne et al. 1996; Alves et al. 1997), DNA analysis (Fitch et al. 1991), or mass spectrometry (Nepomuceno et al. 2004). Substitutions of amino acids or ligands as such do not provide an understanding of the amyloidogenic pathway(s) without considering the associated changes of the molecular environment or conformation. Efforts failed to identify typical structural differences between amyloidogenic variants and normal TTR or non-amyloidogenic variants (Ho¨rnberg et al 2000).
13.3
Microheterogeneity of Human TTR by Conformational Stabilities
The structural differences between soluble native TTR and insoluble aggregates/ fibrils (Blake and Serpell 1996) require transition pathways that are more or less common to normal and variant TTRs, but favored by amyloidogenic variants. All known amyloidogenic amino acid substitutions, as well as all binding sites for physiological ligands, are located within the ordered (crystal) structure (from Cys10 to Thr123) of the TTR tetramer (Kanda et al 1974; Blake et al. 1978). Exposure of normal and variant TTR to gradients of denaturants such as acid, urea, and guanidinium chloride revealed a reduced conformational stability as a characteristic of all tested amyloidogenic variants (Colon and Kelly 1992; Jenne et al. 1996;
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Lashuel et al. 1998; Altland et al. 2007). Different conformational stabilities were found for normal or variant TTR when different ligands were bound to Cys10 (Altland et al. 1999, 2007; Altland and Winter 1999; Zhang and Kelly 2003). This observation is demonstrated by Figs. 13.1 and 13.2.
Fig. 13.1 Urea titration curves of normal human TTR. The patterns in (a) and (b) were stained with Serva-Violet 17 and achieved by IEF with added carrier ampholytes (CA) in a urea gradient and in the absence (a) and presence (b) of a reducing agent (50 mM dithiothreitol (DTT)) to demonstrate the heterogeneity of monomeric conformers by different ligands at Cys10. The numbers and arrows label the curves of monomers with Cys10 bound to cysteine (1), cysteinylglycine (2), glutathione (7), sulfite (8), and with Cys10 in the reduced (4) and oxidized (9) as well as alkylated (X) form. The grid and the vertical arrow at 2.7 M urea were added to assist comparison of the two patterns. Notice the shift of the curves as an indicator of differential conformational stabilities associated with the substitutions at Cys10. The patterns in (c) and (d) were stained with silver and achieved by short (6 h at 5,000 V or 30 kVh) and long (16 h at 5,000 V or 80 kVh), respectively, exposure of TTR from human plasma to IEF in a linear immobilized pH gradient. Notice the changes of the tetramer pattern in (d) indicating a slow decay of tetramers into monomers as well as a reverse aggregation of monomers into tetramers (T1 and T5). The length of the flat part of the titration curves at the left 0 M end of the urea gradient corresponds to the length of the visible zones T1 and T4 indicating that reaggregation to tetramers requires folded monomers. The pI difference between tetramer T1 (created from the major monomer fraction) and T5 (created from the minor monomer fraction) at 0 M urea corresponds to the pI difference between the major and minor monomer fraction at 8 M urea or about a pI difference created by one charge unit. At 0 M urea, the pI difference between T1 and T5 is about one-half of the pI difference between T1 and the curve of the folded cysteinylated monomer (curve 1) indicating a gain of two charge units by the transition from the (cysteinylated) tetramer to the folded (cysteinylated) monomer. A total gain of three positive charge units appears to be associated with the changes from (cysteinylated) tetramers (t1) to unfolded (cysteinylated) monomers. Reproduced from Altland et al. (1999) by permission
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Fig. 13.2 Demonstration of the additive effects of conformational stabilities from genetic and environmental microheterogeneity. The samples were from heterozygotes for normal TTR and the amyloidogenic variant ATTR-I107V (a) and the amyloidogenic variant ATTR-V30M (b). All informative details of the patterns indicate that conformational monomer stability is dependent on the sum of contributions from amino acid substitutions and substitutions at Cys10. This is a general observation reproduced with samples from carriers of more than 30 different amino acid substitutions. Reproduced by permission from Altland and Winter (1999, 2003)
When TTR isolated from human plasma by polyacrylamide gel electrophoresis (PAGE) is submitted to isoelectric focusing (IEF) in a urea gradient, it migrates first as a tetramer to its isoelectric point (pI) between pH 4.7 and 5. Then the tetramers slowly dissociate into monomers which present as continuous S-shaped so-called urea titration curves. At a urea concentration of >6 M, all curves concentrate in two fractions: one ‘‘major monomer fraction’’ with a pI near 5.7 and one ‘‘minor monomer fraction’’ with a pI near 5.45. At a urea concentration between 1 and 5 M, monomers separate into at least 10 individual monomeric urea titration curves which correspond to the microheterogeneity at the Cys10 thiol (Altland et al. 1999; Altland and Winter 1999). There is general agreement that the S-shaped curves achieved by PAGE or IEF in a linear urea gradient represent the continuous changes of the equilibrium between the folded and the unfolded states of single-domain proteins. In the case of human TTR monomers, the two states have a different pI equivalent with the gain or loss of one charge unit. The inclination points of the curves represent the urea concentration, with about 50% of the monomer species in the folded state and 50% in the unfolded state. The difference between the inclination points of any two monomer species on the urea concentration scale corresponds to the difference of conformational stability towards urea. Thus, one can directly read from the presented patterns that the cysteinylated normal monomer (curve 1) is less stable than the S-sulfonated monomer (curve 8) and that the latter is less stable than the monomer with Cys10 oxidized to cysteic acid (curve 9). Similarly, one can read from the patterns in Fig. 13.2 that the variant monomer ATTR-I107V as well
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as the variant monomer ATTR-V30M is less stable than the normal TTR monomer and that the changes by amino acid substitution and ligand substitutions at Cys10 have an additive effect on the monomer conformational stability. This additive effect of changes at two different sites of the monomer structure is a characteristic of cooperativity in the sense that a change at one site has influence on the change at another site. There is experimental evidence that the effect of changes at Cys10 as a fixed site adds to the effect of amino acid substitutions at many different sites of the molecule (Altland et al. 2007). Thus, cooperativity happens between many, if not all, sites of the molecule. The S-shaped titration curves appear to be caused by changing equilibria between two states with different net charges and require that the reversible transition reactions between the two states are frequent and fast. Another important conclusion is that within the transition range of the urea gradient, monomers are always present in the folded as well as in the unfolded state and that there are no intermediate conformers of significant stability. Any stable intermediate in the transition between the two states would disrupt the continuity of the titration curves achieved by IEF. The observed continuity within the monomer titration curves is a strong argument against the existence of any stable, partially unfolded intermediate within the transition reactions between the folded and unfolded states, as has been postulated by others (Lashuel et al. 1998). However, the concept of a cooperative reversible unfolding-refolding reaction does not exclude that refolding of the unfolded state into the native conformation is incomplete by competing in vivo with alternative refolding transitions into intermediates as a source of toxic and/or amyloidogenic aggregates and/or with proteolytic breakdown of the unfolded material. Another aspect of conformational heterogeneity is demonstrated by comparison of the monomer patterns in Fig. 13.2a, b. At the 0 M urea left end of the gradient, the monomer titration curves of normal TTR and variant ATTR-I107V in Fig. 13.2a have identical pI values, while the visible curves of the variant ATTR-V30M start at a pI value closer to that of normal and variant monomers in the unfolded state. This means that in the absence of urea, more than half of the variant cysteinylated ATTR-V30M monomers are in the unfolded state or that the folded variant ATTRV30M monomer is sensitive to the acid environment. Among 28 tested amyloidogenic variants, 24 showed similar effects indicating another aspect of conformational microheterogeneity. The remaining four variants affected the monomer-monomer (ATTR-I68L, ATTR-I107V, and ATTR-V122I) and dimer–dimer contacts (ATTRV20I) (see Table 1 and Figs. 1 and 6–8 in Altland et al. 2007). A comparison of the patterns in Fig. 13.1c, d reveals that the decay of tetramers into monomers is slow but also reversible. The length of the flat part of the monomer titration curves (1 and 2) in Fig. 13.1c corresponds to the length of tetramer (T1) in Fig. 13.1d. Similarly, the length of the flat part of curves (8 and 9) in Fig. 13.1c corresponds to the length of tetramer (T5) in Fig. 13.1d. It is obvious that by prolonged exposure to the electric field, the tetramers between (T1) and (T5) decrease, while (T1) and (T5) remain or increase in density. The most reasonable interpretation is that reassembly of tetramers from monomers has occurred under
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conditions with most of the monomers in the folded state. This reassembly is not observed with ATTR-V30M. In summary, the patterns achieved by IEF in urea gradients permit detection of most of the heterogeneity of TTR by ligand and/or amino acid substitutions as well as the associated microheterogeneity of conformational stability. The patterns visualize the reversible transitions from the assembled tetramer to the unfolded monomer and support the concept of cooperative interactions between many or possibly all sites within the ordered structure of the molecule.
13.4
The Achilles’ Heel of Human TTR: His31
A gain of two positive charges accompanies the dissociation of the human TTR tetramer into the folded monomer and a gain of a third positive charge occurs which is associated with the unfolding of the monomer (Altland et al 1999). This change of charge can be derived from the pattern shown in Fig. 13.1d: At the 8 M end of the urea gradient, the difference in pI between the major monomer fraction containing monomers with Cys10 in the cysteinylated form and the minor monomer fraction containing monomers with Cys10 in the S-sulfonated form (Cys10-S-SO3 ) and Cys10 in the oxidized form (Cys10-SO3 ) corresponds to a difference of one charge unit. This difference in pI also corresponds to that between the folded and unfolded state of TTR monomers, to the difference between tetramers (T1) and (T5), as well as to the difference between the pIs of tetramer (T1) and the folded state of S-sulfonated monomers. This gain of three positive charges has been derived from the crystal structure of human TTR. Blake et al. (1978) have described His88 to be buried in the monomer-monomer contact. Thus, its protonated imidazole contributes to the net charge only after dissociation of dimers into monomers. A molecular explanation for the gain of a second and third positive charges has been proposed by Altland et al. (1999) using the crystal structure presented by Monaco et al. (1995). The second positive charge was gained when the loss of the positive charge of Arg21 adjacent to a hydrophobic patch contributing to the dimer-dimer contact is reversed in the isolated monomer. The third charge gain was derived from protonation of His31.NE2 after disruption of a hydrogen bridge between His31. NE2 and the hydroxyl of Ser46, as shown by Fig. 13.3. While Arg21 ands His88 are found in the sequence of TTR from many other species including wallaby, TTR from wallaby has His31 (human TTR) replaced by lysine and Ser46 (human TTR) replaced by alanine (Brack et al. 1995). A urea titration pattern of TTR from a wallaby in the absence and presence of a reducing agent is shown in Fig. 13.4a and b, respectively. There are four major differences between the patterns of TTR from wallaby and humans (see Fig. 13.1): (1) There is a dimer pattern (wallaby) instead of a tetramer pattern (human); (2) There are no detectable S-shaped titration curves for monomers from wallaby; (3) The transition from dimers to monomers of wallaby TTR is associated with the gain of only one positive charge which could be derived from His88 after dissociation of the dimer;
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Fig. 13.3 Detail from the crystal structure of human TTR showing the close vicinity of His31 and Ser46. The atomic coordinates were taken from Monaco et al. (1995). The distances between ˚ and between His31.NE2 and Ser46.OG of 2.92 A ˚ imply that Ala29.O and His31.ND1 of 2.79 A hydrogen bridges are formed with the hydrogen presented by His31.ND1 and Ser46.OG, respectively. Separation of b-strands B and C has been expected to be accompanied by protonation of His31.NE2 or the gain of a positive charge (Altland et al. 1999). We conclude that decreasing the pH of the aqueous environment weakens the hydrogen bridge between His31.NE2 and Ser46. OG and adds to the risk factors for human but not for wallaby TTR monomers to unfold. Notice that His31 and Ser46 in human TTR correspond to Lys31 and Ala46 in wallaby TTR (Brack et al. 1995)
(4) Dissociation of wallaby TTR dimers into monomers is observed at urea concentrations >3 M while the human patterns contain monomers even in the absence of urea, indicating resistance of wallaby TTR dimers against proton activity within the tested range of pH 5–6. Together with the above-mentioned observation that most of the variability of human TTR monomers by ligand and/or amino acid substitutions is associated with differences detectable at the 0 M urea end of the monomer titration curves, there appears to be a strong argument in favor of the view that a hydrogen bridge between His31 and Ser46 is the pH-sensitive structural element of the folded human monomer whereas protonated His31 characterizes the unfolded state of monomers. The experiments depicted in Fig. 13.5 were performed to study the pH sensitivity of human TTR within the range 7.4–6.5 as observed in the interstitial volume under inflammatory or ischemic conditions. TTR isolated as tetramers by PAGE was incubated for 1 h in the presence of 2% sodium dodecyl sulfate (SDS) under various conditions and subsequently submitted to SDS-PAGE. SDS inhibits (prevents) unfolded conformers from refolding. In the experiment shown in Fig. 13.5a,
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Fig. 13.4 Urea titration of TTR from wallaby by IEF in linear immobilized pH gradients without (a) and with (b) added reducing agent (50 mM DTT). The pH gradient was the same as used to achieve the patterns in Fig. 13.1c, d. There were similar pI levels of the monomers from both species at 8 M urea. However, there are several obvious differences between the patterns from wallaby (this figure) and human TTR (Figs. 13.1 and 13.2): (1) Instead of a tetramer pattern (human) there is a dimer pattern (zones D1 to D3 in a, wallaby); (2) There is only a shift by one charge unit associating the transition from dimers to monomers; (3) There are no S-shaped titration curves visualizing the transitions between the folded and unfolded states of monomers; (4) Within the range of 0 and 3 M urea monomers are detected for human but not for wallaby TTR
one set of samples was incubated for 1 h at pH 7 and 37 C, while an identical set was incubated for 50 min at 37 C followed by incubation for 10 min at 95 C. As expected, the rise of the temperature resulted in the complete denaturation of TTR into monomers, while TTR incubated at 37 C over the total time span of 1 h resulted in the transition of only a minor proportion of the dimer fraction into monomers. The monomer fractions resulting from incubation at both temperatures were indistinguishable. Even the splitting of the monomer fraction from individuals heterozygous for normal TTR and three different amyloidogenic variants was the same after incubation at both temperatures. This splitting represented differential binding of SDS to the denatured variant monomers and reflected the sensitivity of the procedure for minor differences between the monomeric structures. Therefore, we have concluded that the monomers resulting from the incubation of TTR at 37 C are completely unfolded structures in complex with SDS and that the transition from folded dimers to unfolded monomers does not involve any detectable conformational intermediates. The patterns in Fig. 13.5b, c show the dimer to monomer transition at pH levels between 7.4 and 6.5. It appears obvious that the decay of dimers into monomers is very sensitive to minor changes of pH within this range. This sensitivity is significantly increased for dimers from heterozygotes for normal TTR and variant ATTRV30M. Dimers from a heterozygote for normal TTR and the non-amyloidogenic
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a
b
c
Fig. 13.5 SDS PAGE of TTR after preincubation under various conditions. The TTR fractions were isolated from plasma by PAGE. The gel strips containing the TTR fraction were submitted to SDS PAGE after incubation under conditions given at the bottom of the patterns. The patterns of the left and right half in (a) (from Altland and Winter 2003, Fig. 3, by permission) evidently show that the monomer fraction achieved by incubation at 37 C over 1 h in 2% SDS is indistinguishable from the monomer fraction achieved after a standard procedure for preparing denatured protein– SDS complexes (i.e., incubation at 95 C over 10 min in 2% SDS), indicating complete unfolding at both temperatures. From the patterns in (b) we conclude: (1) The decay from dimers to unfolded monomers significantly increases when the pH drops from the physiological plasma level of 7.4–6.5; (2) Dimers from a heterozygote TTR-N/V30M are much more sensitive to denaturation (unfolding) than dimers from normal individuals; (3) Dimers from heterozygotes for normal TTR and the non-amyloidogenic variant TTR-H90N are as stable/unstable as dimers from normal individuals; (4) Dimers from wallaby are stable at all three studied pH levels. The patterns in (c) present a fine tuning for the decay of dimers into monomer within the pH range 7.2–6.8. Notice that at all tested pH levels known to be associated with various forms of interstitial acidosis there is an increasing decay of dimers into unfolded monomers correlating with increasing proton activity. In separate experiments we found that increasing the ionic strength of the incubation solution to near physiological levels decreased but did not eliminate the demonstrated effect of pH on the decay of dimerss
variant TTR-H90N had the same pH sensitivity as normal dimers. Dimers from wallaby TTR, however, remain stable at all tested pH levels. Mixed dimers from compound heterozygotes for the destabilizing variant ATTR-V30M and the stabilizing variant TTR-T119M were found to be more stable than dimers from heterozygotes for normal TTR and ATTR-V30M, indicating a cooperative interaction between two sites on different monomers. Also, S-sulfonation of Cys10 by sulfite or reduction by dithiothreitol (DTT) was found to stabilize the
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dimer. The decay of dimers into monomers obviously varies not only with the pH of the solvent and with most of the inherited amyloidogenic amino acid substitutions, but also with the ligand substitutions at Cys10, in an additive manner (Altland et al 2004; Gales et al 2007). The risk of human TTR unfolding under minor acidifying conditions requires a molecular explanation. Histidines with a pKa between 6 and 7 and the N-terminal amino group with pKa of about 7 represent the only sites for possible effects by differential protonation. Effects on conformational stability are expected only where these amino acids are involved in the maintenance of the structural integrity of a conformer. The N-terminal glycine of human TTR can be excluded, as it belongs to the disordered N-terminal end of all monomers. His88 is buried in the dimer and therefore has no access to the aqueous environment. His90 can be excluded because substitution by asparagine has no effect on pH sensitivity (see Fig. 13.5b). The structural environment of His56 does not present any argument supporting differential contributions of protonation/deprotonation to structural integrity. What remains is His31, where a hydrogen atom from the hydroxyl of Ser46 competes with protons from the solvent for the contact with His31.NE2.
13.5
Conclusions
This is the first compilation of evidence or arguments for His31 representing the most vulnerable site (‘‘Achilles’ heel’’) of human TTR under conditions of mild acidosis as observed in vivo under inflammatory or ischemic conditions (Tannock and Rotin 1989; Helmlinger et al. 1997; Kubasiak et al. 2002; Trevani et al. 1999; Marzouk et al. 2002; Agullo´ et al. 2002; Street et al. 2001). The sensitivity of the hydrogen bridge between His31 from b-strand B and Ser46 from b-strand C for acid adds to the stabilizing/destabilizing effects of mutations and ligand substitutions. The data presented here are consistent with our view that the invariable endpoint of destabilization is the unfolded TTR monomer, which may be considered as the source of any possible refolding product, including the native tetramer and all aggregates detected in patients with TTR amyloidosis. The demonstrated cooperativity of probably all sites within TTR tetramers, dimers, and monomers participating in the folding/unfolding transitions implies that trials to stabilize TTR at the thyroxine binding site (e.g., treatment by diflunisal), Cys10 (e.g., treatment by sulfite), and His31 (e.g., treatment by base, prevention of inflammation and ischemia) should have synergistic beneficial effects for all TTR amyloidosis patients by reducing the load of unfolded material. Unfolded amyloidogenic polypeptides have been demonstrated to react with (–)-epigallocatechin-3-gallate (EGCG) from green teas to form non-amyloidogenic oligomers (Ehrnhoefer et al. 2008). Curcumin inhibits aggregation of Alzheimer’s amyloid b peptides (Ono et al 2004; Yang et al. 2005). Possibly, treatment by EGCG (Hunstein 2007) and curcumin (both with added piperine to increase the bioavailability (Shoba et al. 1998; Altland and Schreiner, unpublished observation (EGCG)) could help to
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prevent the generation of harmful aggregates of TTR and amyloidosis. Furthermore, His31 is found only in human and rabbit TTRs. Residue 31 in nearly all other species of TTR sequenced (including rat and mouse) is Lys (see Richardson 2007). This may be a reason why TTR amyloidosis has not yet been observed in species other than humans.
Acknowledgment We appreciate the technical assistance of Pia Winter.
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Feldin P, Fex G (1984) The increased negative charge of prealbumin in cerebrospinal fluid is acquired in vitro by oxidation of the cysteinylated residue without formation of disulfides. Scand J Clin Lab Invest 44:231–238 Fitch NJS, Akbari MT, Ramsden DB (1991) An inherited non-amyloidogenic transthyretin variant, [Ser6]-TTR, with increased thyroxine-binding affinity, characterized by DNA sequencing. J Endocrinol 129:309–313 Gales L, Saraiva MJ, Damas AM (2007) Structural basis for the protective role of sulfite against transthyretin amyloid formation. Biochim Biophys Acta 1774:59–64 Hack V, Breitkreuz R, Kinscherf R, Ro¨hrer H, Ba¨rtsch P, Taut F, Benner A, Dro¨ge W (1998) The redox state as a correlate of senescence and wasting and as a target for therapeutic intervention. Blood 92:59–67 Helmlinger G, Yuan F, Dellian M, Jain RK (1997) Interstitial pH and pO 2 gradients in solid tumors in vivo: high resolution measurements reveal a lack of correlation. Nat Med 3: 177–182 Ho¨rnberg A, Eneqvist T, Olofsson A, Lundgren E, Sauer-Eriksson AE (2000) A comparative analysis of 23 structures of the amyloidogenic protein transthyretin. J Mol Biol 22:649–669 Holmgren G, Ando Y, Wikstro¨m L, Rygh A, Suhr O (1997) Discordant symptoms in monozygotic twins with familial amyloidotic polyneuropathy (FAP) (TTR met30). Amyloid: J Protein Folding Disord 4:178–180 Hunstein W (2007) Epigallocathechin-3-gallate in AL amyloidosis: a new therapeutic option? Blood 110:2216 (letter) Jacobson DR, McFarlin DE, Kane I, Buxbaum JN (1992) Transthyretin Pro55, a variant associated with early-onset, aggressive, diffuse amyloidosis with cardiac and neurological involvement. Hum Genet 89:353–356 Jenne DE, Denzel K, Bla¨tzinger P, Winter P, Obermaier B, Linke RP, Altland K (1996) A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dime-dimer contacts and causes systemic amyloidosis. Proc Natl Acad Sci USA 93:6302–6307 Jones DP (2006) Extracellular redox state: refining the definition of oxidative stress in aging. Rejuvenation Res 9:169–181 Kanda Y, Goodman DS, Canfield RE, Morgan FJ (1974) The amino acid sequence of human plasma prealbumin. J Biol Chem 249:6796–6805 Kishikawa M, Nakanishi T, Miyasaki A, Shimizu A (1999) A simple and reliable method of detecting variant transthyretins by mutidimensional liquid chromatography coupled with electrospray ionization mass spectrometry. Amyloid: J Protein Folding Disord 6:48–53 Kubasiak LA, Hernandez OM, Bishopric NH, Webster KA (2002) Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci USA 99:12825–12830 Kyle RA, Gertz MA, Linke RP (1992) Amyloid localized to tenosynovium at carpal tunnel release. Immunohistochemical identification of amyloid type. Am J Clin Pathol 97:250–253 Kyle RA, Spittell PC, Gertz MA, Li CY, Edwards WD, Olson LJ, Thibodeau SN (1996) The premortem recognition of systemic senile amyloidosis with cardiac involvement. Am J Med 101:395–400 Lashuel HA, Lai Z, Kelly JW (1998) Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implication for wild-type, V30M, and L55P amyloid fibril formation. Biochemistry 37:17851–17864 Mansoor MA, Svardal AM, Ueland PM (1992) Determination of the in vivo redox status of cysteine, cysteinylglycine, and glutathione in human plasma. Anal Biochem 200:218–229 Marzouk SAM, Buck RP, Dunlap LA, Johnson TA, Cascio WE (2002) Measurement of extracellular pH, K +, and lactate in ischemic heart. Anal Biochem 308:52–60 Mills BJ, Weiss MM, Lang CA, Liu MC, Ziegler C (2000) Blood glutathion and cysteine changes in cardiovascular disease. J Lab Clin Med 135:396–401 Monaco HL, Rizzi M, Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268:1039–1041
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Chapter 14
New Therapeutic Approaches for Familial Amyloidotic Polyneuropathy (FAP) Yukio Ando, Masaaki Nakamura, Mistuharu Ueda and Hirofumi Jono
Abstract Although liver transplantation is the only therapy to halt the clinical manifestations of transthyretin (TTR) related familial amyloidotic polyneuropathy (FAP), the therapy has given rise to several problems. An alternative treatment is needed. After the precursor protein of amyloid fibrils in FAP was established in 1983, several different approaches have been investigated as an essential therapy for FAP: (1) reduction of variant TTR levels in plasma, (2) down regulation of variant TTR gene mRNA, (3) inhibition of amyloid deposition, (4) stabilization of the tetrameric TTR structure and (5) replacement of the variant TTR gene with the normal TTR gene (which can be achieved by liver transplantation or by gene therapy). In this chapter, we introduce these research strategies and trials and discuss the possibility of the optimal treatment for FAP. Keywords Antibody therapy, Gene therapy, Liver transplantation
14.1
Introduction
After Andrade identified patients with familial amyloidotic polyneuropathy (FAP) in Portugal in 1952 (Andrade 1952), many foci of FAP cases have been reported worldwide. As patients with FAP show various serious systemic symptoms, such as cardiac and renal dysfunction, gastrointestinal disorders, ocular disorders, glandular and autonomic dysfunction, and peripheral neuropathy, many trials have attempted to treat these symptoms (Araki 1984; Benson 1989; Benson and Uemichi 1996; Ando et al. 1993; Ando and Suhr 1998). Although liver transplantation has become a well-established treatment for halting the progression of FAP-related clinical Y. Ando (*) Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, Japan e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_14, # Springer‐Verlag Berlin Heidelberg 2009
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symptoms, no truly effective therapy has been designed (Holmgren et al. 1993; Ericzon et al. 1999; Skinner et al. 1994; Takei et al. 1999; Ando et al. 1995; Suhr et al. 1996; Lendoire et al. 1999), and several problems related to its use have arisen (Ando et al. 2001, 2002). We cannot use liver transplantation for these patients as long-term therapy for the following reasons: (1) Medical care before and after the surgery and the surgery itself are extremely expensive; (2) Patients who have received transplants must continue lifelong administration of immunosuppressants after the surgery, and these agents may have adverse side effects; (3) Carriers of the amyloidogenic transthyretin (ATTR) gene who have no clinical symptoms cannot undergo liver transplantation before the onset of the disease; (4) Clinical symptoms of FAP that were present before the surgery continue to occur after liver transplantation; (5) The worldwide shortage of liver donors means that all FAP patients cannot undergo the surgery; and (6) After liver transplantation, patients with FAP ATTR Val30Met or other types of FAP have been reported to develop ocular disorders, caused by vitreous amyloid deposits, and central nervous system symptoms, because of leptomeningeal amyloidosis, as an effect of continuous TTR production by the retina and choroid plexus, respectively. To overcome all these problems, we must develop a new noninvasive effective treatment. The following methods can be considered potential FAP treatment strategies (Table 14.1): (1) reduction of variant TTR levels in the plasma; (2) down regulation of the variant TTR gene mRNA; (3) inhibition of amyloid deposition; (4) stabilization of the tetrameric TTR structure; and (5) replacement of the variant TTR gene with the normal TTR gene (which can be achieved by liver transplantation or by gene therapy) (Table 14.1). Here, we describe new therapeutic approaches for FAP that our group has recently investigated, as well as previously used therapies. We hope that at least one of these approaches will lead to an effective treatment of FAP. Table 14.1 Strategies for treatment of familial amyloidotic polyneuropathy 1. Reduction of variant TTR levels in plasma A. Plasma exchange B. Affinity column chromatography C. TTR absorption column chromatography 2. Downregulation of TTR gene mRNA A. Injection of a large amount of normal TTR 3. Inhibition of amyloid deposition A. Prevention of amyloid formation BSB Antibody therapy B. Dissolution of amyloid fibrils IDOX 4. Stabilization of the tetrameric TTR structure A. NSAIDs B. Cr3+ 5. Replacement of the variant TTR gene with the normal TTR gene A. Liver transplantation B. Gene therapy
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Elimination of Variant TTR in Blood Circulation
Plasma exchange, affinity column chromatography binding with a monoclonal antibody, and use of a special column having a specific affinity for TTR have been considered as possible methods for elimination of ATTR in blood circulation (Sales-Luis et al. 1991; Ikegawa et al. 1988; Regnault et al. 1992; Costa and Costa 1998; Tokuda et al. 1998). Sales-Luis et al. (1991) performed plasma exchange for 1.5–4.5 years with nine patients with FAP ATTR Val30Met, and the effect of this therapy on the disease was compared with the course of the illness in nontreated FAP patients. Progression of the disease was slightly suppressed, and body weight loss and diarrhea were effectively treated (Sales-Luis et al. 1991, Continho et al. 1988). However, Sales-Luis stopped this plasma exchange because of allergic reactions or virus infection (Continho et al. 1988). Ikegawa and colleagues performed plasma exchange once a month with three FAP patients (Ikegawa et al. 1988). They reported that the levels of variant TTR in the plasma decreased from 39 to 19% of the total TTR levels but returned to the values that were seen before treatment (Ikegawa et al. 1988). Objective and subjective evaluations indicated no improvement in laboratory and clinical data. The treatment was very expensive, and the numbers of treated patients were very small. In addition, no follow-up studies have been reported. Costa and colleagues reported application of an affinity column consisting of a monoclonal antibody for ATTR Val30Met to eliminate the variant TTR from the blood stream (Regnault et al. 1992; Costa and Costa 1998). However, the method was unsatisfactory because the rate of elimination was too slight to halt the progression of FAP, and the elevation of plasma TTR levels returned, which was observed just after treatment (Regnault et al. 1992; Costa and Costa 1998). Moreover, vitamin A levels were decreased, and four patients had the TTR–TTR antibody complex in their plasma because of leakage of the antibody from the column to the blood stream (Costa and Costa 1998). Loss of skin hair throughout the whole body and allergic reactions has been reported. Tokuda et al. used the special column PA-01, which showed a specific affinity for TTR, to eliminate ATTR from the blood (Tokuda et al. 1998). However, this therapy had the same defects as did the two trials just mentioned. Just after treatment, plasma ATTR levels decreased by 50%, but they returned to the same values seen before treatment. From these studies, we can conclude that these methods cannot provide sufficient elimination of plasma ATTR, and that regulation of ATTR mRNA production does not seem to be appropriate because TTR itself is a rapid turnover protein whose plasma half-life is about 2 days (Jouquan et al. 1983; Dickson et al. 1982; Ando et al. 1995). FAP patients who want these treatments must go to a hospital every day or every hour of the day to achieve sufficient reduction of plasma ATTR levels. These results also suggest that ordinary antisense or ribosomal therapies that would target variant TTR mRNA would not be advantageous treatments.
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Down regulation of Variant TTR
It is well documented that FAP patients develop systemic disorders induced by amyloid deposition in the viscera in addition to peripheral nerves. As the disease progresses, FAP patients become emaciated, and hypoproteinemia and hypoalbuminemia are often observed. To compensate for the hypoproteinemia and hypoalbuminemia, intravenous injections of fresh frozen plasma (FFP) are often used. FFP contains a significant amount of TTR in addition to various plasma proteins. A surprising finding has been that the variant TTR levels decreased while levels of total TTR, total protein, and albumin in the plasma increased. It is possible that some genetic regulation occurs in response to the FFP injection. We monitored the changes in plasma TTR levels after injection of a large amount of FFP when they treated FAP patients for hypoproteinemia and hypoalbuminemia (Ando et al. 1997). From the 24th to 48th h after injection, the total plasma TTR levels were elevated, and variant TTR levels decreased, accompanied by an elevation of plasma total protein and albumin levels. To elucidate the mechanism of this phenomenon, a large amount of purified normal TTR from normal human plasma was injected intravenously into mice and into patients with FAP ATTR Val30Met. Injection of 3 mg of purified TTR into C57Black6 mice resulted in decreased expression of TTR mRNA from the 6th to the 24th h after injection, and then TTR mRNA expression gradually increased up to the 48th h after injection. Injection of 400 mg of normal human TTR into three FAP patients caused elevated total plasma TTR levels and significantly decreased variant TTR levels from the 24th to the 48th h after injection. These results suggested that this method allowed down regulation of the harmful protein by means of replacement with the normal protein. This phenomenon may explain the mechanism for one of the possible methods for decreasing the amount of harmful protein in the circulation. However, because reduction of the amount of variant TTR in the plasma was not sufficient, and as TTR is a rapid turnover protein as described previously (Jouquan et al. 1983; Dickson et al. 1982; Ando et al. 1995), this treatment cannot be applied as an effective therapy for FAP patients.
14.4
Inhibition of Amyloid Deposition in Tissues
14.4.1 Compounds Binding to Amyloid Fibrils 14.4.1.1
Congo Red
Congo red, with the chemical name 3,30 -[(1,10 -biphenyl)-4,40 -diylbis(azo)] bis(4-amino-1-naphthalene acid) disodium salt, is a commonly used histological dye for amyloid detection. At first, Congo red was introduced in Berlin in 1885 as the first of the economically lucrative direct textile dyes (Steensma 2001). As a
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histological stain, Congo red was applied in 1927 by Belgian neuropathologist, Paul Divry, who noted that Congo red gave a green birefringence to the amyloid deposited in the brain tissue under polarized light (Divry 1927). Although the mechanism of interaction of Congo red with amyloid fibrils is not well understood, it is generally believed that Congo red binding depends on the secondary configuration of the amyloid fibril, consisting predominantly of cross b-pleated sheets. Investigations on the inhibitory effects of Congo red on amyloid formation in different, in vitro models of amyloidosis indicated a complexity of involved mechanisms, from inhibition of protein depositions, through blockade of amyloid-cell interaction, to the modulation of the regulation of intracellular homeostasis (Frid et al. 2007). Increasing lines of evidence indicate that Congo red may prevent Ab amyloid fibril formation by binding and stabilizing native monomers or partially folded intermediates, thus reducing the concentration of oligomers (Gellermann et al. 2006). Contrary to that, when the nucleus had already formed for seeding, Congo red might promote further amyloid formation (Fraser et al. 1992). Studies of the concentration-dependent effects of Congo red were undertaken by Kim et al. (2003), who used a variant of the immunoglobulin light chain. They demonstrated that at low concentrations, Congo red binding promoted the generation of partially folded, aggregation prone forms of the proteins into oligomers and protofilament intermediates, resulting in accelerated fibril formation. At higher concentrations, however, Congo red inhibited fibril formation supporting the denatured state, which is much less prone to aggregation. The data proposed that Congo red exhibited antiamyloidogenic properties. However, it is potentially toxic (Giger et al. 1974). It can reduce serum protein concentration and cause platelet aggregation. Especially if administered orally, Congo red is cleaved by enzymes to benzidine, a highly carcinogenic compound, known to induce hepatocellular and urinary bladder carcinomas. Increased efforts aim to produce analogues that will maintain or enhance the antifibrillar aggregation activities of Congo red, but will not be toxic.
14.4.1.2
Congo Red Derivatives
In systemic amyloidoses such as FAP, AA amyloidosis, AL amyloidosis, and dialysis-related amyloidosis (DRA), biopsy samples are often obtained from the gastrointestinal tract and abdominal fat to make the diagnosis, because amyloid deposits are usually found in these tissues at the early stage of the disease (Guy and Jones 2001; Kaplan et al. 1999; Masouye 1997). However, in certain cases it is sometimes difficult to construct the diagnosis by using only these biopsy samples, because the pattern of amyloid deposition in the body varies in each individual. In addition, a biopsy for diagnostic purposes cannot usually be performed in patients with localized amyloidosis, such as amyloidosis in Alzheimer disease and endocrine amyloidosis (Westermark and Westermark 2000). Diagnosis at the early stage of amyloidosis might be possible if a tool were available that incorporated real-time
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amyloid monitoring, such as radioisotope-labeled scintigraphy (Hawkins et al. 1990; Puille et al. 2002). Among several histopathological methods utilizing stains such as Congo red, fast scarlet, and thioflavine S, Congo red staining is one of the most popular for detection of amyloid deposits in tissues. However, judging the positivity of Congo red-positive lesions is sometimes difficult, and false-positive or false-negative results may be obtained. Moreover, Congo red staining cannot be used for in vivo studies because of its toxic effects in the human body. Chrysamine G, a Congo red derivative with a similar molecular structure used for the same purpose as Congo red, has the same limitations (Klunk et al. 1995, 1998; Dezutter et al. 2001a, b). To overcome these problems, we should have a new tool for examination of amyloid deposition in tissues. (trans, trans)-1-Bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy) styrylbenzene (BSB), which has been found to bind to amyloid plaques in postmortem samples of brains from patients with Alzheimer disease, is also a Congo red derivative and has been the focus of recent attention (Dezutter et al. 1999; Skovronsky et al. 2000; Schmidt et al. 2001; Zhuang et al. 2001). As this compound is lipophilic, it can traverse the blood–brain barrier, and bind itself to amyloid fibrils in senile plaques when it is injected intravenously into transgenic mice with Alzheimer disease. Moreover, this compound can detect cell inclusion bodies derived from a-synclein. However, no studies have addressed the question of whether the compound could become a useful tool for detection of amyloid deposits in systemic amyloidosis such as FAP. Various plasma proteins in the systemic circulation may disturb the binding of BSB to amyloid fibrils in tissues.
14.4.1.3
BSB as a Valuable Therapeutic Drug
We used BSB to detect amyloid fibrils in autopsy and biopsy samples from patients with localized amyloidosis, such as familial Creutzfeldt-Jakob disease, and systemic amyloidosis, such as FAP, AA amyloidosis, AL amyloidosis, and DRA (Ando et al. 2003). Histopathological methods were employed to examine the reactivity of BSB with amyloid fibrils, and the results were compared with those from studies with immunohistochemical and Congo red staining and polarized light. BSB reactivity in vivo was investigated in mice in which AA amyloidosis had been induced. The authors determined the affinity of BSB and Congo red for purified amyloid fibrils by using a highly sensitive 27-MHz quartz crystal microbalance (Okahata et al. 1998; Matsuno et al. 2001). To test the usefulness of BSB as a possible therapeutic agent in FAP, its inhibitory effect on the formation of TTR amyloid in vitro was examined by means of electron microscopy (Ando et al. 2003). BSB showed a significant affinity for amyloid fibrils purified from FAP patients’ samples and the suppressed formation of TTR amyloid in acidic conditions in vitro. BSB may become a valuable therapeutic drug, although further evaluation should be needed to determine therapeutic effects in vivo.
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14.4.2 Drugs Disrupting Amyloid Fibrils 14.4.2.1
IDOX
40 -Iodo-40 -deoxydoxorubicin (IDOX) has been reported to bind to amyloid and lead to the catabolism of amyloid in deposits.This chemical compound was first reported by Merlini et al. as an agent that would bind to amyloid fibrils found in five different types of amyloidosis (Merlini et al. 1995). A multicenter study attempted to develop a dosing schedule to confirm these results. IDOX was administered to AL amyloidosis patients at 15 mg once per week for four consecutive weeks, and this therapy was repeated every 3 months up to four times (Gertz et al. 2002). However, the results were not clear, and no obvious effect on the patients was seen. The authors concluded that the protocol produced insufficient activity at the dose used in the study. It is possible that IDOX does not show the same attraction for amyloid in FAP as that seen in the AL amyloidosis trial. In an in vitro study, Sebastiao et al. observed an interaction of IDOX with ATTR Leu55Pro and reported that monoclinic ATTR Leu55Pro crystals soaked with IDOX undergo rapid dissociation (Sebastiao et al. 2000). Moreover, under the same conditions, the orthorhombic wild-type TTR crystals were quite stable. This result was explained by the different TTR conformations in the crystals of the two proteins: the ATTR Leu55Pro had the necessary conformation for IDOX binding, but the same structure was not present in the crystallized wild-type protein. This study presented a theoretical model of the interaction of ATTR Leu55Pro with IDOX that is consistent with the dissociation of the amyloid-like oligomer. In this model, the IDOX iodine atom was buried in a pocket located between the two beta-sheets of the ATTR Leu55Pro monomer, with the long axis of the IDOX aromatic moiety nearly perpendicular to the direction of the beta-sheets (Sebastiao et al. 2000). This chemical compound, first developed as an anticancer drug, may be one of the most promising drugs for FAP. However, it has nephrotoxic effects, and an IDOX derivative that is less toxic to the kidneys should be sought, because one of the target organs for amyloid deposition in FAP is the kidney, and renal dysfunction often occurs during the course of the illness. In any case, we must wait for further information from in vivo studies of the effect of IDOX to derive conclusions about the usefulness of IDOX for FAP patients.
14.4.2.2
Tetracycline and Its Derivatives
Cardoso et al. showed that tetracycline and its derivatives, doxycycline, rolitetracycline, and minocycline, interfered with TTR amyloid fibril formation in vitro (Cardoso et al. 2003), acting as fibril disrupters. Doxycycline was revealed as the most effective of the four compounds in that study. Doxycycline affects many mammalian cell functions including proliferation, migration, apoptosis, and matrix remodeling (Bendeck et al. 2002). It was also shown that doxycycline had activity
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against amyloid formation of Ab (Forloni et al. 2001). Cardoso et al. also investigated the effect of doxycycline treatment in vivo using transgenic ATTR Val30Met mice (Cardoso and Saraiva 2006). In that study, tissue analysis by Congo red staining indicated that doxycycline was able to disaggregate TTR amyloid fibrils. However, doxycycline was not able to act on nonfibrillar TTR deposits, as the overall TTR load was similar in both treated and untreated groups of transgenic mice. These in vivo results corresponded with the in vitro findings showing that doxycycline was not capable of inhibiting TTR fibril formation till a certain fibril length (Cardoso et al. 2003). 14.4.2.3
Immunotherapies to Promote Clearance of Amyloid Deposition
Antibody Therapy Against Amyloidogenic TTR Gustavsson et al. used various antigenic mapping procedures to determine whether major antigenic sites differ between normal TTR, ATTR, and in situ amyloid fibrils (Gustavsson et al. 1994). Results of this study suggested that the antigenic sites on normal plasma TTR included the AB loop and the CD loop. The amino acid sequences associated with these loops are present on the outside of the TTR molecule. On the other hand, an antiserum against b-strand H (a peptide TTR115–124, which constitutes the monomer-to-monomer interaction areas in the dimer (Blake et al. 1978), reacted only with ATTR in amyloid fibrils but not with normal plasma TTR. Those results suggested that there was an altered configuration of TTR within amyloid fibrils when compared with plasma TTR. The antiserum to TTR115–124, which so far has turned out to be amyloid specific, may serve as a valuable probe and may be useful in antibody therapies. However, Bergstrom et al. (2006) indicated that the epitope TTR115–124 was hidden in ATTR Tyr114Cys both in vitreous aggregates and in mature amyloid deposits in various tissues, by immunochemical means. Differences in the tertiary structure may exist between amyloid fibrils formed by different ATTR variants. A monoclonal antibody, MAb39–44, reacting with high molecular weight aggregates of TTR but not with tetrameric, TTR was also generated and characterized by Goldsteins et al. (Goldsteins et al. 1999). This antibody recognized a cryptic epitope that was expressed in isolated recombinant amyloidogenic mutants and in ex vivo amyloid. MAb39-44 could selectively detect TTR variants in plasma from carriers and patients with amyloidogenic TTR mutations (Palha et al. 2001). MAb39-44 may be useful for detecting amyloidogenic TTR variants and may be a candidate for the antibody in therapies. To determine the usefulness of these antibodies for the immunotherapy, in vivo studies should be performed. Vaccine Therapy A TTR mutant Tyr78Phe, designed to destabilize the native structure, has been shown to expose a cryptic epitope recognized by a monoclonal antibody that reacts
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only with highly amyloidogenic mutants presenting the amyloid fold or with amyloid fibrils (Redondo et al. 2000). Terazaki et al. demonstrated that immunization with TTR Tyr78Phe was powerful in reducing TTR deposition and clearing amyloid deposition in an FAP rodent model transgenic for the human mutant Val30Met with deposition in the gastrointestinal tract (Terazaki et al. 2006). Immunization with TTR Val30Met, an intrinsic protein found in FAP patients, could not induce a high antibody titer in transgenic (Tg) mice possessing the human ATTR Val30Met gene and did not prevent TTR and amyloid deposition in these mice. By contrast, immunization of Tg mice with TTR Tyr78Phe produced a higher titer of antibodies against Tyr78Phe TTR in the serum, than with ATTR Val30Met, suggesting significant structural changes of the Tyr78Phe mutant comparative to the Val30Met mutant, although some epitopes might be shared. Tissue specimens from the Tg mice immunized with Tyr78Phe demonstrated inflammatory cell infiltration and hyperplasia of lymphoid tissues only in or near the areas of usual TTR deposition which were absent in other areas of the tissue. Most cells in the lymphoid tissues immunoreacted predominantly with anti-CD45 (a surface marker of T cells) antibody but not with anti-CD5 (a surface marker of B cells) antibody. Inflammatory cell-infiltrated lesions displayed a reaction for antiMac-1 antibody, indicating tissue infiltration by macrophages. It was difficult to clearly discern the molecular mechanisms underlying the effects observed that can preclude both prevention and clearance of deposition. However, the infiltration of a large number of B cells and macrophages and the detection of IgG in lesions where TTR and amyloid deposits normally appear suggested a clearance mechanism involving FcR-mediated phagocytosis, as earlier reported in the Alzheimer immunization protocols (Bard et al. 2000). The target for this therapy may be applied in FAP ATTR Val30Met patients with amyloid deposition in tissues. In addition, the therapy could be used as a vaccination for healthy TTR variant gene carriers to prevent TTR amyloid deposition in tissues.
14.5
Stabilization of the Tetrameric Structure of TTR
The amyloid formation working hypothesis established by substantial studies, gave rise to the idea that stabilizing tetrameric TTR is a promising method for prevention of amyloid formation (Kelly and Lansbury 1994; Klabunde et al. 2000; Almeida et al. 2005; Johnson et al. 2005). Colon et al. first demonstrated this concept by using recombinant wild-type TTR and variant TTR (Colon and Kelley 1992; McCutchen et al. 1993). Normal TTR behaves as a tetramer and binds to retinol binding protein (RBP) and thyroxine (T4) in plasma (Monaco et al. 1995). Tetrameric TTR is not itself amyloidogenic, but dissociation of the tetramer into a compact nonnative monomer with low conformational stability can lead to amyloid fibril formation (Quintas et al. 1999). Although several of the 50 FAP-associated TTR single-site mutations have a normal tetrameric structure under physiological
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conditions (Connors et al. 2000), these mutations significantly destabilize the tetramer (McCutchen et al. 1993, 1995). Recent biochemical and pathological studies further revealed that instability of the terameric form of TTR by mutation, especially FAP ATTR Val30Met, leads to amyloid formation in the tissues of FAP patients (Saraiva 2001). It is interesting to note that subjects possessing the TTR Thr119Met gene are asymptomatic carriers, and that compound heterozygotes having ATTR Val30Met and TTR Thr119Met genes show very mild FAP symptoms or have no symptoms (Alves et al. 1993). Alves et al. demonstrated by semi denaturing isoelectric focusing that patients possessing TTR Thr119Met or ATTR Val30Met/TTR Thr119Met genes showed marked TTR tetrameric structural stability (Alves et al. 1997; Almeida et al. 1997). Terazaki et al. also reported an interesting late-onset compound heterozygote patient with FAP ATTR Val30Met/ TTR Arg104His who had very mild and slowly progressive clinical symptoms and whose tetrameric TTR stability was greater than that of the TTR from a compound heterozygote ATTR Val30Met/TTR Thr119Met patient (Almeida et al. 2000; Terazaki et al. 1999). These lines of evidence suggest that stabilizing the tetrameric TTR, as a potential therapeutic strategy, is a prerequisite for prevention of amyloid formation. On the basis of this concept, several treatments were proposed and examined (Fig. 14.1).
14.5.1 Various Nonsteroidal Anti-inflammatory Drugs (NSAIDs) Derivatives As thyroxine (T4) is one of the most important molecules for stabilizing TTR in its tetrameric form, T4-based therapeutic drugs have been proposed. Binding of T4 and their derivatives stabilizes the native conformation of TTR and inhibits amyloid formation in vitro and possibly in vivo (Miroy et al. 1996). In fact, it has been proposed that, in the cerebral spinal fluid (CSF) where a high concentration of TTR with bound T4 is found, the protein is in the stable tetrameric conformation and does not lead to detectable TTR amyloid formation in the brain (Miroy et al. 1996). Baures et al. reported that various NSAIDs have the potential for stabilizing the tetramecic TTR (Baures et al. 1999). Several NSAIDs, which bind and inhibit the cyclooxygenases, also bind to TTR as mimic compounds of T4 (Baures et al. 1998, 1999; Munro et al. 1989). The structure of NSAIDs resembles the structure of T4 and these drugs bind to TTR via a T4 binding site (Baures et al. 1999). Peterson et al. demonstrated that the three-dimensional structure of TTR with flufenamic acid (FLU), a derivative of NSAIDs is a more efficacious TTR amyloid inhibitor than the natural ligand T4 (Peterson et al. 1998). Difulnisal and flufenamic acid exhibit notable inhibition of both acid-mediated aggregation and urea-mediated tetramer dissociation of the most common disease associated variants (Johnson et al. 2005; Hammarstro¨m et al. 2003). Tojo et al. reported that therapeutic serum concentrations of difulnisal (100–200 mM) stabilized serum variant TTR tetramer
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Replacement of the variant TTR gene with the normal TTR gene Liver
Liver transplantaon Gene therapy
Down–regulaon of TTR gene mRNA Injecon of a large amount of normal TTR
Stabilizaon of the tetrameric TTR structure NSAIDs Cr3+ Fx-1006 Dissociaon
Tetrameric TTR Reducon of variant TTR levels in plasma Plasma exchange Affinity column chromatography TTR absorpon column chromatography
Misfolding
Monomeric TTR Prevenon of amyloid formaon Dissoluon of amyloid fibrils IDOX Tetracycline
BSB Anbody therapy Radical scavenger
TTR deposits
Polymerizaon
Amyloid Fibrils
Fig. 14.1 Working hypothesis of amyloid formation in FAP and points of intervention for our treatments. Based on the pathogenesis of FAP, various therapeutic approaches can be considered as potential FAP treatment strategies: (1) reduction of variant TTR levels in plasma, (2) down regulation of variant TTR gene mRNA, (3) inhibition of amyloid Deposition, (4) stabilization of the tetrameric TTR structure, and (5) replacement of the variant TTR gene with the normal TTR gene. TTR transthyretin, NSAID nonsteroidal anti-inflammatory drugs, BSB (trans, trans)-1-Bromo2,5-bis-(3-hydroxycarbonyl-4-hydroxy) styrylbenzene, IDOX 40 -Iodo-40 -deoxydoxorubicin
more effectively than those of flufenamic acid (35–70 mM) (Tojo et al. 2006). Sekijima et al also demonstrated that oral administration of difulnisal mediates kinetic stabilization of TTR in human serum (Sekijima et al. 2006). Furthermore, structurally related derivatives of NSAIDs were also exploited to enhance the selectivity and reduce the adverse effect (Adamski-Werner et al. 2004; Reixach et al. 2006; Julius et al. 2007; Almeida et al. 2004). Various reports confirmed these findings and this concept is now widely accepted (Miller et al. 2004; Kingsbury et al. 2008).
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14.5.2 Cr3+ Recent studies suggested that certain metal ions affect amyloidogenesis in several types of amyloidosis (Sato et al. 2006). In FAP, metal ions may influence the stability of the tetrameric form of TTR. Sato et al. therefore investigated whether various metal ions (e.g., Zn2+, Cu2+, Ca2+, Fe3+, Al3+, Cr3+) affect amyloidogenesis of wild-type TTR and ATTR (Sato et al. 2006). Among the metal ions, Cr3+ increased the tetrameric stability of both wild-type and ATTR Val30Met, and suppressed TTR amyloidogenesis. It is unlikely that Cr3+ binds to T4-binding sites of TTR, because of its chemical structure. Cr3+ may increase the thermodynamic stability of TTR tetramer by facilitating the binding of T4. As Cr3+ is a component of health foods that is widely used throughout the world, administration of this metal ion was thought to be a good candidate for therapy. However, at the serum concentration, Cr3+ had no significant effect on TTR stability in serum (Tojo et al. 2006). While serum Cr3+ concentration will increase after Cr3+ supplementation, it is still very low compared to the concentration of TTR, likely explaining why stabilization is not observed. However, Cr3+ could be useful for treating TTR amyloidosis by enhancing the effects of difulnisal, because the effects of T4 and Cr3+ are not competitive but cooperative. Although no obvious effect of Cr3+ on patients was seen, further in vivo evaluation of the effects is needed.
14.5.3 Fx-1006 Tremendous efforts revealed the possibility of stabilization of the tetrameric form of TTR as a potential therapeutic strategy. Ongoing biochemical and pathological studies explored further potentiality of this therapy. It is noteworthy that Fx-1006A, a potent and selective stabilizer for tetrameric TTR, is underway in a multinational Phase II/III clinical study in patients with FAP worldwide (Labaudinie´re et al. 2006; Bulawa et al. 2006; Devit et al. 2006). Moreover, a number of structurally diverse small molecules that bind to TTR, increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis, have been tested. In fact, several small molecules, such as plant-derived flavones and xanthones (Baures et al. 1998; Green et al. 2005; Maia et al. 2005), the synthetic estrogen diethylstilbestrol (Morais-de-Sa´ et al. 2004), 2,4-dinitrophenol (DNP) as well as 3,5-diiodosalicylic acid were shown to have high affinity towards TTR and to inhibit amyloid formation in vitro (Gales et al. 2008).
14.5.4 Controlling Drugs for Endoplasmic Reticulum (ER) Stress In addition, a recent report also suggested that an endoplasmic reticulum (ER) quality control system may differentially regulate the fate of amyloidogenic TTRs
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and their monomers (Sato et al. 2007). Amyloidogenic TTR variants, which exist as tetramer in the ER, could pass the ER quality control system but form aggregates or amyloid fibrils in the extracellular space. ER quality control systems may monitor amyloidogenecity of TTR at monomer level and regulate the secretion of TTR variants. Thus, inhibiting tetramerization of amyloidogenic TTR variants in the ER thereby reducing the secretion of TTR variants from the cells may become a possible therapeutic strategy of FAP. However, although a certain amount of in vitro evidence has been reported, more clinical studies need to be conducted, and the true therapeutic effect for FAP patients remains to be investigated.
14.6
Gene Therapy
As described above, liver transplantation was originally suggested as a treatment that would halt the production of variant TTR in the liver; FAP does not progress when the variant TTR gene is replaced by the normal TTR gene in the liver by the surgery. This fact led to the suggestion that gene therapy which would suppress the variant TTR mRNA expression or correct the TTR gene mutation could become one of the most important methods for ameliorating the clinical symptoms of FAP.
14.6.1 Gene Silencing Tools Ribozymes, antisense methods and small interfering RNAs (siRNAs) are effective gene silencing tools. Ribozymes are based on catalytic RNA originally found in the protozoan tetrahymena (Inoue et al. 1985) and can cleave a targeted RNA sequence or reverse the mRNA to generate correct sequences (Sullenger and Cech 1994). The antisense oligonucleotides were recognized as the therapeutic tool in the 1970s (Zamecnik and Stephenson 1978) and could cause enzymatic degradation of mRNA by activating ribonuclease H1 (Wu et al. 2004). RNA interference (RNAi) was first discovered in Caenorhabditis elegans as a phenomenon of sequencespecific post-transcriptional gene silencing (Fire et al. 1998). The inhibition of specific gene expression by RNAi has also been achieved in mammalian cells by passing the Dicer step and directly introducing synthesized small interfering RNAs (siRNAs) (Elbashir et al. 2001). These technologies are expected to be a powerful tool for gene therapy. Propsting et al. reported specific cleavage of ATTR Val30Met mRNA in a cell culture system by using hammerhead ribozymes (Propsting et al. 1999). They showed that chemically modified nuclease-stable Inosine (15.1)-Hammerhead ribozymes can target ATTR Val30Met mRNA with high specificity at the RNA level (Propsting et al. 1999). In an FAP gene therapy trial, they used the wild-type human normal TTR-expressing cell line HepG2 and a stable transfected cell line with the ATTR Val30Met gene. They cleaved the ATTR Val30Met and wild-type TTR
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mRNAs with specific nuclease-stable chemically modified Inosine (15.1)-Hammerhead ribozymes and analyzed the protein after immunoprecipitation and subsequent Western blotting. They were able to down regulate the TTR concentration by 54.5% (100% = 1.5 mg l1 TTR) and also to specifically target ATTR Val30Met expression in the cell culture system. The therapeutic effect was improved by using cationic liposomes, resulting in a total down regulation of wild-type TTR mRNA and ATTR Val30Met mRNA of 92.1 and 62.7%, respectively. Tanaka et al. reported that another ribozyme targeting a variant TTR (E61 K) degraded the variant mRNA but not the wild-type mRNA (Tanaka et al. 2001). These ribozymes also reduced the amounts of TTR mRNA and protein in HepG2 cells and COS-1 cells transfected with TTR-E61 K cDNA. Benson et al. demonstrated suppression of hepatic TTR mRNA levels and serum TTR levels by as much as 80% in mice transgenic for the human Ile84Ser TTR mutant gene by antisense oligonucleotides (Benson et al. 2006). Kurosawa et al. reported that a siRNA selectively silenced the mutant Val30Met TTR allele in cells expressing both wild type and Val30Met alleles (Kurosawa et al. 2005). Although these data were interesting and the results were clear, this method cannot be applied to treatment of FAP patients, because a perfect ATTR Val30Met gene suppression was not achieved, and, as described previously, TTR is a rapid turnover protein. Continued production of TTR from the liver cannot therefore be sufficiently suppressed. Additional precise in vivo studies and other TTR gene therapy methods should be considered.
14.6.2 Gene Conversion Therapy Chimeric RNA/DNA oligonucleotides (chimeraplasts) have been developed to facilitate correction of single-based mutations of episomal and chromosomal targets in mammalian cells (Yoon et al. 1996; Cole-Strauss et al. 1996). Chimeraplasts consist of short regions of correcting DNA bounded by long stretches of 20 -Omethyl RNA, hairpin loops, and GC clamps. Chimeraplasts were shown to cause a site-specific chromosomal correction or mutation in tissue culture cells and in vivo (Kren et al. 1997, 1998, 1999; Xiang et al. 1997; Alexeev and Yoon 1998). As a permanent and stable gene correction by chimeraplasts was demonstrated by clonal analysis at the level of the genomic sequence, and as shown by protein and phenotypic change (Alexeev and Yoon 1998), chimeraplast-mediated gene repair may be a powerful strategy for treatment of genetic diseases without the use of viral vectors. The strategy of using single-stranded oligonucleotides (SSOs) for gene therapy grew from studies that attempted to characterize and improve chimeraplasty (Gamper et al. 2000a, b). SSOs containing three phosphorothioate bonds at the 30 and 50 termini were more effective than the chimeraplasts in gene repair assays conducted with cell-free extracts and a yeast system (Gamper et al. 2000a, b). SSOs are significantly less expensive than chimeraplasts and far simpler to synthesize and
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purify, so they may be an invaluable resource for treating a variety of genetic diseases. To test the feasibility of gene therapy for FAP, via chimeraplasts and SSOs, that would halt production of variant TTR in the liver and retina, Nakamura et al. first applied chimeraplasts and SSOs to HepG2 cells secreting human wild-type TTR. They then demonstrated gene conversion by SSOs in rabbit eye expressing rabbit wild-type TTR and transgenic murine liver in which the intrinsic wild-type TTR gene was replaced by a TTR Val30Met gene (Fig. 14.2). Their results were promising, although, again, a perfect gene conversion could not be obtained. Although DNA replication plays an important role in achieving significant levels of gene conversion (Olsen et al. 2005; Wu et al. 2005; Engstrom and Kmiec 2007), many potential target tissues in vivo do not have high levels of DNA replication or cell division. To overcome this problem, gene therapy combined with SSOs and partial liver resection might be effective in treating genetic diseases of the liver such as FAP, since modest tissue damage with wound healing would activate DNA replication and cell division processes. These studies have not progressed to the point that a therapeutic agent for FAP can be predicted in the near future. Most important is the development of an animal model to test any new form of therapy including gene therapy in a timely fashion,
Fig. 14.2 Comparison of different gene therapy strategies. (a) Biological activity of antisense oligonucleotides (AS ONs). AS ONs block translation of the mRNA or induce its degradation by RNaseH. (b) Biological activity of ribozyme. Ribozyme possessing catalytic activity cleaves its target RNA. Once the target is cleaved, ribozyme dissociates and recycles itself. (c) Biological activity of siRNA which is produced from cleavage of longer dsRNA precursors by the Dicer or supplied exogenously. The guide strand is incorporated into the RNA-induced silencing complex (RISK), where it guides sequence-specific degradation of the target transcript. (d) Mechanism of targeted gene repair by single-stranded oligonucleotides (SSOs). SSOs create a ‘‘D loop’’ structure with its complement in the target site in the helix. Similar to the chimeraplast, a two-step repair process sequentially converts the targeted base pair
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because FAP is caused by so many different mutations with various phenotypes and usually has a slow but progressive course.
14.7
Other Possible Therapies
14.7.1 Radical Scavengers Advances in immunohistochemistry techniques and the development of new antibodies have increased the possibility of detecting injury caused by oxidative stress (Toyokuni et al. 1994). One of the histopathological hallmarks of Alzheimer’s disease is the occurrence of senile plaques that are found in the neocortex and hippocampus. The main constituent of the core of these plaques is a 40- to 43peptide sequence called ß-amyloid (Aß), which is capable of destabilizing calcium homeostasis and exhibits neurotoxic properties. In a number of studies, the cytotoxicity of Aß(1–40) and its active fragment Aß(25–35) could be suppressed by catalase and antioxidants, e.g., vitamin E and melatonin. Treatment of both cultured hippocampal neurons and synaptosomes with Aß(25–35) produced substantially increased concentrations of the lipid per oxidation product 4-hydroxy-2-nonenal (HNE). The neurotoxic effects of both Aß(25–35) and HNE could be prevented by a treatment with glutathione ethyl ester, whereas the antioxidant propyl gallate was effective against only Aß(25–35)-induced neurotoxicity (Mark et al. 1997). Aßresistant subclones of PC12 cells, unlike the parent cells, do not accumulate peroxides after Aß(25–35) treatment, because of an over expression of antioxidant enzymes (Sagara et al. 1996). Taken together, these data suggest that the neurotoxic effects of Aß are mediated by hydrogen peroxide and HNE. Proteases from polymorphonuclear leukocytes and macrophages are present in amyloid fibrils found in all types of systemic amyloidoses, and the release of free radicals from these cells may be involved in amyloid formation (Skinner et al. 1986; Stone et al. 1993). Hydroxyl radicals readily induce lipid peroxidation, so that products of this reaction should be present in amyloid deposits if free radicals are involved in the process. Several reports of accumulation of aldehydic lipid pe oxidation products during oxidative stress conditions have been published (Benedetti et al. 1980; Siems et al. 1989). HNE is a lipid peroxidation product that exhibits several toxic properties: cell and enzyme stimulation, and enzyme inactivation and modification. By using a purified polyclonal antibody to HNE, this toxic metabolite can be detected immunohistochemically in tissues (Toyokuni et al. 1994; Uchida et al. 1995). We reported that the HNE adduct was present in amyloid deposits in tissues from FAP patients, and that levels of thiobarbituric acid reactive substances (TBARS) and protein carbonyl were elevated in amyloid-rich biopsy samples from FAP patients compared with levels in nonamyloidotic samples (Ando et al. 1997, 1998). Nyhlin et al. reported the presence of advanced glycation end products,
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some of which were formed via oxidative stress (Nyhlin et al. 2000). These results suggest that oxidative stress is significantly involved in the amyloid formation process. In Sweden, 20 FAP Va130Met patients completed the 6-month study period of scavenger treatment (vitamin C, 1 g, three times daily, vitamin E, 0.1 g, three times daily and acetylcysteine, 0.2 g, three times daily). They were evaluated clinically and by immunohistochemical measurement of hydroxynonenal (HNE), a product of lipid peroxidation, in biopsy specimens. Clinically, no differences were found for FAP patients, but an increased nutritional status, measured by a modified body mass index (mBMI) was noted for transplanted patients (Suhr et al. 2001). The results were inconclusive regarding the effect of radical scavengers because the reasonable duration of administration and doses of radical scavengers could not be determined.
14.7.2 Tauroursodeoxycholic Acid (Tudca) TUDCA is a unique natural compound that acts as a potent antiapoptotic and antioxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with TTR deposition in FAP, Macedo et al. investigated the possible TUDCA therapeutical application in this disease. Semi-quantitative immunohistochemistry and western blotting revealed that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER stress markers BiP and eIF2a, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most importantly, TUDCA treatment significantly reduced TTR toxic aggregates by as much as 75%. Since TUDCA has no effect on TTR aggregation in vitro, this finding points to the in vivo modulation of TTR aggregation by cellular responses, such as by oxidative stress, ER stress and apoptosis. They concluded that TUDCA is a candidate as a safe drug in prophylactic and therapeutic measures in FAP.
14.8
Summary
For an optimal effective therapy for TTR related FAP, several projects based on TTR metabolism and amyloid formation mechanisms are now ongoing both in basic research and in clinical trials. While several projects may be promising, we must wait for a while because some are just now beginning and in vivo experiments require more than a year to judge the changes in TTR and amyloid deposition. Although each therapeutic project is progressing independently, combined therapies may have the greatest potential.
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Acknowledgments The authors’ work was supported by grants from the Amyloidosis Research Committee, the Pathogenesis, Therapy of Hereditary Neuropathy Research Committee, the Surveys and Research on Specific Disease, the Ministry of Health and Welfare of Japan, and the Charitable Trust Clinical Pathology Research Foundation of Japan; Research for the Future Program Grant; and Grants-in-Aid for Scientific Research (B) 15390275, (B) 17458276, and from the Ministry of Education, Science, Sports and Culture of Japan.
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beta-pleated sheet structures in postmortem human neurodegenerative disease brains. Am J Pathol 159:937–943 Sebastiao MP, Merlini G, Saraiva MJ et al. (2000) The molecular interaction of 40 -iodo-40 deoxydoxorubicin with Leu-55Pro transthyretin ‘amyloid-like’ oligomer leading to disaggregation. Biochem J 351:273–279 Sekijima Y, Dendle MA and Kelly JW (2006) Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 13:236–249 Siems W, Kowalewski J, Werner A et al. (1989) Radical formation in the rat small intestine during and following ischemia. Free Radic Res Commun 7:347–353 Skinner M, Lewis WD, Jones LA et al. (1994) Liver transplantation as a treatment for familial amyloidotic polyneuropathy. Ann Intern Med 120:133–134 Skinner M, Stone P, Shirahama T et al. (1986) The association of an elastase with amyloid fibrils. Proc Soc Exp Biol Med 181:211–214 Skovronsky DM, Zhang B, Kung MP et al. (2000) In vivo detection of amyloid plaques in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA 97:7609–7614 Steensma DP (2001) ‘‘Congo’’ red: out of Africa? Arch Pathol Lab Med 125:250–252 Stone P, Camistol J, Abraham CR et al. (1993) Neutrophil proteases associated with amyloid fibrils. Biochem Biophys Res Commun 30:130–136 Suhr O, Danielsson A, Rydh A et al. (1996) Impact of gastrointestinal dysfunction on survival after liver transplantation for familial amyloidotic polyneuropathy. Dig Dis Sci 41:1909–1914 Suhr OB, Lang K, Wikstrom L et al. (2001) Scavenger treatment of free radical injury in familial amyloidotic polyneuropathy: a study on Swedish transplanted and non-transplanted patients. Scand J Clin Lab Invest 61:11–18 Sullenger BA and Cech TR (1994) Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing. Nature 371:619–622 Takei Y, Ikeda S, Hashikura Y et al. (1999) Partial-liver transplantation to treat familial amyloid polyneuropathy: Follow-up of 11 patients. Ann Intern Med 131:592–595 Tanaka K, Yamada T, Ohyagi Y et al. (2001) Suppression of transthyretin expression by ribozymes: A possible therapy for familial amyloidotic polyneuropathy. J Neurol Sci 183:79–84 Terazaki H, Ando Y, Fernandes R et al. (2006) Immunization in familial amyloidotic polyneuropathy: counteracting deposition by immunization with a Y78F TTR mutant. Lab Invest 86: 23–31 Terazaki H, Ando Y, Misumi S et al. (1999) A novel compound heterozygote (FAP ATTR Arg104His/ATTR Val30Met) with high serum transthyretin (TTR) and retinol binding protein (RBP) levels. Biochem Biophys Res Commun 264:365–370 Tojo K, Sekijima Y, Kelly JW et al. (2006) Diflunisal stabilizes familial amyloid polyneuropathyassociated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis. Neurosci Res 56:441–449 Tokuda T, Kondo T, Hanaoka N et al. (1998) A selective transthyretin-adsorption column for the treatment of patients with familial amyloid polyneuropathy. Amyloid 5:111–116 Toyokuni S, Uchida K, Okamoto K et al. (1994) Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate. Proc Natl Acad Sci USA 91:2616–2620 Uchida K, Itakura K, Kawakishi S et al. (1995) Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies. Arch Biochem Biophys 324:241–248 Westermark GT and Westermark P (2000) Endocrine amyloid—a subject of increasing interest for the next century. Amyloid 7:19–22 Wu H, Lima WF, Zhang H et al. (2004) Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. J Biol Chem 279:17181–17189 Wu XS, Xin L, Yin WX et al. (2005) Increased efficiency of oligonucleotide-mediated gene repair through slowing replication fork progression. Proc Natl Acad Sci USA 102:2508–2513 Xiang Y, Cole-Strauss A, Yoon K et al. (1997) Targeted gene conversion in a mammalian CD34enriched cell population using a chimeric RNA/DNA oligonucleotide. J Mol Med 75:829–835
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Chapter 15
Liver Transplantation for Transthyretin Amyloidosis Bo-Go¨ran Ericzon, Erik Lundgren and Ole B. Suhr
Abstract Liver transplantation has until now proved to be the only treatment available that halts the progression of hereditary transthyretin (TTR) associated amyloidosis. The rationale behind the procedure is to replace the liver producing variant TTR with one that produces wild type TTR only, and thereby cease the production of amyloidogenic TTR (ATTR). Even though the transplantation does not improve the patient’s symptoms, the progression of the disease comes to a halt for a majority of patients. However, unforeseen complications after the transplantation have emerged, in particular a continuous amyloid formation in the heart observed in non-ATTR Val30Met mutations. Thus, combined liver and heart transplantation has been performed in selected cases. Since the ATTR liver functions normally apart from a synthesis of the variant TTR, utilisation of ATTR-amyloid patients’ livers for transplantation of liver disease patients has been performed. In a few patients, development of amyloid disease has been reported, but the procedure remains an important source of organs, especially for patients with hepatocellular cancer. Keywords Transthyretin, Liver transplantation, Heart complication, Amyloidosis
Abbreviations TTR ATTR FAP PND-score
Transthyretin Amyloidogenic variant transthyretin Familial amyloidotic polyneuropathy Polyneuropathy disability score
O.B. Suhr (*) Department of Medicine, Section for Gastroenterology and Hepatology, Umea˚ University Hospital, SE 901 85 Umea˚, Sweden e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_15, # Springer‐Verlag Berlin Heidelberg 2009
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FAPWTR mBMI ECG MIBG 99 Tc-DPD SSA SAP
15.1
Familial amyloidotic polyneuropathy world transplant registry Modified body mass index Electrocardiogram Iodine-123 metaiodobenzylguanidine scintigraphy 99 Tc-3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy Senile systemic amyloidosis Serum amyloid P component
Background
The identification of mutated transthyretin (TTR) as the amyloidogenic protein in hereditary TTR amyloidosis (Saraiva et al. 1984), and the knowledge that the circulating TTR was mainly synthesised by the liver initiated the proposal that a liver transplantation should cease the production of the amyloidogenic mutant TTR and replace it with the wild type TTR, and thereby halt amyloid formation. The suggestion to try liver transplantation as a treatment for a familial amyloidotic polyneuropathy (FAP) patient was put forward by the late Prof. Go¨sta Holmgren and Dr Steen from Umea˚ University Hospital, Sweden to Prof. Carl Gustaf Groth and Bo-Go¨ran Ericzon at Karolinska Institute in Stockholm, Sweden in 1989. Several Swedish FAP-patients suffering from the most common neuropathic TTR mutation, ATTR Val30Met, were considered as candidates for the procedure, and the first patient was accepted and underwent liver transplantation in 1990. Later the same year, a FAP patient of Portuguese descent underwent transplantation at Sahlgrenska Hospital, Go¨teborg, Sweden. Both transplantations were successful. Several patients also underwent transplantation over the following years with encouraging results (Holmgren et al. 1993), including almost complete disappearance of mutated TTR from the circulation (Holmgren et al. 1991). Until 1995, practically all patients with the FAP ATTR Val30Met who presented without any contraindications for the operation were accepted for transplantation. However, it soon became obvious that patients in a depleted nutritional status with severe gastrointestinal complications were poor candidates with an unacceptable high mortality and morbidity (Suhr et al. 1995) as serious circulatory complications during the transplantation accounted for several fatalities (Suhr et al. 1997b). Therefore the transplantation policy changed, and patients in a depleted nutritional status were not accepted, and patients with severe autonomic dysfunction were regarded as high risk patients especially if they suffered from advanced disabling disease. The initial hope of an improvement of the patients’ neuropathy and gastrointestinal complications after transplantation was not fulfilled. Several investigations disclosed that no improvement of the patient’s symptoms was noted after transplantation. Therefore, an early intervention was advocated, before the patient’s neurological status had deteriorated and other complications had supervened
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(Adams et al. 2000; Bittencourt et al. 2002; Parrilla et al. 1997; Perdigoto et al. 2003; Sharma et al. 2003; Suhr et al. 1995; Yamamoto et al. 2007b). In addition, it soon became obvious that the outcome after transplantation varied with genotype, and thus cardiomyopathy appeared to progress in spite of the transplantation in several ATTR-mutations (Pomfret et al. 1998; Stangou et al. 1998). A combined heart and liver transplantation was favorable for some genotypes to halt a progressing cardiomyopathy (Sharma et al. 2003). Today, 18 years after the first transplantation, the outcome for an individual patient is still difficult to predict. Even though the initial hopes of improvement for ATTR-amyloidosis after liver transplantation have not been fulfilled, the procedure has proved to be the only available treatment in which the progression of the disease in the majority of patients has been halted. With time, better selection of patients and better understanding of the special complications encountered during and after transplantation of ATTR-amyloid patients have also improved survival (Fig. 15.1), with a five year survival time approaching 90% according to the FAP World Transplant Registry (FAPWTR,: www.fapwtr.org). Long-term follow-up and the initial experience of transplanting patients with multiple complications, has clearly indicated that patients should be transplanted at an early stage, before they are marked by multiple complications, not only to ensure a reasonable chance of survival, but also to ensure an acceptable quality of life (Jonse´n et al. 2001).
15.2
Patient Selection and Outcome After Transplantation
Since TTR amyloidosis is a systemic amyloidosis, a pre-transplant evaluation of the patient needs to include an examination of several organ functions in addition to those normally performed. Thus, patients with predominantly peripheral neurological
Actuarial survival, %
100 80 60
1990-94 (n=146) 1995-99 (n=463) 2000-04 (n=555) 2005-2007 (n=277) Overall (n=1441)
40 20 0 0
1
2
3
4
5
Years after transplantation
Fig. 15.1 Patients’ survival after liver transplantation. Overall survival and survival in relation to era. From the survival curves it is evident that results have improved with time, reflecting an initial learning phase. Factors contributing to the improvements are probably better patient selection as well as an improved post-transplant care (with permission from the FAPWTR)
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symptoms will often also have other organ systems such as the autonomic nervous system, the heart, gastrointestinal tract, kidneys and bladder affected. Serious unexpected complications can arise after transplantation if the pre-transplant examinations have failed to take the systemic aspects of the disease into account.
15.2.1 Demographic Data and ATTR-Mutation Expectedly, the age of the patient was clearly related to long-term, but not to shortterm (<6 months) survival of Swedish transplanted patients (Suhr et al. 2002). Duration of disease has expectedly also had an impact on survival (Fig. 15.2), whereas gender was not found to have an impact on the outcome. The survival rate of non-ATTR Val30Met transplanted patients is significantly inferior to that of ATTR Val30Met patients according to the FAPWTR registry (Fig. 15.3) and several reports (Dubrey et al. 1997; Singer et al. 2005; Stangou et al.
Actuarial survival, %
100 80 p< 0.001
60 40
Dur ≤7yrs (n=1045) Dur >7yrs (n=133)
20 0 0
2
4 6 8 Years after transplantation
10
Fig. 15.2 Patient survival in relation to duration of symptoms. All mutations (with permission from the FAPWTR)
Actuarial survival, %
100 80 60
p<0.001
40
Val30Met (n=1198)
20
Non Val30Met (n=175) 0 0
2
4 6 8 Years after transplantation
10
Fig. 15.3 Patient survival for ATTR Val30Met and non-ATTR Val30Met mutations (with permission from the FAPWTR)
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1998). The concern for those patients has especially been heart complications after transplantation (Herlenius et al. 2004).
15.2.2 Amyloid Deposits We always secure the diagnosis of TTR amyloid disease by detection of amyloid deposits in Congo red stained biopsy specimens examined in polarised light. This is not required in all centres, but with the knowledge of the high number of asymptomatic carriers in the Swedish population (Holmgren et al. 1994), there is a risk of transplanting a carrier with a polyneuropathy caused by another disease, if the diagnosis is not substantiated by a positive biopsy. For patients with suspected heart amyloidosis, heart biopsy may be needed to secure the diagnosis (O’Hara and Falk 2003). Amyloid scintigraphy, utilising Serum Amyloid P component (SAP) has been carried out before and after transplantation, and regression of amyloid deposits has been noted in up to 50% of the patients (Holmgren et al. 1993; Rydh et al. 1998). However, the heart is not visualised by SAP-scintigraphy, and the technique appears to be less sensitive for TTR-amyloidosis than for amyloid A protein (AA) and amyloid light chain protein (AL)-amyloidosis; diagnostic sensitivity 90% for AL and AA, 48% for TTR amyloidosis (Hazenberg et al. 2006). In two studies of biopsy specimens from peripheral tissues, stabilisation and for some patients regression of amyloid deposits, was noted after transplantation (Haagsma et al. 2007; Tsuchiya et al. 2008). Since SAP-scintigraphy has a relatively low sensitivity in TTR amyloidsis and does not visualise amyloid deposits in the heart, in our experience its value in pre-transplant evaluation of the patients is limited. In the follow up of the patients, we have not found a relationship between scintigraphic amyloid regression and clinical improvement.
15.2.3 Neuropathy 15.2.3.1
Peripheral Neuropathy
The first manifestations of peripheral neuropathy are the affects on the small myelinated and unmyelinated nerve fibres that regulate temperature and sensation of pain. Later on, affects on the larger myelinated fibres are noted. Tests of pain and sensation of temperature with quantitative sensory testing are useful to substantiate loss of nervous function, before more conventional electrophysiological techniques such as nerve velocity conduction studies, which indicate that large-fibre involvement has become abnormal (Heldestad and Nordh 2007). In a recent study, sympathetic skin response testing was useful to detect early neuropathy in ATTR Val30Met carriers. The test had a positive and negative predictive
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value of 0.83, and appears to be a useful indicator of early disease (Conceicao et al. 2008). For diagnosis and follow-up on the development of nerve damage, electrophysiological studies of nerve function are useful; however, for evaluation of the patients’ suitability for transplantation their value is limited. The clinical evaluation of patients’ functional status will determine whether he/she is a candidate for transplantation. The degree of peripheral neuropathy was not related to patient survival in an examination of transplanted Swedish patients (Suhr et al. 2002). Their neuropathy was graded according to a neurological performance scale, the PND-score, going from zero that denotes a patient who walks without any difficulties to IV: a patient bedridden or in a wheelchair. In a series of transplanted patients of French origin, another score, the Norris score, was used and found to predict survival. This is a neurological score that primarily has been utilised to grade patients with amyotrophic lateral sclerosis (ALS), and the score is mainly focused on motor function. The Norris score had a significant correlation to survival after transplantation, and a score below 55 has been regarded as a contraindication for transplantation (Adams et al. 2000). It is generally accepted that patients with advanced neuropathy, PNDscore of more than IIIA (walking with help of two sticks or crutches) are not suitable candidates for transplantation. Long-term evaluation of neuropathy after transplantation for ATTR Val30Met and other mutations such as the ATTR Tyr71Ala has disclosed that for the majority of patients, the progression of nerve damage comes to a halt (Adams et al. 2000; de Carvalho et al. 2002; Kobayashi et al. 2003; Ramirez et al. 2000; Shimojima et al. 2008; Tashima et al. 1999). A report on reinnervations (Ikeda et al. 1997) also reports of progression after liver transplantation, especially for those with an advanced stage of the disease (Adams et al. 2000; Takei et al. 2005; Yamamoto et al. 2007b). For non-ATTR Val30Met mutations the outcome has varied (GarciaHerola et al. 1999). So far, it has not been possible to identify those individuals at risk for progression with certainty, but advanced polyneuropathy appears to be a risk factor.
15.2.3.2
Autonomic Neuropathy
Autonomic disturbances with orthostatic hypotension, urinary bladder dysfunction and impotence are commonly found in FAP patients, and an examination of bladder emptying should be performed. Urinary incontinence has been suggested to be a contraindication for transplantation (Adams et al. 2000). Defective regulation of heart rate and blood pressure are serious manifestations of the disease, and may give rise to fatal hypotension during transplantation. An examination of heart rate variability and blood pressure in supine and upright position gives important information on the autonomic nervous system regulation of heart rate and blood pressure. Scintigraphic examination by Iodine-123 metaiodobenzylguanidine (MIBG) of the heart has been utilised to detect sympathetic denervation of the heart (Coutinho et al. 2004; Delahaye et al. 1999).
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In our experience, investigation of the autonomic regulation of heart rate and blood pressure is needed before transplantation, and a combination of autonomic dysfunction and conduction disturbances (SA and AV block II – III) on Holter ECG recordings is an indication for insertion of a permanent pacemaker. Autonomic dysfunction is not a contraindication in our experience, but a risk factor, and should be taken into account in the global assessment of the patient for transplantation. The reported outcome after transplantation has varied, but most report a status quo. Long-term evaluation of heart rate variability has not shown any improvement, and disturbances caused by arrhythmia has often prevented long term evaluation (Delahaye et al. 2006; Hornsten et al. 2008; Suhr et al. 1997a). In a Japanese report, MIBG heart scintigraphy for sympathetic heart denervation showed signs of reinnervations after transplantation, but a later study was not able to confirm this finding (Ando et al. 1995; Delahaye et al. 2006). No systematic examination of bladder function or sexual function has been published, but a report of successful pregnancies as well as fatherhood of transplanted patients has emerged (Holmgren et al. 2004).
15.2.4 Heart Complications Heart complications in ATTR amyloidosis are primarily those of a restrictive cardiomyopathy, but often a pronounced hypertrophy of the heart walls is noted, which is difficult to differentiate from a hypertrophic cardiomyopathy (Morner et al. 2005). Echocardiography is a valuable tool to detect heart dysfunction in amyloid related cardiomyopathy, and has been utilised extensively for evaluation of patients for liver transplantation (Delahaye et al. 2006). The identification of a restrictive pattern is typical for amyloid heart disease. However, restrictive pattern and functional impairment may be difficult to visualise with routine echo-cardiographic techniques. Thus, right sided heart catherisation with volume expansion unmasked a latent restrictive pattern in 56% of patients submitted for liver transplantation in a French series (Algalarrondo et al. 2008), and appears to be a valuable tool to detect impeding heart failure in patients submitted for liver transplantation. It also demonstrates the need for improved diagnostic tools. Several techniques that appear promising have emerged. Echo-cardiography with one and two-dimensional strain measurements has proven to be a sensitive tool to detect early abnormalities in the heart, before septal thickness or other abnormalities were noted (Bennani et al. 2008; Lindqvist et al. 2008; Lindqvist et al. 2006). Magnetic resonance examination showing a late gadolinium enhancement is a typical finding in heart amyloidosis (Perugini et al. 2006; Vogelsberg et al. 2008). Likewise, good results have also been achieved with 99Tc-3,3-diphosphono1,2-propanodicarboxylic acid scintigraphy (99Tc-DPD) (Perugini et al. 2005; Puille et al. 2002), that visualised amyloid deposits in the heart. However the place of the techniques in the evaluation of heart complications during and after transplantation for TTR-amyloidosis has not been settled.
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B-natriuretic peptide (BNP) was elevated in patients with abnormal echo-findings, and appears to be a sensitive tool to diagnose latent or mild heart failure. It has proven to be valuable for follow-up purposes after treatment for AL amyloidosis, and may be of similar value for liver transplanted TTR-amyloid patients (Palladini et al. 2003; Suhr et al. 2008). Troponin T was not a sensitive marker in ATTR Val30Met amyloidosis in our experience, compared with findings in AL-amyloidosis (Suhr et al. 2008). Since liver transplantation may fail to halt the progression of heart complications, cardiomyopathy is a serious complication, and a heart transplantation must be considered in patients with cardiomyopathy, especially for non-ATTR Val30Met mutations (Arpesella et al. 2003; Nardo et al. 2004; Rapezzi et al. 2006). For patients in whom heart transplantation is considered, a heart biopsy to substantiate the diagnosis is required at our centre. Conduction disturbances with atrio-ventricular and/or sino-atrial blocs are common, and may lead to sudden death. Thus, an investigation for heart arrhythmia is mandatory before transplantation, and since the arrhythmia may be intermittent, a 24 h Holter ECG-recording is required. Conduction disturbances can successfully be treated by a pacemaker. It should be noted, that the combination of autonomic disturbances and conduction disturbances probably increase the risk for fatal arrhythmia, since a decreased sympathetic function diminishes the autonomic response to bradycardia. Thus, the presence of AV-block grade II or sino-atrial blocks are an indication for the insertion of a permanent pacemaker, even without symptoms of arrhythmia. However, in patients without conduction disturbances, insertion of a permanent pacemaker before transplantation is not required, but a temporary pacemaker can be of value to treat hypotension during transplantation, since the sympathetic denervation of the heart may diminish the response to inotrope drugs. ECG abnormalities, mimicking those of coronary heart disease can be found in heart amyloidosis, as well as the classical low-voltage ECG of a hypertrophic cardiomyopathy. Even though coronary heart disease is rarely found in FAP-patients, coronary angiography is necessary if coronary heart disease is suspected because neither exercise ECG (Juneblad et al. 2004) nor scintigraphy, in our experience, are able to distinguish between coronary heart disease and amyloid cardiomyopathy. However, exercise ECG gives important information on the patients’ ability to increase their heart rate and blood pressure during exercises, which are important markers of the autonomic nervous system’s regulation of the cardiovascular function. In summary, a 24-h Holter ECG is required to detect arrhythmia, as well as echocardiography to disclose diastolic and/or systolic heart failure and hypertrophic cardiomyopathy. Exercise ECG gives valuable information on the patient’s working capacity and the ability to increase heart rate and blood pressure during stress. If coronary heart disease is suspected, a coronary angiography is in our experience the only investigation that can distinguish coronary heart disease from cardiomyopathy with pseudo-infarct ECG-pattern. An isolated heart transplantation has been carried out in a patient homozygous for the ATTR Val122Ile mutation without progression of cardiomyopathy after a follow-up of 3 years (Hamour et al. 2008). However, rapid amyloid cardiomyopathy was observed in a Danish patient who underwent isolated heart transplantation for
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the ATTR Leu111Met mutation (personal communication Svendsen, I, Copenhagen, Denmark). For patients with ATTR amyloid cardiomyopathy and who suffer from heart failure, a combined or sequential heart and liver transplantation should be considered (Pilato et al. 2007). One of the main concerns after liver transplantation for ATTR amyloidosis has been the development of cardiomyopathy. The initial reports were all on non-ATTR Val30Met patients (Dubrey et al. 1997; Stangou et al. 1998), but later it was also observed in ATTR Val30Met patients (Olofsson et al. 2002). In selected cases, a combined liver and heart transplantation appears to be a reasonable solution, and good results have recently been reported from several centres (Arpesella et al. 2003; Grazi et al. 2003; Pilato et al. 2007; Sharma et al. 2003; Svendsen et al. 1999). Another unexpected post-transplant complication has been the development of heart arrhythmia after transplantation (Delahaye et al. 2006; Hornsten et al. 2004). In long term follow-up of our patients, up to 50% had developed heart arrhythmia necessitating pacemaker treatment (Hornsten et al. 2004). Investigations into the mechanism behind post-transplant development of cardiomyopathy indicate, that wild type TTR in some cases continuously assembles into amyloid fibrils (Liepnieks and Benson 2007; Yazaki et al. 2000, 2007). Why this is the case for some ATTR mutations and also for some ATTR Val30Met is not understood. It can be speculated that advanced age is a risk factor for cardiomyopathy, as non-mutated TTR has a propensity to form amyloid in experimental systems and in vivo late in life. Senile systemic amyloidosis (SSA) affects elderly individuals with cardiac deposits, in which the peptide is truncated. This form is due to non-mutated TTR (Christmanson et al. 1991; Westermark et al. 1990). It would be interesting to see whether such cleaved TTR peptides also occur in posttransplant aggregates. Another possibility is that ATTR deposits form due to a nucleation process. This is well known to occur for several other forms of amyloidosis, and even crossseeding might occur (Westermark 2005). However, in the case of TTR, seeding experiments in vitro or in mouse models have failed to demonstrate nucleation of TTR under the experimental conditions used (Hurshman et al. 2004; Wei et al. 2004). Because of its clinical importance, continued studies are necessary to understand whether post-transplant TTR deposits are formed on existing deposits, or whether the mechanism for amyloid formation involved in SSA is accelerated after liver transplantation and post-transplant treatment.
15.2.5 Gastrointestinal Complications Malnutrition is a well-known complication of FAP. The mechanism behind the disturbances is not known with certainty, but is generally suggested to be caused by autonomic disturbances. However, even though autonomic denervation is known to affect ventricular emptying and defecation, a denervated small intestine has a well preserved capacity to absorb nutrients, as is the case for small intestinal transplanted
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patients; thus autonomic denervation alone cannot explain the severe malabsorption found in FAP-patients. Delayed ventricular emptying, with symptoms of nausea and vomiting, as well as altered bowel habits with constipation or diarrhoea are common findings especially in FAP ATTR Val30Met (Steen and Ek 1983). In the evaluation of patients for transplantation, an investigation of ventricular emptying with scintigraphy or endoscopy is important. In addition, bacterial contamination of the small bowel, giving rise to intermittent severe diarrhoea, can be evaluated by small intestinal culture or by breath tests such as the hydrogen breath test. Severe malabsorbtion of fat and bile acids can be examined by the faecal fat test and 75Se Homocholic-acidtaurine (SeHCAT) scintigraphy, respectively. Identification of these complications is important, since they can be alleviated by antibiotic treatment, bile acid sequestrates and/or diet. In addition, the presence of marked gastrointestinal dysfunction is a predictor for post-transplant complications, in our experience (Suhr et al. 1996; Suhr et al. 2003). An examination of the first non-selected 20 transplanted Swedish FAP-patients revealed that nutritional status, measured by the modified body mass index (mBMI) was a strong predictor for survival; thus only one patient with a mBMI below 600 survived the procedure during the follow-up period of 5 years (Suhr et al. 1995). mBMI that is calculated by multiplying body mass index by s-albumin in grams per litre has also been shown to be a good predictor for survival for non-transplanted patients (Suhr et al. 1994). In the evaluation of the patients for transplantation, an examination of gastric emptying is useful, as it can be compromised without overt symptoms, and may delay recovery after transplantation (Suhr et al. 2003). From our experience, gastrointestinal disturbances with marked malabsorption, and especially a low mBMI (<600), are predictors of a poor survival after transplantation, and are, at our centre, a contraindication for transplantation. From the FAPWTR (www.fapwtr.org), malnutrition was also found to be related to decreased survival after transplantation. Few studies of gastrointestinal function after transplantation have emerged. We found a normalisation of enteric neuroendocrine cell depletion after transplantation, but it was not associated with any symptomatic improvement (Anan et al. 2000). Even though nutritional status and symptoms for many patients have improved after transplantation, objective measurements have shown a stabilisation of the patients’ gastrointestinal function, and generally no improvement (Lang et al. 2000; Suhr et al. 2003).
15.2.6 Kidney Complications Amyloid deposits in the kidneys are nearly always found in TTR-amyloidosis. Proteinuria and decreased kidney function are common expressions of the disease (Lobato 2003; Lobato et al. 1998). However, even though nearly 30% of the kidney
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function is lost during the transplantation procedure, the long term outcome has been favourable (Nowak et al. 2005; Snanoudj et al. 2004). An assessment of the kidney function by clearance determination by Chrome EDTA- or Iohexol- clearance is mandatory at our centre. We only perform kidney biopsy if a kidney transplant is under consideration to substantiate the diagnosis of amyloid kidney disease. With clearance below 30 ml min 1 m 2 a combined liver and kidney transplantation should be considered (Stangou and Hawkins 2004). The liver transplantation procedure, and the following immune-suppression leads to an initial deterioration of the kidney function, but thereafter a stabilisation is noted, and the amyloid content in kidney biopsies remains unchanged (Snanoudj et al. 2004). Reduction of protein loss has been reported (Carvalho et al. 2000), and transplanted FAP-patients kidney function appeared even to remain more stable than that of patients transplanted for liver diseases (Nowak et al. 2005). It is likely that the transplantation halts amyloid formation in the kidney, and thereby leads to improved kidney function that counteracts the toxicity of immunosuppressive treatment.
15.2.7 Central Nervous and Eye Complications A few patients having rare mutations with central nervous manifestations such as ATTR Tyr69His have had their liver transplanted. However, the choroid plexus of the CNS produces TTR, thus, progression of CNS symptoms, such as dementia and cerebral hemorrhage have continued after transplantation in mutations such as ATTR Leu12Pro and Gly53Glu (De Carolis et al. 2006; Otto et al. 2008). Even though favourable results have been reported for CNS-manifestations in patients with the ATTR Tyr114Cys mutation (Yamashita et al. 2008), patients suffering from CNSamyloidosis are generally not considered candidates for liver transplantation. Expectedly, amyloid deposits in the eyes (Fig. 15.4), caused by the continuous variant TTR production by the retina have been reported (Ando et al. 1996). Amyloid deposition in the vitreous body of the eye can successfully be removed, but a concern is the development of glaucoma, often asymptomatic, and induced by amyloid deposits in the anterior chamber. Thus, a regular control of the eyes’ pressure is recommended (Kimura et al. 2003; Sandgren et al. 2008).
15.2.8 Summary and a Proposed Algorithm for Patient Selection Liver transplantation for TTR-amyloidosis is an accepted procedure, but caution must be exerted when patients are selected for the procedure. An algorithm for selection of patients has been presented by Dr Arie Stangou (Fig. 15.5) (Stangou et al. 2008). It emphasises the problems in patient selection with regard to cardiomyopathy, neurological impairment and ATTR mutation. However, from our point of view, patients over the age of 60, without overt cardiomyopathy (inter-ventricular
Fig. 15. 4 Amyloid deposits in the vitreous body of the eye (foto Ola Sandgren) Diagnosis of FAP
TTR genotype Non–ATTR Val30Met
ATTR Val30Met
Echo, BNP, histology Age Early onset < 50 years
Late onset > 50 years
Cardiac amyloid
Echo, BNP
No cardiac amyloid
No cardiac amyloid
Systemic disease
Cardiac amyloid
Age < 60 PND score £ IIIB
Consider LT
Yes
Age > 60 PND score ³ IIIB No transplant
Consider combined or sequential LHT
No
Yes
No
Age < 60 PND score £ IIIB Vigilant annual monitoring Consider LT but make provisions for possible sequential HTx Consider heart Tx (Ile 122)
Fig. 15.5 Adopted from Algorithm of the King’s College Hospital proposed guidelines for liver transplantation in the management of familial amyloid polyneuropathy. Presented at the VII International symposium on Familial Amyloid polyneuropathy, London, UK 2008 (with permission from Arie Stangou). TTR transthyretin, ATTR amyloidogenic transthyretin variant, BNP B-natriuretic peptide, PND polyneuropathy disability score, LT liver transplantation, LHT liver and heart transplantation
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septal thickness below 15 mm), and well preserved systolic and diastolic function) may well be candidates for liver transplantation. Besides, cardiomyopathy may develop after transplantation in patients under the age of 50, even without signs of heart amyloidosis before transplantation; this has been the case for ATTR Val30Met patients and several other patients carrying non-ATTR Val30Met mutations such as the ATTR Glu42Gly and Gly47Glu (Olofsson et al. 2002; Otto et al. 2008; Stangou et al. 1998). With careful selection of patients for transplantation, their expected survival is better than that of non-transplanted historical controls (Suhr et al. 2005). The identification of different types of amyloid fibrils in ATTR Val30Met patients and their relationship with age at onset and presence of cardiomyopathy raises the hope, that the type of amyloid fibril in the individual patient could be linked to the development of post-transplant cardiomyopathy, and that it may be an important tool for prediction of the outcome of transplantation. However, this needs to be substantiated in future studies (Ihse et al. 2008).
15.3
The Transplantation Procedure
Most commonly, liver transplantation is performed in patients with end stage liver disease having typical complications of liver cirrhosis such as; portal hypertension with varices, ascites and failing metabolic liver function. Common indications are hepatitis C cirrhosis, primary biliary cirrhosis and metabolic liver disorders causing damage to the liver such as alfa-1 antitrypsin deficiency, Wilson’s disease and thyrosinemia. Some of the metabolic liver disorders do not cause end stage liver disease, but may result in death of the patient due to complications elsewhere, thus familial hypercholesterolemia may cause early and significant cardiovascular disease and primary hyper oxalosis may result in end stage renal failure. Familial amyloidotic polyneuropathy belongs to the latter. As mentioned above, liver transplantation for FAP should be performed early in the course of the disease in order to optimise survival after transplantation, and for the patient to benefit more from the procedure since liver transplantation mainly halts the progress of the disease (Ericzon et al. 1995; Suhr et al. 2005). In the early era of liver transplantation for FAP, the negative impact on the progress of the disease during waiting time was often underestimated, and the patient was usually waiting longer than other patients with end stage liver disease. This was in particular seen when allocating organs according to the MELD-system (Forman and Lucey 2001; Wiesner et al. 2003). The FAP patient would not easily qualify for transplantation since they would lack signs of progressing end stage liver disease. Therefore, it is generally accepted that patients suffering from FAP need to be evaluated for timing of transplantation separately from patients with cirrhosis, for instance. Surgically, liver transplantation for FAP is straight forward and a relatively easy procedure, since signs of cirrhosis with portal hypertension and severe coagulopathy is absent. The intra-operative challenge is rather related to the FAP patient’s
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autonomic dysfunctions such as arrhythmia and cardiomyopathy, and the difficulties to maintain an adequate myocardial function during the procedure (Suhr et al. 1997b; Viana Jda et al. 1999). These patients often lack the ability to compensate for hypotension with increasing heart-rate and most commonly a pacemaker is used temporarily during the operation procedure in order to be able to increase the pace of the heart. Some patients already have a permanent pacemaker placed preoperatively. In traditional liver transplantation i.e. orthotopic liver transplantation the native diseased liver is removed together with a segment of the retro-hepatic vena cava. With this technique the inferior vena cava is clamped during the anhepatic phase of the transplant procedure. This can readily be done in patients with pre-existing portal hypertension with several collaterals developed for the return of the blood from the lower part of the body to the heart. In order to minimise the effect of this caval clamping in recipients with minimal or no collaterals, a veno-venous by-pass pump is routinely used at many centres, pumping the blood from the splanicus via a portal vein catheter and from the inferior vena cava via the saphenous vein into the jugular or auxiliary vein and the heart. An alternative technique is to leave the retro-hepatic vena cava in place when removing the native liver, thus maintaining the caval blood flow during the anhepatic phase (Lerut et al. 1989). In many patients today and especially in patients without end stage liver disease such as TTR-amyloidosis, this technique is preferred. With this technique the supra hepatic vena cava of the graft is anastomosed to the hepatic vein entrance of the recipient, or alternatively the donor vena cava is anastomosed side to side to the recipient’s vena cava using temporary partial occlusion of the recipient’s vena cava. With this so called ‘piggy back’ technique no veno-veno by-pass pump is necessary. However, if the FAP liver is going to be used in a domino procedure (see below) it is generally believed that the use of a by pass pump is preferred. Normally the portal vein anastomoses is performed end to end as is the arterial anastomoses. The bile duct reconstruction is usually performed by an end to end cholodocho-choledocho stomi with or without a temporary stent in the biliary duct. The gallbladder is routinely removed from the transplanted liver. Post-operatively the patient will need to remain on life long immunosuppressant based on either Tacrolimus or Ciclosporin A in combination with steroids and sometimes Azatioprin or Mycofenylate Mofetyl. Rejection usually occurs in about 1/3 of the patients during the first months after transplantation. It is most often successfully treated by elevation of the base line immuno-suppression, and/or a bolus dose of steroids. The patients are usually sent home at the end of the first month after transplantation. The recovery process after liver transplantation for end stage liver disease is usually experienced very positively by the patients with a feeling of new strength and well-being, with improved capacity to handle daily work. The situation for the FAP patient is different since he or she first will suffer from a major surgical procedure, and later on only be able to return to the pretransplant condition, with the hope that progression of the disease symptoms has been halted.
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Domino Liver Transplantation
The domino procedure means that a liver that has to be removed from a patient who needs to undergo liver transplantation, in turn can be used for another patient with a liver disease necessitating liver transplantation. This procedure was first carried out in Portugal in 1995 (Furtado et al. 1997; Furtado 2003). In FAP the liver morphology and function are normal apart from the production of variant TTR. In spite of being inherited, FAP clinical symptoms never develop before the age of 15 and often only after the age of 30–50. In some individuals with the genetic trait, the disease will never manifest during the normal life span (Hellman et al. 2008; Holmgren et al. 1994). Once the disease starts to develop, the survival is often 10–13 years. Therefore, in theory, by correct selection of liver recipients, many patients should benefit from being transplanted with a domino FAP liver with minimal or no risk to have the disease transferred during their expected life span. In the early era of domino liver transplantation using FAP livers, mainly patients with extended liver tumours were considered. Today approximately 50% of domino recipients have a malignant disorder and the remaining have end stage liver disease. The survival of Domino recipients transplanted for liver tumour or other liver diseases are displayed in Fig. 15.6. As can be seen from the figure, survival mainly depends on whether the patient was transplanted for a malignant disease or not. Apart from the risk of transferring the disease, there are several advantages of using FAP livers for domino transplantation. First of all it is the advantage of a whole organ live donor graft. Minimal ischemic damage therefore takes place during the organ retrieval procedure, since the donor is well evaluated, the circulation is stable and cold ischemia time can be kept to a minimum, when the domino transplant is performed shortly after organ retrieval. One of the challenges in using the domino technique is that the donor and the recipient of the domino liver graft have to share a short segment of the suprahepatic caval vein. Several suggestions on how to handle this problem have been proposed (Furtado et al. 1997; Nishida and Tzakis 2002). At our institution we prefer to leave the longer caval segment in the domino donor,
Actuarial survival, %
100 80 60
p<0.001
40
Malignancy (n=296) Non malignancy (n=316)
20 0 0
2
4 6 8 Years after transplantation
10
Fig. 15.6 Outcome after Domino liver transplantation in recipients with cancer vs. non-cancer indication for liver transplantation (with permission from the FAPWTR)
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Number of patients
100 80 60 40 20 0
1
2
3
4 5 6 7 8 9 10 11 12 13 14 15 Years after transplantation
Fig. 15.7 Number of patients presently alive after domino transplantation who are approaching the time after transplantation when disease transmission has been seen to occur in a minority of patients, i.e. 7–8 years post-transplantation (with permission from the FAPWTR)
i.e. in the FAP patient, in order not to increase the operative risk for the donor. So far we have always managed to repair the cava of the domino graft, making it usable for implantation into the recipient. It was long believed that at least 15 years would elapse before any transmission of disease would be seen following domino transplantation. However, of more than 600 domino recipients reported to the FAPWTR, two patients developed FAP symptoms 7 and 8 years, respectively, after liver transplantation (Stangou et al. 2005; Takei et al. 2007). One patient was retransplanted and the symptoms disappeared. So far about 5% of the patients who reached 7 years after transplantation have developed symptoms of FAP (Yamamoto et al. 2007a). In Fig. 15.7, the number of living domino liver recipients and the time they have had their graft is displayed (data from the FAPWTR). It is still too early to calculate the exact risk of the domino procedure using FAP livers. Since the numbers of domino livers are relatively small in comparison with the deceased donor organs, it should be relatively easy to select recipients in whom the risk to develop FAP disease from the graft is much less than the risk of graft loss for other causes (Ericzon 2007; Ericzon et al. 2008; Wilczek et al. 2008). Therefore, it is generally believed that the domino procedure should continue by careful selection of recipients and by world wide collaboration in order to accumulate data, not only for the ATTR Val30Met mutation, but also for rare ATTR variants.
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Jonse´n E, Suhr OB, Tashima K, Athlin E. Early liver transplantation is essential for familial amyloidotic polyneuropathy patients’ quality of life. Amyloid 2001;8:52–57. Juneblad K, Naslund A, Olofsson BO, Suhr OB. Outcome of exercise electrocardiography in familial amyloidotic polyneuropathy patients, Portuguese type, under evaluation for liver transplantation. Amyloid 2004;11:208–213. Kimura A, Ando E, Fukushima M, Koga T, Hirata A, Arimura K, et al. Secondary glaucoma in patients with familial amyloidotic polyneuropathy. Arch Ophthalmol 2003;121:351–356. Kobayashi S, Morita H, Asawa T, Takei Y, Hashimoto T, Ikegami T, et al. Peripheral nerve function in patients with familial amyloid polyneuropathy after liver transplantation. Amyloid 2003;10:17–24. Lang K, Wikstrom L, Danielsson A, Tashima K, Suhr OB. Outcome of gastrointestinal complications after liver transplantation for familial amyloidotic polyneuropathy. Scand J Gastroenterol 2000;35:985–989. Lerut J, Gertsch P, Blumgart LH. ‘‘Piggy back’’ adult orthotopic liver transplantation. Helv Chir Acta 1989;56:527–530. Liepnieks JJ, Benson MD. Progression of cardiac amyloid deposition in hereditary transthyretin amyloidosis patients after liver transplantation. Amyloid 2007;14:277–282. Lindqvist P, Morner S, Suhr O, Waldenstrom A, Henein MY. Reduced left atrial function in familial amyloidosis: a myocardial strain rate study (abstract). VIIth International Symposium on Familial Amyloid Polyneuropathy. Kings Collage, London: Liver Unit & Amyloid treatment Programme, Kings Collage Hospital, London, 2008, pp P47–P48. Lindqvist P, Olofsson BO, Backman C, Suhr O, Waldenstrom A. Pulsed tissue Doppler and strain imaging discloses early signs of infiltrative cardiac disease: a study on patients with familial amyloidotic polyneuropathy. Eur J Echocardiogr 2006;7:22–30. Lobato L. Portuguese-type amyloidosis (transthyretin amyloidosis, ATTR V30M). J Nephrol 2003;16:438–442. Lobato L, Beirao I, Guimaraes SM, Droz D, Guimaraes S, Grunfeld JP, et al. Familial amyloid polyneuropathy type I (Portuguese): distribution and characterization of renal amyloid deposits. Am J Kidney Dis 1998;31:940–946. Morner S, Hellman U, Suhr OB, Kazzam E, Waldenstrom A. Amyloid heart disease mimicking hypertrophic cardiomyopathy. J Intern Med 2005;258:225–230. Nardo B, Beltempo P, Bertelli R, Montalti R, Vivarelli M, Cescon M, et al. Combined heart and liver transplantation in four adults with familial amyloidosis: experience of a single center. Transplant Proc 2004;36:645–647. Nishida S, Tzakis AG. Pitfalls of domino transplant. Transplantation 2002;73:1009–1010. Nowak G, Suhr OB, Wikstrom L, Wilczek H, Ericzon BG. The long-term impact of liver transplantation on kidney function in familial amyloidotic polyneuropathy patients. Transplant Int 2005;18:111–115. O’Hara CJ, Falk RH. The diagnosis and typing of cardiac amyloidosis. Amyloid 2003;10:127–129. Olofsson BO, Backman C, Karp K, Suhr OB. Progression of cardiomyopathy after liver transplantation in patients with familial amyloidotic polyneuropathy, Portuguese type. Transplantation 2002;73:745–751. Otto G, Post F, Hoppe-Luichius M, Greif-Higer G, Linke RP, Altland K, et al. Liver transplantation in patients with familial amyloid polyneuropathy: comparison of different transthyretin mutations (abstract). VIIth International Symposium on Familial Amyloid Polyneuropathy. Kings Collage, London: Liver Unit & Amyloid treatment Programme, Kings Collage Hospital, London, 2008, p P20. Palladini G, Campana C, Klersy C, Balduini A, Vadacca G, Perfetti V, et al. Serum N-terminal pro-brain natriuretic peptide is a sensitive marker of myocardial dysfunction in AL amyloidosis. Circulation 2003;107:2440–2445. Parrilla P, Ramirez P, Andreu LF, Bueno SF, Robles R, Miras M, et al. Long-term results of liver transplantation in familial amyloidotic polyneuropathy type I. Transplantation 1997;64: 646–649.
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Perdigoto R, Furtado AL, Furtado E, Oliveira FJ, Geraldes B, Mota O, et al. The Coimbra University Hospital experience in liver transplantation in patients with familial amyloidotic polyneuropathy. Transplant Proc 2003;35:1125. Perugini E, Guidalotti PL, Salvi F, Cooke RM, Pettinato C, Riva L, et al. Noninvasive etiologic diagnosis of cardiac amyloidosis using 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy. J Am Coll Cardiol 2005;46:1076–1084. Perugini E, Rapezzi C, Piva T, Leone O, Bacchi-Reggiani L, Riva L, et al. Non-invasive evaluation of the myocardial substrate of cardiac amyloidosis by gadolinium cardiac magnetic resonance. Heart 2006;92:343–349. Pilato E, Dell’Amore A, Botta L, Arpesella G. Combined heart and liver transplantation for familial amyloidotic neuropathy. Eur J Cardiothorac Surg 2007;32:180–182. Pomfret EA, Lewis WD, Jenkins RL, Bergethon P, Dubrey SW, Reisinger J, et al. Effect of orthotopic liver transplantation on the progression of familial amyloidotic polyneuropathy. Transplantation 1998;65:918–925. Puille M, Altland K, Linke RP, Steen-Muller MK, Kiett R, Steiner D, et al. 99mTc-DPD scintigraphy in transthyretin-related familial amyloidotic polyneuropathy. Eur J Nucl Med Mol Imaging 2002;29:376–379. Ramirez P, De Mingo P, Andreu F, Munar M, Hernandez Q, Munitiz V, et al. Long-term results of liver transplantation in four siblings from the same family with familial amyloidotic polyneuropathy type I TTR Ala-71. Transplant Int 2000;13(Suppl 1):S171–S173. Rapezzi C, Perugini E, Salvi F, Grigioni F, Riva L, Cooke RM, et al. Phenotypic and genotypic heterogeneity in transthyretin-related cardiac amyloidosis: towards tailoring of therapeutic strategies? Amyloid 2006;13:143–153. Rydh A, Suhr O, Hietala SO, Ahlstrom KR, Pepys MB, Hawkins PN, et al. Serum amyloid P component scintigraphy in familial amyloid polyneuropathy: regression of visceral amyloid following liver transplantation. Eur J Nucl Med 1998;25:709–713. Sandgren O, Kjellgren D, Suhr OB. Ocular manifestations in liver transplant recipients with familial amyloid polyneuropathy. Acta Ophthalmol 2008;86(5):520–524. Saraiva MJ, Birken S, Costa PP, Goodman DS. Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of molecular abnormality in transthyretin (prealbumin). J Clin Invest 1984;74:104–119. Sharma P, Perri RE, Sirven JE, Zeldenrust SR, Brandhagen DJ, Rosen CB, et al. Outcome of liver transplantation for familial amyloidotic polyneuropathy. Liver Transplant 2003;9:1273–1280. Shimojima Y, Morita H, Kobayashi S, Takei Y, Ikeda S. Ten-year follow-up of peripheral nerve function in patients with familial amyloid polyneuropathy after liver transplantation. J Neurol 2008;255(8):1220–1225 Singer R, Mehrabi A, Schemmer P, Kashfi A, Hegenbart U, Goldschmidt H, et al. Indications for liver transplantation in patients with amyloidosis: a single-center experience with 11 cases. Transplantation 2005;80:S156–S159. Snanoudj R, Durrbach A, Gauthier E, Adams D, Samuel D, Ferlicot S, et al. Changes in renal function in patients with familial amyloid polyneuropathy treated with orthotopic liver transplantation. Nephrol Dial Transplant 2004;19:1779–1785. Stangou AJ, Hawkins PN. Liver transplantation in transthyretin-related familial amyloid polyneuropathy. Curr Opin Neurol 2004;17:615–620. Stangou AJ, Hawkins PN, Heaton ND, Rela M, Monaghan M, Nihoyannopoulos P, et al. Progressive cardiac amyloidosis following liver transplantation for familial amyloid polyneuropathy: implications for amyloid fibrillogenesis. Transplantation 1998;66:229–233. Stangou AJ, Heaton ND, Hawkins PN. Transmission of systemic transthyretin amyloidosis by means of domino liver transplantation. N Engl J Med 2005;352:2356. Stangou AJ, Mathias CJ, Rela M, O’Grady J, Wendon J, Brass P, et al. Liver transplantation for familial amyloidosis; the Kings Collage Hospital selection criteria (abstract). VIIth International Symposium on Familial Amyloid Polyneuropathy. Kings Collage, London: Liver Unit & Amyloid treatment Programme, Kings Collage Hospital, London, 2008, p 25.
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Steen L, Ek B. Familial amyloidosis with polyneuropathy. A long-term follow-up of 21 patients with special reference to gastrointestinal symptoms. Acta Med Scand 1983;214:387–397. Suhr O, Danielsson A, Holmgren G, Steen L. Malnutrition and gastrointestinal dysfunction as prognostic factors for survival in familial amyloidotic polyneuropathy. J Intern Med 1994; 235:479–485. Suhr O, Danielsson A, Rydh A, Nyhlin N, Hietala SO, Steen L, et al. Impact of gastrointestinal dysfunction on survival after liver transplantation for familial amyloidotic polyneuropathy. Dig Dis Sci 1996;41:1909–1914. Suhr OB, Anan I, Ahlstrom KR, Rydh A. Gastric emptying before and after liver transplantation for familial amyloidotic polyneuropathy, Portuguese type (Val30Met). Amyloid 2003;10: 121–126. Suhr OB, Anan I, Backman C, Karlsson A, Lindqvist P, Morner S, et al. Do troponin and B-natriuretic peptide detect cardiomyopathy in transthyretin amyloidosis?. J Intern Med 2008; 263:294–301. Suhr OB, Ericzon BG, Friman S. Long-term follow-up of survival of liver transplant recipients with familial amyloid polyneuropathy (Portuguese type). Liver Transplant 2002;8:787–794. Suhr OB, Friman S, Ericzon BG. Early liver transplantation improves familial amyloidotic polyneuropathy patients’ survival. Amyloid 2005;12:233–238. Suhr OB, Holmgren G, Steen L, Wikstrom L, Norden G, Friman S, et al. Liver transplantation in familial amyloidotic polyneuropathy. Follow-up of the first 20 Swedish patients. Transplantation 1995;60:933–938. Suhr OB, Wiklund U, Ando Y, Ando E, Olofsson BO. Impact of liver transplantation on autonomic neuropathy in familial amyloidotic polyneuropathy: an evaluation by spectral analysis of heart rate variability. J Intern Med 1997a;242:225–229. Suhr OB, Wiklund U, Eleborg L, Ando Y, Backman C, Birgersdotter V, et al. Impact of autonomic neuropathy on circulatory instability during liver transplantation for familial amyloidotic polyneuropathy. Transplantation 1997b;63:675–679. Svendsen IH, Mortensen SA, Kirkegaard P, Suhr OB, Tashima K, Ohlsson P-I, et al. Combined heart and liver transplantation for familial amyloid cardiomyopathy (ATTR Leu111Met) (abstract). In: Ericzon B-G, Holmgren G, Lundgren E and Suhr OB, editors. The 4th International Symposium on Familial Amylodotic Polyneuropathy and Other Transthyretin Related Disorders & The 3rd International Workshop on Liver Transplantation in Familial Amyloid Polyneuropathy, Umea˚, Sweden 1999, p 76. Takei Y, Gono T, Yazaki M, Ikeda S, Ikegami T, Hashikura Y, et al. Transthyretin-derived amyloid deposition on the gastric mucosa in domino recipients of familial amyloid polyneuropathy liver. Liver Transplant 2007;13:215–218. Takei Y, Ikeda S, Ikegami T, Hashikura Y, Miyagawa S, Ando Y. Ten years of experience with liver transplantation for familial amyloid polyneuropathy in Japan: outcomes of living donor liver transplantations. Intern Med 2005;44:1151–1156. Tashima K, Ando Y, Terazaki H, Yoshimatsu S, Suhr OB, Obayashi K, et al. Outcome of liver transplantation for transthyretin amyloidosis: follow-up of Japanese familial amyloidotic polyneuropathy patients. J Neurol Sci 1999;171:19–23. Tsuchiya A, Yazaki M, Kametani F, Takei Y, Ikeda S. Marked regression of abdominal fat amyloid in patients with familial amyloid polyneuropathy during long-term follow-up after liver transplantation. Liver Transplant 2008;14:563–570. Viana Jda S, Bento C, Vieira H, Neves S, Seco C, Elvas L, et al. Haemodynamics during liver transplantation in familial amyloidotic polyneuropathy: study of the intraoperative cardiocirculatory data of 50 patients. Rev Port Cardiol 1999;18:689–697. Wei L, Kawano H, Fu X, Cui D, Ito S, Yamamura K, et al. Deposition of transthyretin amyloid is not accelerated by the same amyloid in vivo. Amyloid 2004;11:113–120. Westermark P. Aspects on human amyloid forms and their fibril polypeptides. FEBS J 2005;272:5942–5949.
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Westermark P, Sletten K, Johansson B, Cornwell GGd. Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc Natl Acad Sci USA 1990;87:2843–2845. Wiesner R, Edwards E, Freeman R, Harper A, Kim R, Kamath P, et al. Model for end-stage liver disease (MELD) and allocation of donor livers. Gastroenterology 2003;124:91–96. Wilczek HE, Larsson M, Yamamoto S, Ericzon BG. Domino liver transplantation. J Hepatobiliary Pancreat Surg 2008;15:139–148. Vogelsberg H, Mahrholdt H, Deluigi CC, Yilmaz A, Kispert EM, Greulich S, et al. Cardiovascular magnetic resonance in clinically suspected cardiac amyloidosis: noninvasive imaging compared to endomyocardial biopsy. J Am Coll Cardiol 2008;51:1022–1030. Yamamoto S, Wilczek HE, Iwata T, Larsson M, Gjertsen H, Soderdahl G, et al. Long-term consequences of domino liver transplantation using familial amyloidotic polyneuropathy grafts. Transplant Int 2007a;20:926–933. Yamamoto S, Wilczek HE, Nowak G, Larsson M, Oksanen A, Iwata T, et al. Liver transplantation for familial amyloidotic polyneuropathy (FAP): a single-center experience over 16 years. Am J Transplant 2007b;7:2597–2604. Yamashita T, Ando Y, Ueda M, Nakamura M, Okamoto S, Zeledon ME, et al. Effect of liver transplantation on transthyretin Tyr114Cys-related cerebral amyloid angiopathy. Neurology 2008;70:123–128. Yazaki M, Mitsuhashi S, Tokuda T, Kametani F, Takei YI, Koyama J, et al. Progressive wild-type transthyretin deposition after liver transplantation preferentially occurs onto myocardium in FAP patients. Am J Transplant 2007;7:235–242. Yazaki M, Tokuda T, Nakamura A, Higashikata T, Koyama J, Higuchi K, et al. Cardiac amyloid in patients with familial amyloid polyneuropathy consists of abundant wild-type transthyretin. Biochem Biophys Res Commun 2000;274:702–706.
Chapter 16
Mouse Models of Transthyretin Amyloidosis Sadahiro Ito and Shuichiro Maeda
Abstract Transthyretin (TTR) amyloidosis is caused by mutations in the TTR gene. Several observations, however, suggest the presence of factors, other than a mutation in the TTR gene, which affect TTR amyloid deposition. Although liver transplantation is the only curative treatment for TTR amyloidosis, its donor pool faces shortage, and TTR amyloid deposition continues in many patients after transplantation. Thus, some effective therapeutic strategies other than liver transplantation need to be developed. Mouse models of TTR amyloidosis would facilitate defining factors that accelerate amyloid deposition and would aid in developing effective treatments. Here, we summarize studies of transgenic mouse models of TTR amyloidosis in which questions were addressed about the role of various risk factors in the molecular pathogenesis of this intractable disease. Keywords Amyloid, Animal model, Genetically altered mouse, Knockout mouse, Serum amyloid P component, Transgenic mouse, Transthyretin amyloidosis
16.1
Introduction
Transthyretin (TTR) amyloidosis is the most common form of autosomal dominantly inherited amyloidosis. More than 100 distinct mutations in the TTR gene have been found and the majority are associated with TTR amyloidosis, the most common being the substitution of Met for Val at position 30 (TTR Met30). S. Maeda (*) Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_16, # Springer‐Verlag Berlin Heidelberg 2009
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Because most of the TTR mutations cause severe peripheral and autonomic neuropathy as well as systemic amyloidosis, they have been named familial amyloidotic polyneuropathy (FAP) (Benson and Kincaid 2007). Several observations, however, suggest the presence of factors, other than a mutation in the TTR gene, which affect amyloid deposition. For example, the same Val to Met substitution at position 30 of TTR is responsible for the Japanese, Portuguese, and Swedish types of FAP, but the mean age at onset in Swedish patients is significantly later than in Japanese and Portuguese patients. The patients in Sweden often develop symptoms at a later age and have a more prolonged illness than such patients living in the U.S. and descended from Swedish immigrants (Benson and Cohen 1977). Differences in the mean age at onset and some clinical symptoms have been also noted between monozygotic twins (Holmgren et al. 2004). Although liver transplantation is the only curative treatment for TTR amyloidosis, it is expensive, its donor pool faces shortages, patients with a transplant must receive life-long administration of immune suppressants, and TTR amyloid deposition continues in many patients after transplantation (Bergethon et al. 1996; Holmgren et al. 1993; Olofsson et al. 2002; Suhr et al. 1995). Thus, an effective therapeutic strategy other than liver transplantation needs to be developed. If one could define factors that accelerate amyloid deposition, one might be able to protect individuals with the mutant TTR gene against TTR amyloidosis, by protecting them from the risk factors. Mouse models of TTR amyloidosis would facilitate defining such factors and aid in developing effective treatments.
16.2
Transgenic Mice Carrying a Human Mutant TTR Gene
An animal model is crucial for studying the pathogenesis of TTR amyloidosis and testing new therapeutic strategies for the disease. To establish the animal model, several groups generated transgenic mice carrying a human mutant TTR gene that is responsible for TTR amyloidosis. We first generated two lines of transgenic mice carrying the human amyloidogenic mutant TTR Met30 gene, one carries a DNA fragment containing the human mutant gene with its own 0.6 kb upstream region (0.6-hTTRMet30), and another carries a fragment in which the promoter region of the mouse metallothionein-I gene was ligated to the entire human mutant gene (MT-hTTRMet30). 0.6-hTTRMet30 was expressed only in the liver, whereas MThTTRMet30 was expressed in various organs. Although the serum levels of human TTR in the two transgenic lines were similar (1.5–4.8 mg dl1), amyloid deposits were more prominent in the MT-hTTRMet30 line than in the 0.6-hTTRMet30 line (Tables 16.1and 16.2). In the former line, human TTR-derived amyloid deposits were first observed in the esophagus, stomach, and kidney when the mice were 6 months of age (Table 16.1). With advancing age, amyloid deposits extended to various other tissues. The tissue distribution of amyloid deposition in transgenic mice carrying the human mutant TTR Met30 gene was similar to that seen in TTR
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amyloidosis patients, except that they had no amyloid deposits in the peripheral and autonomic nervous systems, the characteristic sites of amyloid deposition in the patients, and they never developed neuropathy (Table 16.1) (Shimada et al. 1989; Wakasugi et al. 1987; Yi et al. 1991). TTR is a tetramer composed of four identical subunits. Analysis of 125I-thyroxine (T4) binding proteins in sera of the transgenic mice carrying the human mutant TTR Met30 gene provided evidence for the presence of mouse/human hybrid TTR tetramers. Mouse/human hybrid TTR tetramers probably form in the liver of transgenic mice prior to being secreted into the plasma. Thus, the serum level of homotetramers composed of human variant TTR was expected to be much higher in the MT-hTTRMet30 line than in the 0.6-hTTRMet30 line, because a significant fraction of the human TTR produced in the former derived from organs in which TTR is not normally synthesized. Our findings suggested that the human variant homotetramer might be more amyloidogenic than the mouse/human hybrid TTR tetramers (Kohno et al. 1997). We then generated transgenic mice carrying the human mutant TTR Met30 gene with its own 6.0 kb upstream region (6.0-hTTRMet30) (Takaoka et al. 1997). The serum level of human TTR Met30 in this line was 13.7–17.1 mg dl1, i.e., more than fivefold higher than 0.6-hTTRMet30 line. In good correlation with the serum levels of human variant TTR, amyloid deposits were more prominent in the former line than in the latter (Table 16.1). Several other research groups also generated transgenic animals carrying distinct human TTR gene constructs (Table 16.2) (Benson et al. 2006; Buxbaum et al. 2003; Sasaki et al. 1986; Sousa et al. 2002; Takaoka et al. 2004; Teng et al. 2001; Ueda et al. 2007). The serum levels of human TTR in the animals varied from 0.25 to 350 mg dl1. Sasaki et al. (1986) generated transgenic mice carrying the mouse MT-I promoter-human mutant TTR Met30 fusion gene. The serum level of human TTR in the mice was low (0.25–1.40 mg dl1) and the mice never developed amyloid deposition (Table 16.2) (Sasaki et al. 1986). Teng et al. (2001) generated transgenic mice carrying either a wild-type human TTR or a mutant human TTR (TTR Pro55) gene with its own 3 kb upstream region. Human TTR Pro55 is one of the most aggressive TTR amyloidosis-related mutations. The serum levels of normal human TTR and TTR Pro55 in two lines of transgenic mice were 100– 350 mg dl1 and 1–3 mg dl1, respectively. In good correlation with the serum levels of human TTR, amyloid deposits were detected only in the line transgenic for wild-type TTR gene (Table 16.2). The mice carrying the human wild-type TTR gene developed human TTR deposits mainly in the heart and kidney. In most of the mice, the deposits were nonfibrillar and non-Congophilic, but some mice older than 18 months of age developed cardiac TTR amyloid deposits (Teng et al. 2001). Their findings suggested that the nonfibrillar and non-Congophilic deposits might represent a precursor of the amyloid fibril. Sousa et al. (2002) generated a transgenic mouse line carrying a DNA fragment in which the promoter region of the sheep MT-I gene was ligated to the human mutant TTR Pro55 cDNA (MT-hTTRPro55). The serum level of TTR Pro55 in the MT-hTTRPro55 line was 5–20 mg dl1. The mice developed nonfibrillar and non-Congophilic TTR deposits in the
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Table 16.1 Tissue distribution of amyloid deposits in transgenic mice carrying a mutant human TTR gene Mouse and Organs Months of Age 6
12
15
21
MT-hTTRMet30 Brain Choroid plexus Sciatic nerve Heart Lung Liver Spleen Kidney Pancreas Upper alimentary tract Lower alimentary tract Lymph node
+ + +
+ + ++ ++
++ + ++ ++
++ +++ ++ +++
0.6-hTTRMet30 Brain Choroid plexus Sciatic nerve Heart Lung Liver Spleen Kidney Pancreas Upper alimentary tract Lower alimentary tract Lymph node
+
+ + ++ +++ +
6.0-hTTRMet30 Brain Choroid plexus Sciatic nerve Heart + + + Lung + Liver +++ Spleen ++ Kidney + +++++ Pancreas Upper alimentary tract ++ +++ ++ ++++ Lower alimentary tract +++ +++ Lymph node The data are from Wakasugi et al. (1987), Simada et al. (1989), Yi et al. (1991), and Takaoka et al. (1997). Amyloids are absent, ; limited to the wall of small blood vessels, ; observed in the wall of small blood vessels and their surrounding regions, +; moderate in the wall of small blood vessels and in the interstitium, ++; marked in the interstitium and parenchyma, +++
Val30Met (authentic 6.0 kb upstream region) Wild-type (authentic 3.0 kb upstream region) Leu55Pro (authentic 3.0 kb upstream region) Leu55Pro (sheep MT-I)
Val30Met (authentic 0.6 kb or 6.0 kb upstream region) Val30Met (MT-I)
Val30Met mouse metallothionein-I (MT-I) Val30Met (MT-I)
4.8
22–79
100–350 (wild type)
1–3 (Leu55Pro)
5–20 5–20
C57BL/6
C57BL/6 129/Sv//Ev
C57BL/6 DBA/2
C57BL/6 DBA/2
C57BL/6 C57BL/6 129/Sv//Ev
13.7–17.1 (6.0 kb)
1.5–3.0 (0.6 kb)
2.1–4.8
C57BL/6
C57BL/6
0.25–1.40
C57BL/6 C3H BALB/c
+
+
+
+
+
+
+
A(), B(+) A(+), B(+)
A(), B()
A(+), B(+)
A(+), B(ND)
A(+), B(ND)
A(+), B(ND)
A(+), B(ND)
None Mouse Ttr
None
None
Serum amyloid P component (SAP) Mouse Ttr
None
None
None
#
$
$
(continued )
Sousa et al. (2002)
Teng et al. (2001)
Wakasugi et al. (1987) Yi et al. (1991) Shimada et al. (1989) Takaoka et al. (1997) Murakami et al. (1992) Tashiro et al. (1991) Kohno et al. (1997)
Sasaki et al. (1986)
Table 16.2 Summary of the phenotype of transgenic murine models of TTR amyloidosis, factors tested, and effects of the factors on the TTR deposition described in this review Human TTR Murine strain Serum level Endogenous TTR amyloid Factor tested Effects of the Refs Transgene of human murine Ttr (A) or TTR factor on TTR (promoter) TTR (mg/dl) deposit (B) amyloid (A) or TTR deposit (B)
Val30Met (authentic 6.0 kb upstream region) Val30Met (authentic 6.0 kb upstream region) Ile84Ser (authentic 7.0 kb upstream region) Val30Met (mouse albumin) Val30Met (authentic 6.0 kb upstream region) Val30Met (authentic 6.0 kb upstream region) ND Not Determined
Leu55Pro (authentic 3.0 kb upstream region) Val30Met (authentic 7.0 kb upstream region) Wild -type (authentic 3.0 kb upstream region) Val30Met
3.8–7.8
70–300 (wild type)
22–79
C57BL/6
C57BL/6 DBA/2
C57BL/6 129/Sv// Ev C57BL/6 129/Sv// Ev
22–79
85
1–4
22–79
22–79
C57BL/6 129/Sv// Ev
C3H DBA C57BL/6
DA rat
C57BL/6 129/Sv// Ev
C57BL/6 129/Sv// Ev BALB/c
22–79
1–3
Serum level of human TTR (mg/dl)
C57BL/6 DBA/2
Table 16.2 (continued) Human TTR Murine strain Transgene (promoter)
+
+
A(+), B(+)
A(ND), B(+)
A(), B(+)
A(), B(ND)
A(+), B(+)
A(+), B(+)
A(+), B(ND)
A(+), B(+)
A(+), B(ND)
A(+), B(+)
TTR amyloid (A) or TTR deposit (B)
+ or
+
+
Endogenous murine Ttr
ND
#
#
$
$
necessary
#
Effects of the factor on TTR amyloid (A) or TTR deposit (B)
An anti-apoptotic agent; # Tauroursodeoxycholic acid Heat shock # transcription factor 1
None
Antisense oligonucleotide
Immunization with TTR Y78F
Doxycycline
TTR amyloid
TTR amyloid
TTR Cys10
Mouse Ttr
Factor tested
Santos et al. (2008)
Ueda et al. (2007) Macedo et al. (2008)
Benson et al. (2006)
Cardoso and Saraiva (2006) Terazaki et al. (2006)
Wei et al. (2004)
Tagoe et al. (2004)
Takaoka et al. (2004)
Buxbaum et al. (2003)
Refs
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gastrointestinal tract at age 1–3 months. They, however, never developed Congophilic TTR amyloid deposition (Table 16.2). The tissue distribution and age at onset of nonfibrillar and non-Congophilic TTR deposition in the MT-hTTRPro55 line was similar to that seen in the 6.0-hTTRMet30 line. However, deposition of Congophilic TTR amyloid was only observed in the 6.0-hTTRMet30 line older than 19 months of age in their laboratory (Sousa et al. 2002). In our earlier study, deposition of TTR amyloid was first observed in the 6.0-hTTRMet30 line when the mice were 9–12 months of age (Table 16.1) (Takaoka et al. 1997). The difference in age at onset and progression of TTR amyloid deposition between the same transgenic mice kept in different facilities might be caused by the effect of environmental factors. Noguchi et al. reported that the 6.0-hTTRMet30 line, when kept under specific pathogen-free conditions, never developed deposition of TTR amyloid (Noguchi et al. 2002). We and others, however, noted that deposition of TTR amyloid has been significantly suppressed in the recent generations of transgenic mouse models relative to the earlier generations of the same models kept under the similar environmental conditions, with TTR amyloid depositing at a later age in successive generations (Buxbaum et al. 2003; Kohno et al. 1997; Wei et al. 2004). The observation suggests that presently unknown genetic factor(s) might also affect the TTR amyloid deposition in the transgenic mouse models. Ueda et al. (2007) generated transgenic rats carrying the mouse albumin promoter-human mutant TTR Met30 cDNA fusion gene to analyze the metabolism of human amyloidogenic TTR Met30 after liver transplantation. The serum level of human TTR in the rat was low (1–4 mg dl1) and the rat never developed amyloid deposition (Table 16.2) (Ueda et al. 2007). Takaoka et al. generated three lines of transgenic mice carrying one of the three distinct human mutant TTR cDNAs, i.e., TTR Cys10/Met30, Cys10/Val30, and Ser10/Met30 cDNAs to analyze the role of –SH side chain of Cys at position 10 of human TTR in amyloidogenesis. Their results suggested that the –SH residue might play an important role in TTR Met30 amyloidogenesis in vivo (Table 16.2) (Takaoka et al. 2004). Benson et al. generated transgenic mice carrying and expressing the human amyloidogenic mutant TTR Ser84 gene. Treatment of the transgenic mice with TTR-specific antisense oligonucleotides significantly reduced the serum level of human TTR in the mice. Although the serum level of transgenic mice was 85 mg dl1, the mice never developed TTR-derived amyloid deposition (Table 16.2) (Benson et al. 2006). The reason the transgenic mice failed to develop amyloidosis is unknown. Various factors, such as tissue specific expression of the human TTR transgene, human TTR level in serum, conformation of the human variant TTR, genetic background of the mice, and environmental factors may affect human TTR-derived amyloid deposition in the transgenic mice. Although all the above transgenic animals carrying the distinct human amyloidogenic TTR gene constructs have no amyloid deposits in the nervous system and they never develop neuropathy, the animals facilitated defining factors important for amyloid deposition and will aid in developing appropriate treatments.
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16.3
S. Ito and S. Maeda
Disruption of the Ttr Gene in Mice
To understand thephysiological function of TTR, we generated Ttr-deficient (Ttr/) mice through gene targeting. The mutant mice, although they show significantly depressed levels of serum retinol, retinol-binding protein (RBP), and T4, display no obvious phenotypic abnormalities and their fertility is normal (Episkopou et al. 1993). The levels of retinol and retinyl esters in liver, testis, kidney, spleen, and eye cups were equivalent in the Ttr/ mice and in the wild-type (Ttr+/+) mice. Plasma all-trans-retinoic acid levels of the Ttr/ mice were 2.3-fold higher than those of Ttr+/+ mice (Wei et al. 1995). The free T4 and triiodothyronine levels in the serum of Ttr/ mice were normal (Palha et al. 1994). Because Ttr/ mice displayed no obvious phenotypic abnormalities and appeared to have normal life span, suppression of amyloidogenic TTR synthesis might be considered for use as a therapeutic agent for TTR-derived amyloid deposition. A possible reason for the difference in the pattern of amyloid deposition between transgenic mice carrying the human mutant TTR Met 30 gene and TTR amyloidosis patients might be that the endogenous normal mouse TTR affects amyloid deposition by altering conformation of the human variant TTR, through formation of hybrid tetramers. To study the effect of endogenous mouse TTR on human TTR Met 30-derived amyloid deposition, we introduced the 6.0-hTTRMet30 into the Ttr/ as well as Ttr+/+ mice. The transgene, 6.0-hTTRMet30, was expressed in the liver, choroid plexus of brain, kidney, retina, and visceral yolk sac, exactly the same tissues as for the endogenous mouse Ttr gene, and the levels of expression were equivalent to those of the endogenous mouse Ttr gene (Nagata et al. 1995). We compared the onset, progression, and tissue distribution of amyloid deposition of the Ttr/ transgenic mice expressing only 6.0-hTTRMet30 with that of the Ttr+/+ transgenic mice expressing both the endogenous mouse Ttr and 6.0-hTTRMet30 (Kohno et al. 1997).
16.4
Transgenic Mouse Models of TTR-Associated Homozygous Amyloidosis
In the Ttr/ transgenic mice expressing 6.0-hTTRMet30, human TTR Met30derived amyloid deposits were first observed in the esophagus and stomach when the mice were 11 months of age, and with advancing age, these deposits extended to various other tissues. Because the serum level of homotetramers composed of human TTR Met30 should be significantly higher in the Ttr/ transgenic mice expressing 6.0-hTTRMet30 than in the Ttr+/+ transgenic mice expressing 6.0-hTTRMet30, we expected that amyloid deposits would be more prominent in the former than in the latter. However, no significant difference was detected in the onset, progression, and tissue distribution of amyloid deposition between the Ttr/ and Ttr+/+ transgenic mice (Kohno et al. 1997). No amyloid deposits were detected
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in the peripheral nervous tissues of Ttr/ as well as Ttr+/+ transgenic mice expressing 6.0-hTTRMet30 up to age 24 months. Our observation was consistent with the finding that clinical symptoms of TTR amyloidosis patients homozygous for the TTR Met30 mutation are similar to those of the TTR amyloidosis patients heterozygous for the mutation (Holmgren et al. 1992). The Ttr/ mouse facilitated studies on the function of another human variant TTR associated with amyloidosis. Sousa et al. assessed the onset, progression, and tissue distribution of TTR deposition of the Ttr/ transgenic mice expressing only human TTR Pro55 gene (MT-hTTRPro55) and that of the Ttr+/+ transgenic mice expressing both the endogenous mouse Ttr and MT-hTTRPro55, in comparison with the 6.0-hTTRMet30 line (Sousa et al. 2002). Contrary to our findings, they reported that the deposition of both non-Congophilic and Congophilic TTR deposition were significantly retarded in the Ttr+/+ transgenic mice relative to the Ttr/ transgenic mice, suggesting that human variant TTR homotetramers are more amyloidogenic than the wild-type mouse/variant human hybrid TTR heterotetramers present in the Ttr+/+ transgenic mice expressing both the endogenous wildtype mouse Ttr and human MT-hTTRPro55 or 6.0-hTTRMet30. (Table 16.2) (Sousa et al. 2002). The contradiction between their findings and ours suggests that some environmental factors may affect the deposition of human TTR of the same transgenic mice housed in different animal facilities. Buxbaum et al. also reported that when they crossed the transgenic mice carrying mutant human TTR Pro55 gene with Ttr/ mice, the frequency of human TTR-derived deposits increased threefold (Table 16.2) (Buxbaum et al. 2003). However, the serum level of human TTR Pro55 in the transgenic mice was 1–3 mg dl1, i.e., much lower than the 6.0-hTTRMet30 line (22–79 mg dl1) (Kohno et al. 1997). We observed no correlation in the 6.0-hTTRMet30 line between the serum levels of human variant TTR (22–79 mg dl1) and the onset, extent, or tissue distribution of amyloid deposition. We also compared the onset and progression of amyloid deposition between two independent lines of MT-hTTRMet30 transgenic mice. The serum level of human TTR Met30 in one line was about 5 mg dl1, i.e., fivefold higher than the other. In good correlation with the serum levels of human variant TTR, amyloid deposits were more prominent in the former line than in the latter (Yamamura et al. 1990). The serum levels of human TTR Met30 were significantly higher in the 6.0-hTTRMet30 transgenic mice (22–79 mg dl1) than in either of MT-hMet30 lines (1 or 5 mg dl1), and there was no correlation between amyloid deposition and the serum levels of human TTR Met30 in the 6.0-hTTRMet30 line (Kohno et al. 1997; Yamamura et al. 1990). These findings suggest that although amyloidogenic variant TTR in the form of homotetramer or mouse/human hybrid TTR heterotetramer is necessary for amyloid deposition, above a threshold serum level, amyloid deposition is not further enhanced by increased concentrations of human variant TTR. The introduction of 6.0-hTTRMet30 into the Ttr/ mice significantly increased their depressed serum levels of total T4 and RBP, suggesting that human TTR Met30 binds T4 and RBP in vivo. The T4-binding ability of human TTR Met30 was confirmed by analysis of 125I-T4-binding proteins in sera of Ttr/ transgenic mice expressing 6.0-hTTRMet30 (Kohno et al. 1997).
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S. Ito and S. Maeda
Analysis of the Role of Various Factors in Amyloid Deposition
16.5.1 Serum Amyloid P Component Serum amyloid P component (SAP, encoded by APCS gene) is a plasma glycoprotein with a characteristic pentameric organization of five identical subunits of 25.5 ˚ in diameter with a hole in its kDa arranged as an annular pentagonal disc 95 A center. SAP binds to many ligands such as glycosaminoglycans, pyruvate acetal of galactose, DNA, chromatin, complement components, lipopolysaccharide (LPS) and is involved in recognition of pathogens and damaged tissues (Garlanda et al. 2005; Pepys et al. 1997). As SAP binds to amyloid fibrils, it accumulates in all types of amyloid deposits, including Ab deposits of Alzheimer’s disease. SAP inhibits proteolysis of amyloid fibrils in vitro and is thereby speculated to contribute to persistence of amyloid in vivo (Tennent et al. 1995). We and others therefore attempted to elucidate the role of SAP in the pathogenesis of amyloidoses using transgenic and knockout mice. The genetically altered mice have provided new insights into the role of SAP in amyloid deposition.
16.5.1.1
Generation of Doubly Transgenic Mice Carrying Both a Human Mutant TTR Gene and the Human APCS Gene Encoding SAP
To examine whether human SAP enhanced the human TTR-derived amyloid deposition in the transgenic mouse model of TTR amyloidosis, a transgenic mouse line carrying the human APCS gene encoding SAP was first generated. The serum level of human SAP in the transgenic mice was higher than that in human serum (Iwanaga et al. 1989). The transgenic mice were mated with the transgenic mouse model of TTR amyloidosis (MT-hTTRMet30 line) to generate doubly transgenic mice carrying both the human mutant TTR gene and the human APCS gene (Fig. 16.1a). The age at onset, progression, and tissue distribution of amyloid deposition were equivalent in the doubly transgenic mice and in the mice carrying only the human mutant TTR gene (Table 16.2) (Tashiro et al. 1991). These results suggested that SAP might not play an important role in TTR-derived amyloid deposition.
16.5.1.2
Induction of Mouse Sap Synthesis in a Transgenic Mouse Model of TTR Amyloidosis (MT-hTTRMet30 line) by Injection of Escherichia coli Lipopolysaccharide (LPS)
Sap is a major acute-phase reactant in mice, whereas in human serum it is constitutively present (Pepys et al. 1997). Thus, we asked if sustained high serum levels of Sap induced by repeated intraperitoneal injections of E. coli LPS enhanced
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b
c
MT–hTTRMet30
LPS
Sap– deficient
emulsion
Fig. 16.1 Analysis of the role of SAP in the pathogenesis of TTR amyloidosis using genetically altered mice. (a) We introduced human APCS gene encoding SAP into the transgenic mice carrying the human mutant TTR gene (MT-hTTRMet30) that is responsible for TTR amyloidosis. (b) We examined whether sustained high serum levels of mouse Sap induced by repeated intraperitoneal injections of E. coli LPS enhanced the TTR-derived amyloid deposition in the transgenic mouse carrying the human mutant TTR gene (MT-hTTRMet30). (c) We asked if experimental AA amyloidosis can be induced in the Apcs/ mice by a single intraperitoneal emulsion injection
TTR-derived amyloid deposition in the MT-hTTRMet30 line (Murakami et al. 1992). Male mice were given 1 mg of LPS/g body weight intraperitoneally. There was a progressive increase in the serum level of Sap and a peak was reached within 48 h after inducing an acute inflammation. At the maximum level, the serum level of Sap was 50-fold higher than that of the control level. The elevated level then gradually decreased, but it had not returned to the original unstimulated level even 4 days later. Based on this observation, we injected LPS intraperitoneally into the transgenic mice every 5–6 days, from the age of 2 months (Fig. 16.1b). Because transcription of the human mutant TTR gene in these transgenic mice was under the control of the mouse MT-I gene promoter, the serum level of human TTR, the precursor protein of amyloid fibril, in these transgenic mice also increased up to 16-fold within 10 h after inducing an acute inflammation. After repeated LPS injections and at 5.5, 7, 13, and 18 months of age, we examined the mice for distribution and degree of amyloid deposits. To compare the distribution and degree of amyloid deposition between the LPS-stimulated and unstimulated transgenic mice, we also histochemically examined unstimulated transgenic mice of the same litter for amyloid deposition. No difference was detected in the age at onset, progression, and tissue distribution of amyloid deposition between the LPS-stimulated and unstimulated transgenic mice (Tashiro et al. 1991). Contrary to our expectation, all these findings suggested that high serum levels of human SAP or mouse endogenous Sap did not affect the age at onset and extent of human TTR-derived amyloid deposition in the transgenic mouse model of TTR amyloidosis. However, since normal endogenous serum levels of mouse Sap might be sufficient to promote amyloid deposition, these data did not rule out a crucial role for SAP in the deposition. Thus, we approached this question by analyzing the induction of experimental amyloid A (AA) amyloidosis in Sap-deficient mice generated through gene targeting (Togashi et al. 1997).
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Induction of AA Amyloidosis in the Sap-Deficient Mice
We and another research group independently generated a unique strain of mice carrying a null mutation at Apcs locus encoding Sap through gene targeting (Botto et al. 1997; Togashi et al. 1997). The resultant Sap-deficient (Apcs/) mice displayed no obvious phenotypic abnormalities and their fertility was normal. Thus, Sap is not essential for fetal development, postnatal viability or fertility in mice. We asked if experimental AA amyloidosis can be induced in the Apcs/ mice by a single intraperitoneal emulsion injection (Fig. 16.1c) (Togashi et al. 1997). The mice were killed 18 days after the injection, and examined for the occurrence of AA amyloid deposits in the spleen. No difference was noted in the degree of splenic AA amyloid deposition in the mice of both genotypes. We then compared the kinetics of induction of AA amyloidosis in the Apcs/ and Apcs+/+ mice, testing for splenic amyloid deposits at 11, 12, 14, 18, and 29 days after emulsion injection. The induction of AA amyloidosis was significantly retarded in Apcs/ mice relative to Apcs+/+ mice (Togashi et al. 1997). Botto et al. also reported that AA amyloid deposition was delayed in their Apcs/ mice (Botto et al. 1997). Our experiments presented compelling evidence that, although not essential in the deposition of AA amyloid, Sap significantly accelerates the reaction. Although the induction of AA amyloidosis is very rapid, the result suggests that SAP may promote amyloid deposition in late-onset human amyloidoses, including TTR amyloidosis and Alzheimer’s disease in which it takes many years for amyloidogenic precursors to deposit. The role of Sap in the pathogenesis of lateonset human amyloidoses can be assessed by crossing the transgenic mouse models of human amyloidoses and Apcs/ mice. It was, however, reported that Apcs/ mice with a mixed genetic background (129/Sv x C57BL/6; F2) spontaneously developed antibody to nuclear antigens (ANA) and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus (SLE). Thus, Sap was speculated to suppress the development of antinuclear autoimmunity (Bickerstaff et al. 1999). However, the Apcs gene encoding Sap on mouse chromosome 1 is located in a region harboring several genes reported to be associated with SLE susceptibility in multiple strain combinations (Marrack et al. 2001, Wakeland et al. 2001). This suggested that ignoring the confounding effect of genes around the disrupted Apcs gene in the knockout mice might lead to misinterpretation of the role of Sap in autoimmunity. Furthermore, although Apcs/ mice generated from 129/Sv//Ev-derived CCE embryonic stem cells in our laboratory and backcrossed into C57BL/6 for 4 generations (129/Sv//Ev x C57BL/6; F4) also produced significantly higher titers of ANA than did control Apcs+/+ littermates, yet they displayed no abnormalities in development, fertility, or longevity and did not develop severe glomerulonephritis (Soma et al. 2001). Although the reason for the discrepancy between data of other authors and our data is not clear, this may reflect minor genetic differences between the two lines of mutant mice used. Thus, we carefully assessed the effects of genetic background and genes linked to the disrupted Apcs gene on the development of antinuclear
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autoimmunity (Tamaoki et al. 2005). Our data suggested that autoimmunity in the Apcs/ 129/Sv//Ev x C57BL/6 mice might not be caused by Sap-deficiency in itself but rather the expression of 129/Sv//Ev-type genes, including a candidate lupus-susceptibility Ifi202 gene (Rozzo et al. 2001) on a C57BL/6 background. In accordance with the findings, it was reported that C57BL/6 mice congenic for a 26-cM 129-type chromosome 1 segment around the Apcs gene developed high titers of ANA independent of Sap (Bygrave et al. 2004). The results indicate that we must eliminate the effect of 129/Sv//Ev-type genes linked to the disrupted Apcs gene to elucidate the role of Sap in autoimmunity using the mice with targeted disruption of the Apcs gene. Our data clearly showed that contrary to documented data, the Apcs/ mice do not develop severe autoimmune disease (Soma et al. 2001; Tamaoki et al. 2005). Thus, the Apcs/ mice should have immense significance for understanding the role of SAP in the pathogenesis of human amyloidoses.
16.5.1.4
Could Inhibition of Binding of SAP to Amyloid Fibrils be Effective as a Treatment For Human Amyloidoses? Contribution of Sap to the Persistence of Mouse AA Amyloid
Pepys et al. reported that a low molecular weight sugar that is a SAP ligand inhibits binding of SAP to amyloid fibrils and dissociates bound SAP from the amyloid deposits. In view of the protective effect of human SAP against proteolysis of amyloid fibrils in vitro, and the delayed AA amyloid deposition in Apcs/ mice, they proposed that such ligands might be useful as drugs for the otherwise intractable human amyloidoses (Pepys et al. 1997; Pepys et al. 2002; Tennent et al. 1995). However, the role of SAP in the pathogenesis of late-onset human amyloidoses has not yet been elucidated. If SAP has a crucial role in the pathogenesis of human amyloidoses, such ligands or related compounds may be useful as drugs for the diseases, especially since Sap inactivation does not appear to induce morbidity in mice. To elucidate the contribution of Sap to persistence of murine AA amyloid and to assess potential ways of treating individuals with amyloidosis, we examined the regression of splenic AA amyloid fibrils in Apcs/ and Apcs+/+ mice (Usui et al. 2001). Amyloid was induced in the Apcs/ and Apcs+/+ mice by daily subcutaneous injections of 5% casein. Partial splenectomy was done for mice by giving casein injections daily for 9 weeks. Although significant individual differences in the degree of splenic AA amyloid deposition were noted, the mean degree of amyloid deposition in 8 Apcs+/+ mice was much the same as that in 8 Apcs/ mice (Table 16.3). All the mice were killed 60 days after the biopsy and the degree of amyloid deposition in the autopsied spleen was compared with that in the biopsied spleen of the same mouse. The levels of serum amyloid A, the precursor protein of AA amyloid, of all the 16 autopsied mice were normal, indicating no inflammation at the time of autopsy. The degree of amyloid deposition was reduced in 2 out of 8 Apcs+/+ mice, while it was reduced in 1 out of 8 Apcs/ mice. In the remaining
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Table 16.3 Effect of Sap on the persistence of experimental mouse AA amyloid deposits (Usui et al. 2001) Histlogical grade of amyloid deposition Genotype Partial splenectomy (Biopsy) Autopsy SAP-deficient (Apcs/)
2 2 2 3 3 3 4 4
2 2 1 3 3 3 4 4
Wild-type (Apcs+/+)
1 1 1 1 2 2 2 2 3 2 4 4 4 4 5 4 The grading of amyloid deposition expressed as follows: Grade 0, no amyloid deposit; Grade 1, small nodular or lineal amyloid deposit in the perifollicular zone; Grade 2, appearance of rings of amyloid deposits in the perifollicular zone; Grade 3, broad bands of amyloid deposits in the perifollicular zone and occasional amyloid deposits in the red pulp; Grade 4, amyloid deposits in the red pulp is more pronounced and the follicles are reduced to feebly staining reticulum cells; Grade 5, complete loss of structure and the spleen consists mainly of amyloid deposits
mice, the degree of amyloid deposition did not change during the 60 days (Table 16.3). These results indicated that lack of Sap in AA amyloid deposits did not enhance regression of the deposits in vivo and suggested that dissociation of bound Sap from AA amyloid deposits would not significantly accelerate regression of the deposits in vivo (Usui et al. 2001). Our findings suggest that the low molecular weight sugar SAP ligands or related compounds should probably be applied before amyloid begins to deposit, if one is to evaluate their clinical relevance to amyloidoses. Our experiments presented compelling evidence that, although not essential in the deposition of mouse AA amyloid, Sap significantly accelerates the reaction. Thus, SAP may play an important role in the pathogenesis of human amyloidoses. However, further investigation needs to be carried out to elucidate the role of SAP in the pathogenesis of human amyloidoses and to evaluate the efficacy of targeted pharmacological depletion of SAP from amyloid deposits for treatment of human amyloidoses. Transgenic mouse models of human amyloidoses and the Apcs/ mice should be useful in the evaluation.
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16.5.2 Analysis of the Role of Factors Other than SAP in TTR Amyloid Deposition 16.5.2.1
TTR Amyloid Fibrils
Some amyloidoses were shown to be transmissible or infectious, namely, administration of one type of amyloid results in deposition of the same amyloid in vivo. For example, prion disease in humans and domestic animals, and mouse senile amyloidosis are known to be transmissible (Sigurdsson et al. 2002). On the other hand, families with a TTR Met30-associated amyloidosis exhibit genetic anticipation, with TTR Met30-amyloid depositing at an earlier age in successive generations. The molecular bases of genetic anticipation remained to be determined. We asked if administration of TTR amyloid fibrils (ATTR) extracted from the heart of a TTR Met30-associated amyloidosis patient would accelerate ATTR deposition in the transgenic mice expressing the human amyloidogenic TTR Met30 gene (6.0-hTTRMet30 line) and indeed the administration did accelerate deposition of apolipoprotein A-II amyloid fibrils (AApoAII), but not ATTR. Our experiments presented evidence that the degree of inducibility of ATTR is low relative to AApoAII (Wei et al. 2004). Tagoe et al. also reported that TTR amyloid deposition was neither accelerated nor enhanced by injections of preformed TTR fibrils in mice transgenic for wild-type human TTR (Tagoe et al. 2004). Thus, administration of ATTR may not explain the genetic anticipation which occurs in TTR amyloidosis.
16.5.2.2
Doxycycline
The drug doxycycline is able to disrupt TTR fibrils in vitro. Cardoso and Saraiva assessed the efficacy of doxycycline in vivo in the Ttr/ transgenic mice expressing 6.0-hTTRMet30. No differences were detected in nonfibrillar TTR deposition between the drug-administrated and unadministrated mice. However, Congophilic TTR deposition was absent in the administrated mice, being observed only in the unadministrated mice. These results suggested that doxycycline might be capable of disrupting TTR amyloid deposits (Cardoso and Saraiva 2006).
16.5.2.3
TTR Y78F
A variant TTR with phenylalanine replacing tyrosine at position 78 (TTR Y78F) has been shown to expose a cryptic epitope recognized by a monoclonal antibody that reacts only with highly amyloidogenic mutants presenting the amyloid fold or with amyloid fibrils. Thus, Terazaki et al. immunized the Ttr/ transgenic mice expressing the 6.0-hTTRMet30 with TTR Y78F. They found that immunization of the mice with TTR Y78F reduced human TTR deposition and cleared TTR amyloid deposits in the gastrointestinal tract (Terazaki et al. 2006). The immunotherapy
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with the use of human TTR Y78F may be a useful therapeutic tool for TTR amyloidosis.
16.5.2.4
An Antiapoptotic Agent
Tauroursodeoxycholic acid, a nontoxic hydrophilic bile acid acts as a potent antiapoptotic and antioxidant agent. Because oxidative stress, apoptosis, and inflammation are associated with TTR amyloidosis, Macedo et al. (2008) investigated the effect of tauroursodeoxycholic acid on the human TTR deposition in the Ttr/ transgenic mice expressing the 6.0-hTTRMet30. They showed that the administration of tauroursodeoxycholic acid to the transgenic mice decreased apoptotic and oxidative biomarkers usually associated with TTR deposition and significantly reduced non-Congophilic TTR toxic aggregates (Macedo et al. 2008). These data suggested that tauroursodeoxycholic acid might be valuable as an effective drug for TTR amyloidosis.
16.6
Attempts to Generate a Closer Mouse Model of TTR Amyloidosis
The transgenic mice expressing the human mutant TTR Met 30 gene have proved to be useful in defining factors that affect amyloid deposition and will aid in developing effective treatments. However, these mice have no amyloid deposits in peripheral and autonomic nervous tissues, and do not develop neuropathy; the most common symptom of TTR amyloidosis. One of the challenges we face is to generate a closer mouse model of TTR amyloidosis which develops neuropathy. Amyloid deposition in the gastrointestinal tract is more prominent in transgenic mice than in TTR amyloidosis patients and, in the stomach of transgenic mice, it is more prominent in the nonglandular side than in the glandular side (Yi et al. 1991). The difference in the pattern of amyloid deposition between transgenic mice and TTR amyloidosis patients might reflect the necessity of a specific interaction between variant TTR and component(s) of tissues that may be species-specific. Introduction of amyloidogenic point mutations into the mouse endogenous Ttr gene will aid in testing this hypothesis. We developed a novel gene targeting procedure to introduce point mutations efficiently into the mouse Ttr gene (Horie et al. 1995). We used this procedure to generate a mutant mouse line having the Val to Met substitution at position 30 of endogenous mouse Ttr (Ito et al.). The mutant mouse proved to be useful for the analysis of targeted Ttr gene repair in vivo (Nakamura et al. 2004). We suggest that this procedure may be used to introduce various TTR mutations associated with TTR amyloidosis efficiently into mouse genes.
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TTR deposits in the tissues of TTR amyloidosis patients lead to cellular stress and increased proinflammatory molecules. Thus a possible defense mechanism against the TTR deposits may be a stress response. Disruption of the response pathway, therefore, might accelerate TTR deposition. To test the hypothesis Santos et al. generated a new transgenic mouse line expressing 6.0-hTTRMet30 in a heat shock transcription factor 1 (Hsf1) null background. The lack of HSF1 led to an extensive and earlier nonfibrillar human TTR deposition, evolving into fibrillar deposits in distinct organs including the peripheral nervous system. Inflammatory stress and a reduction in the number of unmyelinated nerve fibers were observed in the new transgenic mouse model, as in human patients (Santos et al. 2008). Although the new mouse model has not yet developed neuropathy, these mice will aid in testing new therapeutic strategies and in studying the pathogenesis of TTR amyloidosis.
16.7
Conclusions
Mouse models of TTR amyloidosis have facilitated the understanding of the pathogenesis of this intractable disorder and have aided in evaluating and developing novel therapeutic strategies. However, to date, ideal transgenic mouse models of TTR amyloidosis which mimic TTR amyloidosis patients in the degree and tissue-distribution of Congophilic TTR amyloid deposition, in the symptoms, and in the pathological lesions have not yet been established. Thus, it is very important to generate an ideal or closer animal model of TTR amyloidosis to conquer the intractable disorder.
Acknowledgments This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan; Grants-in-aids for Scientific Research (to SM); and by grants from the Ministry of Health, Labour and Welfare, Japan; the Amyloidosis Research Committee, Surveys and Research on Specific Diseases (to SM). We thank all our collaborators for their invaluable help and contributions.
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Bergethon PR, Sabin TD, Lewis D et al. (1996) Improvement in the polyneuropathy associated with familial amyloid polyneuropathy after liver transplantation. Neurology 47:944–951 Bickerstaff MC, Botto M, Hutchinson WL et al. (1999) Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity. Nat Med 5:694–697 Botto M, Hawkins PN, Bickerstaff MCM et al. (1997) Amyloid deposition is delayed in mice with targeted deletion of the serum amyloid P component gene. Nature Med 3:855–859 Buxbaum J, Tagoe C, Gallo G et al. (2003) The pathogenesis of transthyretin tissue deposition: Lessons from transgenic mice. Amyloid 10(Suppl 1):2–6 Bygrave AE, Rose KL, Cortes-Hernandez J et al. (2004) Spontaneous autoimmunity in 129 and C57BL/6 mice-implications for autoimmunity described in gene-targeted mice. PLoS Biol 2:e243 Cardoso I, Saraiva MJ (2006) Doxycycline disrupts transthyretin amyloid: Evidence from studies in a FAP transgenic mice model. FASEB J 20:234–239 Episkopou V, Maeda S, Nishiguchi S et al. (1993) Disruption of the transthyretin gene results in mice with depressed levels of plasma retinol and thyroid hormone. Proc Natl Acad Sci USA 90:2375–2379 Garlanda C, Bottazzi B, Bastone A et al. (2005) Pentraxins at the crossroads between innate immunity inflammation matrix deposition and female fertility. Annu Rev Immunol 23:337–366 Holmgren G, Bergstrom S, Drugge U et al. (1992) Homozygosity for the transthyretin-Met30-gene in seven individuals with familial amyloidosis with polyneuropathy detected by restriction enzyme analysis of amplified genomic DNA sequences. Clin Genet 41:39–41 Holmgren G, Ericzon B-G, Groth C-G et al. (1993) Clinical improvement and amyloid regression after liver transplantation in hereditary transthyretin amyloidosis. Lancet 341:1113–1116 Holmgren G, Wikstro¨m L, Lundgren HE et al. (2004) Discordant penetrance of the trait for familial amyloidotic polyneuropathy in two pairs of monozygotic twins. J Intern Med 256:453–456 Horie K, Maeda S, Nishiguchi S et al. (1995) A replacement vector used to introduce subtle mutations into mouse genes. Gene 166:197–204 Iwanaga T, Wakasugi S, Inomoto T et al. (1989) Liver-specific and high-level expression of human serum amyloid P component gene in transgenic mice. Dev Genet 10:365–371 Kohno K, Palha JA, Miyakawa K et al. (1997) Analysis of amyloid deposition in a transgenic mouse model of homozygous familial amyloidotic polyneuropathy. Am J Pathol 150:1497–1508 Macedo B, Batista AR, Ferreira N et al. (2008) Anti-apoptotic treatment reduces transthyretin deposition in a transgenic mouse model of Familial Amyloidotic Polyneuropathy. Biochim Biophys Acta 1782:517–522 Marrack P, Kappler J, Kotzin BL (2001) Autoimmune disease: Why and where it occurs. Nat Med 7:899–905 Murakami T, Yi S, Maeda S et al. (1992) Effect of serum amyloid P component level on transthyretin-derived amyloid deposition in a transgenic mouse model of familial amyloidotic polyneuropathy. Am J Pathol 141:451–456 Nagata Y, Tashiro F, Yi S et al. (1995) A 6-kb upstream region of the human transthyretin gene can direct developmental tissue-specific and quantitatively normal expression in transgenic mouse. J Biochem 117:169–175 Nakamura M, Ando Y, Nagahara Set al. (2004) Targeted conversion of the transthyretin gene in vitro and in vivo. Gene Ther 11:838–846 Noguchi H, Ohta M, Wakasugi S et al. (2002) Effect of the intestinal flora on amyloid deposition in a transgenic mouse model of familial amyloidotic polyneuropathy. Exp Anim 51:309–316 Olofsson BO, Backman C, Karp K et al. (2002) Progression of cardiomyopathy after liver transplantation in patients with familial amyloidotic polyneuropathy Portuguese type. Transplantation 73:745–751 Palha JA, Episkopou V, Maeda S et al. (1994) Thyroid hormone metabolism in a transthyretin-null mouse strain. J Biol Chem 269:33135–33139 Pepys MB, Booth DR, Hutchinson WL et al. (1997) Amyloid P component. A critical review. Amyloid 4:274–295
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Wei L, Kawano H, Fu X et al. (2004) Deposition of transthyretin amyloid is not accelerated by the same amyloid in vivo. Amyloid 11:113–120 Wei S, Episkopou V, Piantedosi R et al. (1995) Studies on the metabolism of retinol and retinolbinding protein in transthyretin-deficient mice produced by homologous recombination. J Biol Chem 270:866–870 Yamamura K, Tashiro F, Wakasugi S et al. (1990) Transgenic mouse model of autosomal dominant disease: Familial amyloidotic polyneuropathy. In: Beyreuther K Schttler G (ed) Molecular Mechanisms of Aging (pp 146–154) Springer-Verlag, Heidelberg. Yi S, Takahashi K, Naito M et al. (1991) Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene. Pathologic similarity to human familial amyloidotic polyneuropathy type I. Am J Pathol 138:403–412
Chapter 17
What Have We Learned from TTR-Null Mice: Novel Functions for TTR? Joa˜o Carlos Sousa and Joana Almeida Palha
Abstract Ablation of the gene encoding for transthyretin (TTR) in mice showed that, in the adult, TTR is not necessary for thyroid hormone and vitamin A delivery to tissues despite the decreased levels of serum thyroxine and vitamin A. Surprisingly, the absence of TTR resulted in behavioral alterations. Specifically, TTR-null mice display decreased signs of depressive-like behavior, increased exploratory activity, and impaired spatial learning, which may have implications in disorders such as Alzheimer’s disease. The present chapter discusses the lines of evidence implicating TTR in behavior, and whether these are directly or indirectly related to well described or novel TTR functions. Keywords Transthyretin knock-out, Mice, Hypothyroxinemia, Behavior, Vitamin A
Abbreviations AD Ab APP CSF ES RBP T3 T4
Alzheimer’s disease amyloid b peptide amyloid precursor protein cerebrospinal fluid embryonic stem retinol-binding protein triiodothyronine thyroxine
J.A. Palha (*) Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710–057 Braga, Portugal e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_17, # Springer-Verlag Berlin Heidelberg 2009
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TBG TTR TTR-null
thyroxine-binding globulin transthyretin mice with targeted disruption of the transthyretin gene
Fifteen years ago, when the first article on mice with targeted disruption of the transthyretin gene (TTR-null) was published (Episkopou et al. 1993), it was certainly with some surprise that the authors, then working in embryonic development, found that these mice were not only viable and fertile, but also did not display any gross abnormality. This was unexpected because transthyretin (TTR) is a major plasma and cerebrospinal fluid (CSF) carrier of thyroxine (T4) and retinol, both known to be essential for normal development. While at the time the lack of an overt phenotype in TTR-null mice suggested a redundant role for TTR, time and several experiments have challenged this view. In this chapter, we review the contribution of TTR-null mice in addressing the previously known TTR functions but also in uncovering possible new roles for this protein.
17.1
Transthyretin-Null Mice
TTR-null mice were generated by the technique of targeted gene disruption in embryonic stem (ES) cells (Capecchi 2005). Briefly, a marker for positive selection, the bacterial neomycin-resistance gene was introduced into exon 2 of a mouse Ttr gene fragment containing exons 1–3. After transfection in ES cells and homologous recombination, ES clones containing the mutant Ttr gene were injected into MF1 blastocytes and implanted in MF1 foster mothers who transmitted the mutated allele to their progeny (Episkopou et al. 1993). Ttr gene ablation did not seem to cause any major developmental impairment, at least with respect to mice viability and fertility (Episkopou et al. 1993). While the initial studies were performed in TTR-null mice in mixed (129Sv/ MF1) genetic background (Episkopou et al. 1993), backcrosses with inbred mice from diverse strains originated the currently available TTR-null mice models in 129Sv (Palha et al. 1994), C57Bl6 (Wati et al. 2008), or mixed 129Sv/C57Bl6 (Richardson et al. 2007; Brouillete and Quirion 2008) backgrounds.
17.2
Thyroid Hormones
Thyroid hormones, T4 and triiodothyronine (T3), are synthesized in the thyroid gland from where they are secreted into the blood. In circulation, 99.9% of the thyroid hormones are bound to carrier proteins: thyroxine-binding globulin (TBG), TTR, albumin and lipoproteins. TBG is the principal thyroid hormone plasma carrier in humans, transporting as much as 65% of T4, due to its higher affinity to the hormone (Schreiber 2002). In man, TTR transports only around 15% of total serum T4, but in rodent it is the major serum T4 carrier (Davis et al. 1970). In the
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CSF, TTR is the major T4 carrier in humans (Hagen and Solberg 1974) and seems to be the unique carrier in mice (Palha et al. 2000). Of the three plasma thyroid hormone transport proteins, albumin presents the lowest affinity to T4 and T3. Nevertheless, because of its high concentration in serum, albumin transports about 20% of thyroid hormones in humans (Schussler 2000). The role of carrier proteins in thyroid hormone transport and distribution to tissues has been a subject of great study and controversy (Palha 2002). Studies in analbuminemic rats and in humans with total TBG deficiency excluded an essential role for albumin and TBG, respectively, in mediating thyroid hormone transfer to tissues (Mendel et al. 1989; Refetoff 1989). It took longer to recognize that TTR, as well, was not essential for thyroid hormone homeostasis. In fact, the TTR ontogenic and phylogenetic expression patterns, together with its abundance in the choroid plexus and in the CSF, and the lack of its natural absence in man or in other animals, have suggested a major role for TTR in thyroid hormone homeostasis – particularly in T4 transfer into the brain (Pardridge 1981; Divino and Schussler 1990). To reach the brain T4 must cross either the blood–brain barrier and/or the choroid plexus– CSF barrier (Dratman et al. 1991). Because TTR synthesized by the choroid plexus is unidirectionally secreted into the CSF (Dickson et al. 1986), and since intravenous injections of radiolabeled T4 showed that radioactivity first appeared in the choroid plexus, then in the CSF, and only later in the cortex and striatum of the brain (Dickson et al. 1987), it was suggested that T4 transport from the blood into the brain involved TTR in the choroid plexus and CSF (Schreiber et al. 1990; Chanoine et al. 1992). Other studies however, questioned this view. Specifically, 125 I-T4 injected intraventricularly resided essentially in the spaces occupied by the CSF; and 125I-T4 injected intravenously accumulated mainly in the brain parenchyma (Dratman et al. 1991). The controversies described above on the role of TTR in thyroid hormone metabolism were extensively studied in the TTR-null mouse. Detailed thyroid hormone metabolism analysis of the TTR-null mice revealed a 50% decrease in serum total T4 levels that were not reflected in changes in total T3, free T4, or free T3 (Episkopou et al. 1993; Palha et al. 1994). Thyroid-stimulating hormone circulating levels, brain deiodinase type 2 activity, and the brain expression levels of the gene encoding for neurogranin, all known to respond to hypothyroidism or hyperthyroidism, were normal in TTR-null mice (Palha et al. 1994, 2000). In further support of TTR-null mice being euthyroid was the observation that both the mRNA and protein levels of TBG, well recognized as influenced by the thyroid status (Savu et al. 1989), were normal in the absence of TTR, which also showed that TBG was not compensating for the absence of TTR in the serum (Palha et al. 1994). While these studies suggested that TTR, per se, was not necessary for thyroid hormone homeostasis, great interest still remained on the transfer of T4 into the tissues, particularly into the brain, given the fact that TTR represents 20% of the total protein synthesis by the choroid plexus (Dickson et al. 1986; Schreiber et al. 1990; Southwell et al. 1993). In fact, T4 content in TTR-null mouse brain is only 70% of that in wild-type mice (Palha et al. 1997). However, this seems to reflect solely the absence of TTR in the choroid plexus since T4 distributes equally well within the
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brain parenchyma of TTR-null and control mice, as revealed by autoradiographic brain analysis upon intravenous administration of radiolabeled T4 and by measurement of T4 levels in various regions of the brain parenchyma (Palha et al. 2000, 2002). The evidence that in the absence of TTR mice were euthyroid did not exclude the possibility that TTR participated in thyroid hormone homeostasis under conditions of increased/decreased hormone demand, where circulating TTR could serve as an extrathyroidal T4 reservoir and/or buffer, releasing hormone to tissues under conditions of increased hormone need or protecting tissues against excessive circulating hormone concentrations. In fact, standard laboratory housing conditions are certainly not the best settings when compared to the conditions rodents face in the wild, where stressful and metabolically demanding situations require varied thyroid hormone contribution for several cellular processes. Experiments inducing mild (cold exposure) and severe (thyroid gland ablation) conditions of increased thyroid hormone demand were investigated in TTR-null mice (Sousa et al. 2005). In response to exposure to cold for 1 month, both TTR-null and control mice similarly increased the expression of uncoupling protein 1, a protein that promotes thermogenesis and deiodinase type 2 in brown adipose tissue, to provide the necessary additional T3. This was associated with equal increases in food intake. In addition, both TTR-null and wild-type mice exposed to cold maintained T4 and T3 serum and tissue levels identical to those of animals kept at room temperature. Similarly, when the thyroid was ablated, both TTR-null and control mice displayed a rapid decrease in thyroid hormone circulating and tissue levels that were not further aggravated by the absence of TTR (Sousa et al. 2005). Thus, TTR does not seem to be necessary as a reservoir for thyroid hormones, even in conditions of increased hormone need. While studies in TTR-null mice have excluded an essential role for TTR in hormone delivery to tissues even in conditions of increased hormone demand, its possible role as a buffer for thyroid hormones in conditions of increased hormone load, such as those found in hyperthyroidism, remains to be investigated. Altogether, the studies performed to investigate the role of TTR in thyroid hormone metabolism are in agreement with the free hormone hypothesis for T4 tissue uptake, which states that the biological activity of hormones is a function of their free concentration (Mendel 1989), which is normal in TTR-null mice. However, the meaning of ‘‘physiological’’ euthyroid hypothyroxinemia is far from consensual, particularly when this occurs during pregnancy, which is always the case in TTR-null dams. Thyroid hormones are essential for the proper development of the central nervous system, and thyroid hormone deficiency in specific periods during development has irreversible severe to subtle changes that remain later in life (Morreale de Escobar et al. 2004; Bernal 2005). Of note, several studies with rodents showed that the offspring of hypothyroxinemic dams display alterations in neuronal migration (Lavado-Autric et al. 2003; Auso et al. 2004; Cuevas et al. 2005), synaptic structure, and behavior (Gilbert and Sui 2006; Opazo et al. 2008). Whether these observations relate to the behavioral phenotype observed in TTR-null mice will be revisited later in the text.
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Retinoids
Retinoids are obtained from the diet and delivered to the liver, its major storage organ, as chylomicron retinyl esters (Ross and Zolfaghari 2004). Distribution of liver retinol stores to other body tissues is accomplished by liver secretion of retinol bound to the retinol-binding protein (RBP). TTR forms a complex with RBP, and so participates in the plasma transport of retinol. Binding to TTR prevents RBP glomerular filtration and renal catabolism (reviewed in Gottesman et al. 2001). Similar to the discussion on thyroid hormones, the literature has long debated whether TTR is needed for the liver secretion of the RBP-retinol complex (Goodman and Blaner 1984; Melhus et al. 1992) and for the distribution of retinol to tissues (Vogel et al 1999). Studies in TTR-null and also in RBP-null mice have given further insights on the role of these carrier proteins in retinoid metabolism. TTR-null mice present retinol plasma levels lower than 6% of those in wild-type mice (Episkopou et al. 1993; Wei et al. 1995). This drop in circulating retinol levels is not explained by deficient dietary retinoid uptake in TTR-null mice since total liver retinol (retinol + retinyl esters) is identical to that in control animals (Wei et al. 1995). Interestingly, TTR-null mice have increased liver RBP levels and solely 5% of the circulating levels found in wild-type, mice suggesting that TTR participates in RBP–retinol liver secretion (Wei et al. 1995). The observation that the TTR–RBP complex is formed in the endoplasmic reticulum of a hepatocyte cell line (Bellovino et al. 1996) provided additional evidence that TTR does in fact mediate, at least in part, RBP–retinol secretion from the liver. An explanation for the decreased RBP and retinol levels found in TTR-null mice could result from increased RBP filtration in the kidney in the absence of TTR. In agreement, despite normal urine RBP levels (Wei et al. 1995), TTR-null mice present an increased rate of RBP–retinol renal filtration (van Bennekum et al. 2001). In spite of the increased RBP liver levels, tissue retinol levels are similar in wildtype and TTR-null mice (Wei et al. 1995). Since RBP is the only plasma specific transport protein for retinol (Gottesman et al. 2001), TTR-null mice might have developed an alternative pathway for retinol delivery to the tissues. One possibility is retinyl ester transport in association with lipoproteins. Another compensatory mechanism could be an increase in tissue uptake of plasma retinoic acid, thus diminishing the need for retinol to retinoic acid oxidation in tissues. Supporting this hypothesis is the fact that TTR-null mice present a 2.3-fold increase in retinoic acid plasma levels when compared to wild-type (Wei et al. 1995). Similar to TTR-null mice, ablation of the mouse RBP gene resulted in viable and fertile animals. RBP-null mice present decreased levels of plasma retinol (12.5% of wild-type) but, surprisingly, have no apparent alternative retinol transport mechanisms (Quadro et al. 1999). However, RBP-null mice are unable to mobilize retinoid stores from hepatocytes and have impaired visual function as a consequence of low eye retinol content. Recovery of vision at around 5 months of age (Quadro et al. 1999) strongly suggests the existence of another mechanism of retinol delivery to
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the eye, independent of the retinol–RBP–TTR pathway. Because TTR-null mice show no visual impairment (Bui et al. 2001), the RBP levels present in TTR-null mice, even though strongly diminished, seem sufficient to provide adequate supply of retinoids for vision. Interestingly, RBP expression was reported to occur in the choroid plexus of rodents (Duan and Schreiber 1992) and RBP mRNA in the brain is 1–3% of the levels found in the liver (Soprano et al. 1986). The relevance of RBP expression in the choroid plexus and its local interaction with TTR for retinol supply to the brain remains to be clarified. Despite the possible relevance of this choroid plexus route for delivery of retinol to the brain, the retinol concentration in the cortex and cerebellum of TTR-null mice does not differ from that found in wild-type mice. Similarly, the expression of retinoic acid nuclear receptors is not affected in adult TTR-null mice brain (unpublished data). Therefore, TTR does not seem, likewise, necessary for retinoid delivery into the brain. Even though the data gathered to date in TTR-null mice do not suggest a major impact of the absence of TTR in retinoid metabolism, recent evidence has brought further interest on the subject, since behavioral alterations observed in adult TTRnull mice are recovered after administration of retinoic acid (Brouillete and Quirion 2008), as will be discussed next.
17.4
Behavioral Disorders
Abnormal thyroid hormone and retinoid availability impacts on brain development. In humans, several reports have associated TTR with behavior disorders such as depression (Sullivan et al. 1999), and neurodegenerative disorders such as Alzheimer’s disease (AD) (Serot et al. 1997). Whether the role of TTR in such diseases relates to its role as a thyroid hormone and retinol carrier or to another function of the protein is still debatable. TTR-null mice have contributed to strengthening evidence on the involvement of TTR in behavior and in shedding some light into which mechanisms might be involved in this process.
17.4.1 Depression A possible involvement of TTR in depression was first devised in a study with lumbar CSF from psychiatric patients. TTR was found to be 7.5% increased in depressed patients (Jorgensen 1988). On the contrary, subsequent studies found significantly lower TTR concentrations in the CSF from depressed patients when compared to a control group (Hatterer et al. 1993; Sullivan et al. 1999, 2006). Sullivan et al. (1999) suggested that decreased CSF TTR concentration could result
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in an insufficient supply of thyroid hormones to the brain leading to a state of hypothyroidism in the central nervous system. This hypothesis is in agreement with several observations regarding thyroid hormones and depression: abnormal thyrotropin response to thyrotropin releasing hormone stimulation is observed in 25% of depressed patients (Loosen and Prange 1982) and supplementation of antidepressant medication with T3 improves the response rate of patients refractory to narcoleptic treatment (Aronson et al. 1996). Nevertheless, data on thyroid hormone content in serum from depressed patients is still a matter of some controversy, namely due to the effects of antidepressant treatment (Premachandra et al. 2006; Eker et al. 2008). The diminished CSF TTR could result from the effect of psychotropic medication (Sullivan et al. 1999) which is in agreement with a report on the effects of lithium administration in diminishing choroid plexus TTR mRNA expression (Pulford et al. 2002). Therefore, the direction and significance of TTR concentration changes in depression is far from clearly understood. Of notice, behavioral characterization of TTR-null mice revealed that in the absence of TTR, mice display reduced signs of depressive-like behavior as evaluated in the Porsolt forced swim test, increased exploratory activity in the open field test, and no signs of anxiety phenotype as evaluated using the elevated plus maze (Sousa et al. 2004). These observations are concordant with increased levels of noradrenaline in the limbic forebrain of TTR-null mice (Sousa et al. 2004). Still remaining to be investigated are the reasons for such noradrenaline increase, since they do not result from increased synthesis or decreased degradation of the neurotransmitter. One possibility may be an increase in synaptic density in the amygdala of TTR-null mice as suggested (Pego et al. 2003). It is reasonable to speculate that some of the observed changes may originate from developmental impairment caused by hypothyroidism and/or hypovitaminosis A, which should be further investigated.
17.4.2 Alzheimer’s Disease, Aging, and Memory Impairment Possibly the most exciting observations on the role of TTR in behavior regard its involvement in memory and cognition, which has implications for AD. Alzheimer’s disease is a neurodegenerative disorder characterized by the deposition, in the brain parenchyma and in the cerebrovasculature, of the amyloid b peptide (Ab) and by the formation of neurofibrilary tangles composed of aggregates of the tau protein (Dickson 2004). Discussion on Ab deposition has been divided on whether it is a consequence of increased production and/or decreased clearance of Ab out of the brain (Selkoe 2001). TTR seems to be involved in both. The first evidence of the involvement of TTR in AD came from the demonstration that TTR has the ability to bind Ab in vitro (Schwarzman et al. 1994). Therefore, a role for TTR in sequestering Ab in the CSF and in preventing amyloid formation was suggested. Studies with transgenic Caenorhabditis elegans coexpressing human Ab and human TTR presented reduced amyloid deposition which
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further supported a role for TTR in inhibiting amyloid fibril formation (Link 1995). Interestingly, Ab and TTR colocalize and coimmunoprecipitate in the kidney of AD patients (Tsuzuki et al. 1996, 2000). Recently, it was also shown that TTR is capable of proteolytically cleave Ab and thus abrogates the toxicity of the peptide (Costa et al. 2008), in this way contributing to decreased Ab availability. Studies in the CSF of AD patients reported decreased TTR levels, which would be in accordance with a decreased capacity for Ab binding and sequestration and, therefore, for increased availability of free circulating Ab for amyloid formation (Riisoen 1988; Serot et al. 1997; Merched et al. 1998; Castano et al. 2006). However, the literature is still not unanimous on the TTR levels in the CSF of AD patients, since in other patients no alterations were found (Sampaolo et al. 2005). Additional data emerged from studies in animal models of aging and AD. In aged rats, insulin growth factor I was shown to induce clearance of Ab out of the brain, a process suggested to be mediated by TTR (Carro et al. 2002, 2005, 2006). A protective role for TTR, as a sequester for Ab, was also suggested in studies in a transgenic mice model (tg2576 strain) overexpressing a mutant form of human amyloid precursor protein (APP). In spite of the higher brain levels of Ab, the mild neurodegenerative phenotype of these mice was attributed to increased levels of TTR in the hippocampus (Stein and Johnson 2002; Stein et al. 2004). Similarly, the neuroprotective effects against AD on aged rats feed on an n-3 polyunsaturated fatty acid diet (Puskas et al. 2003) or with Ginkgo biloba extracts (Watanabe et al. 2001), was attributed to their ability to increase the expression of Ttr in the hippocampus. Altogether, these studies support the view that increasing the expression of the Ttr gene and/or the TTR protein levels is neuroprotective in AD. A note is necessary, at this point, on the sites of TTR synthesis within the brain, since gross dissection of brain regions such as the hippocampus carries on contamination with choroid plexus, in which the Ttr gene is highly expressed. A detailed study on gene expression in brain regions collected by laser microdissection clearly showed that Ttr is not expressed in the brain parenchyma in wild-type mice or in transgenic mouse models of AD (Sousa et al. 2007a). Therefore, in the context of AD, it is important to consider the choroid plexus and the CSF as the most probable origin of the TTR present in the brain parenchyma. More on the possible involvement of TTR in AD comes from studies in transgenic mice models of AD in the TTR-null background. As expected when considering TTR as a relevant sequester of Ab, mice overexpressing mutated human APP, APPswe/PS1deltaE9 strain (Choi et al. 2007), and APP23 strain (Buxbaum et al. 2008), in a TTR-null background displayed increased amyloid load in the brain, which was associated with cognitive impairment. In accordance, when APP-overexpressing mice were backcrossed with human TTR-overexpressing mice, both biochemical and behavioral effects of Ab were reverted (Buxbaum et al. 2008). These observations, however, were recently challenged by Wati et al. (2008) who showed that eliminating TTR by backcrossing TTR-null mice with APP transgenic mice (tg2576 strain) did not significantly affect Ab load or tau phosphorylation. Strikingly, Ab vascular deposition was even reduced in the
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absence of TTR. Clarification on the role of TTR in Ab peptide deposition is therefore needed. One other aspect that should be considered is the fact that both ligands transported by TTR, thyroid hormone and retinol, are also related to AD. Thyroid hormones are reported to decrease APP expression, through a functional thyroid response element in the gene, and affect splicing and secretion of APP isoforms in a neuronal cell line (Belandia et al. 1998, Latasa et al. 1998). Retinoic acid also regulates APP expression (Konig et al. 1990). Moreover, it was suggested that impaired functioning of retinoid transport and signalling pathways may be involved in the disease state (Goodman and Pardee 2003). This involvement was recently demonstrated in a dietary vitamin A deficient adult rat model that presented increased deposition of Ab and downregulation of genes involved in retinoid action (Corcoran et al. 2004). Since TTR is a carrier of T4 and retinol, it is certainly interesting to further investigate whether the decreased T4 and vitamin A levels in TTR-null mice have implications toward increased susceptibility to develop AD. Irrespective of the precise mechanisms implicated, studies in TTR-null mice do suggest the involvement of TTR in learning and memory. At 5 months of age, TTR-null mice take longer than wild-type mice to learn the platform position (a hippocampus-dependent task) in the Morris water maze test – a test that addresses deficits in spatial memory. While in control mice a decline in spatial memory function is observed with age, the impairment observed in TTR-null mice at 5 months of age is not further aggravated at 18 months of age. When the platform position in the maze is changed (a prefrontal cortex dependent task), both groups of animals behave similarly in the acquisition of the new platform location (Sousa et al. 2007b). Taken together these results indicate that the absence of TTR seems to anticipate the cognitive deficits normally observed in aged subjects; and that the observed alterations in behavior are related to hippocampal dysfunction. The spatial memory impairment in adult (7-month-old) TTR-null mice was confirmed by Brouillette and Quirion (2008). Evaluation of gene expression differences in the hippocampus (including choroid plexus) of aged memory-unimpaired and aged memory-impaired rats found decreased expression of Ttr in the latter group. Interestingly, the spatial memory deficits were reversed when TTR-null mice were injected with retinoic acid during the days of the test (Brouillette and Quirion 2008), which again brings into discussion that some of the behavioral phenotype of TTR-null mice may be only indirectly attributed to TTR. One of the well described behavioral consequences of dysfunction in thyroid hormone and retinoid metabolism and/or signaling is alteration in cognitive function, namely memory impairment (Cocco et al. 2002; Rivas and Naranjo 2007). Hence, whether the memory impairment phenotype observed in TTR-null mice is specifically related to the thyroid hormone and/or retinoid function deserves further studies.
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Others: Energy Metabolism
Hypothyroxinemia and hypovitaminosis A may also interfere with general energetic metabolism. Both thyroid hormones and retinoids influence cellular metabolism through binding to their respective nuclear receptors that, in turn, regulate the expression of several genes involved in energy production/expenditure. To evaluate if the absence of TTR could result in impaired energy metabolism, glucose and lipid metabolisms were studied in TTR-null mice. Comparison of morphometric parameters such as body weight and number and volume of adipocytes revealed no differences between TTR-null and control mice (Marques et al. 2007). Quantification of serum glucose, triglycerides, cholesterol, and leptin levels in the fed state were similar in both mutant and control strains. In addition, glucose- and insulin-tolerance tests revealed that TTR-null mice were equally able to remove glucose from circulation as wild-type animals. Analysis, in the liver and white adipose tissue, of gene expression for several nuclear receptors that modulate the expression of genes involved in lipid and glucose metabolisms showed no influence of the absence of TTR (Marques et al. 2007). These results indicate that the reported increased brain levels of the orexigenic hormone neuropeptide Y in TTR-null mice (Nunes et al. 2006) do not result in a deregulation of lipid and glucose metabolism. Interestingly, NPY is negatively regulated by retinoic acid (Magni et al. 2000) and therefore this effect on NPY levels may again be solely indirectly related to TTR. In any case, altered NYP signalling may underlie some of the behavior changes observed in TTRnull mice since NPY is reported to influence anxiety- and depressive-like behavior, and spatial learning (Heilig 2004; Thorsell et al. 2000).
17.6
Final Remarks
TTR is well described as a carrier for thyroxine and retinol. While studies in TTRnull mice have excluded a major role for TTR in thyroxine and retinol delivery into tissues, it is still uncertain whether the observed hypothyroxinemia and hypovitaminoses A induce neurodevelopmental changes that result in emotional and cognitive impairment, as observed in TTR-null mice. In order to further analyze this, future studies should address specific neurodevelopment processes such as neuronal proliferation, migration, and apoptosis [notably, it was recently shown that apoptosis is diminished in the subventricular zone of adult TTR-null mice (Richardson et al. 2007)], as well as myelinization in TTR-null mice when compared to animals born from dams in which conditions of hypothyroxinemia and/or hypovitaminosis A are induced.
Acknowledgment The more recent work from our laboratory presented in this chapter was funded by Grant POCTI/SAU-NEU/56618/2004 from Fundaca˜o para a Cieˆncia e Tecnologia (Portugal)/FEDER.
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Mendel CM (1989) The free hormone hypothesis: A physiologically based mathematical model. Endocr Rev 10:232–274 Mendel CM, Cavalieri RR, Gavin LA, Pettersson T, Inoue M (1989) Thyroxine transport and distribution in Nagase analbuminemic rats. J Clin Invest 83:143–148 Merched A, Serot JM, Visvikis S, Aguillon D, Faure G, Siest G (1998) Apolipoprotein E, transthyretin and actin in the CSF of Alzheimer’s patients: Relation with the senile plaques and cytoskeleton biochemistry. FEBS Lett 425:225–228 Morreale de Escobar G, Obregon MJ, Escobar del Rey F (2004) Role of thyroid hormone during early brain development. Eur J Endocrinol 151(Suppl 3):U25–U37 Nunes AF, Saraiva MJ, Sousa MM (2006) Transthyretin knockouts are a new mouse model for increased neuropeptide Y. FASEB J 20:166–168 Opazo MC, Gianini A, Pancetti F, Azkcona G, Alarcon L, Lizana R, Noches V, Gonzalez PA, Porto M, Mora S, Rosenthal D, Eugenin E, Naranjo D, Bueno SM, Kalergis AM, Riedel CA (2008) Maternal hypothyroxinemia impairs spatial learning and synaptic nature and function in the offspring. Endocrinology 149:5097–5106 Palha JA (2002) Transthyretin as a thyroid hormone carrier: Function revisited. Clin Chem Lab Med 40:1292–1300 Palha JA, Episkopou V, Maeda S, Shimada K, Gottesman ME, Saraiva MJ (1994) Thyroid hormone metabolism in a transthyretin-null mouse strain. J Biol Chem 269:33135–33139 Palha JA, Fernandes R, de Escobar GM, Episkopou V, Gottesman M, Saraiva MJ (2000) Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: Study in a transthyretin-null mouse model. Endocrinology 141:3267–3272 Palha JA, Hays MT, Morreale de Escobar G, Episkopou V, Gottesman ME, Saraiva MJ (1997) Transthyretin is not essential for thyroxine to reach the brain and other tissues in transthyretinnull mice. Am J Physiol 272:E485–E493 Palha JA, Nissanov J, Fernandes R, Sousa JC, Bertrand L, Dratman MB, Morreale de Escobar G, Gottesman M, Saraiva MJ (2002) Thyroid hormone distribution in the mouse brain: The role of transthyretin. Neuroscience 113:837–847 Pardridge WM (1981) Transport of protein-bound hormones into tissues in vivo. Endocr Rev 2:103–123 Pego JM, Cerqueira JJ, Palha J, Sousa N (2003) Morphological changes in the basolateral division of the amygdala of TTR-null mice. In: Sixth IBRO World Congress of Neuroscience, Prague, p 207 Premachandra BN, Kabir MA, Williams IK (2006) Low T3 syndrome in psychiatric depression. J Endocrinol Invest 29:568–572 Pulford DJ, Henry B, Adams F, Harries DN, Mallinson DJ, Reid IC, Stewart CA (2002) Chronic administration of lithium chloride down-regulates transthyretin mRNA expression in rat brain. Society for Neuroscience 32nd Annual Meeting, Washington, DC Puskas LG, Kitajka K, Nyakas C, Barcelo-Coblijn G, Farkas T (2003) Short-term administration of omega 3 fatty acids from fish oil results in increased transthyretin transcription in old rat hippocampus. Proc Natl Acad Sci USA 100:1580–1585 Quadro L, Blaner WS, Salchow DJ, Vogel S, Piantedosi R, Gouras P, Freeman S, Cosma MP, Colantuoni V, Gottesman ME (1999) Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein. EMBO J 18:4633–4644 Refetoff S (1989) Inherited thyroxine-binding globulin abnormalities in man. Endocr Rev 10: 275–293 Richardson SJ, Lemkine GF, Alfama G, Hassani Z, Demeneix BA (2007) Cell division and apoptosis in the adult neural stem cell niche are differentially affected in transthyretin null mice. Neurosci Lett 421:234–238 Riisoen H (1988) Reduced prealbumin (transthyretin) in CSF of severely demented patients with Alzheimer’s disease. Acta Neurol Scand 78:455–459 Rivas M, Naranjo JR (2007) Thyroid hormones, learning and memory. Genes Brain Behav 6 (Suppl 1):40–44
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Ross AC, Zolfaghari R (2004) Regulation of hepatic retinol metabolism: Perspectives from studies on vitamin A status. J Nutr 134:269S–275S Sampaolo S, Campos-Barros A, Mazziotti G, Carlomagno S, Sannino V, Amato G, Carella C, Di Iorio G (2005) Increased cerebrospinal fluid levels of 3,30 ,50 -triiodothyronine in patients with Alzheimer’s disease. J Clin Endocrinol Metab 90:198–202 Savu L, Vranckx R, Maya M, Gripois D, Blouquit MF, Nunez EA (1989) Thyroxine-binding globulin and thyroxine-binding prealbumin in hypothyroid and hyperthyroid developing rats. Biochim Biophys Acta 992:379–384 Schreiber G (2002) The evolutionary and integrative roles of transthyretin in thyroid hormone homeostasis. J Endocrinol 175:61–73 Schreiber G, Aldred AR, Jaworowski A, Nilsson C, Achen MG, Segal MB (1990) Thyroxine transport from blood to brain via transthyretin synthesis in choroid plexus. Am J Physiol 258: R338–R345 Schussler GC (2000) The thyroxine-binding proteins. Thyroid 10:141–149 Schwarzman AL, Gregori L, Vitek MP, Lyubski S, Strittmatter WJ, Enghilde JJ, Bhasin R, Silverman J, Weisgraber KH, Coyle PK, et al. (1994) Transthyretin sequesters amyloid beta protein and prevents amyloid formation. Proc Natl Acad Sci USA 91:8368–8372 Selkoe DJ (2001) Clearing the brain’s amyloid cobwebs. Neuron 32:177–180 Serot JM, Christmann D, Dubost T, Couturier M (1997) Cerebrospinal fluid transthyretin: Aging and late onset Alzheimer’s disease. J Neurol Neurosurg Psychiatr 63:506–508 Soprano DR, Soprano KJ, Goodman DS (1986) Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat. J Lipid Res 27:166–171 Sousa JC, Cardoso I, Marques F, Saraiva MJ, Palha JA (2007a) Transthyretin and Alzheimer’s disease: Where in the brain? Neurobiol Aging 28:713–718 Sousa JC, de Escobar GM, Oliveira P, Saraiva MJ, Palha JA (2005) Transthyretin is not necessary for thyroid hormone metabolism in conditions of increased hormone demand. J Endocrinol 187:257–266 Sousa JC, Grandela C, Fernandez-Ruiz J, de Miguel R, de Sousa L, Magalhaes AI, Saraiva MJ, Sousa N, Palha JA (2004) Transthyretin is involved in depression-like behaviour and exploratory activity. J Neurochem 88:1052–1058 Sousa JC, Marques F, Dias-Ferreira E, Cerqueira JJ, Sousa N, Palha JA (2007b) Transthyretin influences spatial reference memory. Neurobiol Learn Mem 88:381–385 Southwell BR, Duan W, Alcorn D, Brack C, Richardson SJ, Kohrle J, Schreiber G (1993) Thyroxine transport to the brain: Role of protein synthesis by the choroid plexus. Endocrinology 133:2116–2126 Stein TD, Anders NJ, DeCarli C, Chan SL, Mattson MP, Johnson JA (2004) Neutralization of transthyretin reverses the neuroprotective effects of secreted amyloid precursor protein (APP) in APPSW mice resulting in tau phosphorylation and loss of hippocampal neurons: Support for the amyloid hypothesis. J Neurosci 24:7707–7717 Stein TD, Johnson JA (2002) Lack of neurodegeneration in transgenic mice overexpressing mutant amyloid precursor protein is associated with increased levels of transthyretin and the activation of cell survival pathways. J Neurosci 22:7380–7388 Sullivan GM, Hatterer JA, Herbert J, Chen X, Roose SP, Attia E, Mann JJ, Marangell LB, Goetz RR, Gorman JM (1999) Low levels of transthyretin in the CSF of depressed patients. Am J Psychiatry 156:710–715 Sullivan GM, Mann JJ, Oquendo MA, Lo ES, Cooper TB, Gorman JM (2006) Low cerebrospinal fluid transthyretin levels in depression: Correlations with suicidal ideation and low serotonin function. Biol Psychiatr 60:500–506 Thorsell A, Michalkiewicz M, Dumont Y, Quirion R, Caberlotto L, Rimondini R, Mathe AA, Heilig M (2000) Behavioral insensitivity to restraint stress, absent fear suppression of behavior and impaired spatial learning in transgenic rats with hippocampal neuropeptide Y overexpression. Proc Natl Acad Sci USA 97:12852–12857
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Tsuzuki K, Fukatsu R, Hayashi Y, Yoshida T, Sasaki N, Takamaru Y, Yamaguchi H, Tateno M, Fujii N, Takahata N (1996) Amyloid beta protein and transthyretin, sequestrating protein colocalize in normal human kidney. Neurosci Lett 222:163–166 Tsuzuki K, Fukatsu R, Yamaguchi H, Tateno M, Imai K, Fujii N, Yamauchi T (2000) Transthyretin binds amyloid beta peptides, Abeta1–42 and Abeta1–40 to form complex in the autopsied human kidney – Possible role of transthyretin for abeta sequestration. Neurosci Lett 281: 171–174 van Bennekum AM, Wei S, Gamble MV, Vogel S, Piantedosi R, Gottesman M, Episkopou V, Blaner WS (2001) Biochemical basis for depressed serum retinol levels in transthyretindeficient mice. J Biol Chem 276:1107–1113 Vogel S, Gamble MV, Blaner WS (1999) Biosynthesis, absorption, metabolism and transport of retinoids. In: Nau H, Blaner WS (eds) The retinoids. Springer Verlag, Heidelberg, pp 31–96 Watanabe CM, Wolffram S, Ader P, Rimbach G, Packer L, Maguire JJ, Schultz PG, Gohil K (2001) The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba. Proc Natl Acad Sci USA 98:6577–6580 Wati H, Kawarabayashi T, Matsubara E, Kasai A, Hirasawa T, Kubota T, Harigaya Y, Shoji M, Maeda S (2008) Transthyretin accelerates vascular Abeta deposition in a mouse model of Alzheimer’s disease. Brain Pathol 19:48–57 Wei S, Episkopou V, Piantedosi R, Maeda S, Shimada K, Gottesman ME, Blaner WS (1995) Studies on the metabolism of retinol and retinol-binding protein in transthyretin-deficient mice produced by homologous recombination. J Biol Chem 270:866–870
Chapter 18
Transthyretin Null Mice: Developmental Phenotypes Julie A. Monk and Samantha J. Richardson
Abstract Transthyretin (TTR) is an extracellular thyroid hormone (TH) distributor protein. The TH distributor proteins ensure the adequate distribution of THs throughout the body, buffer against excess TH uptake into cells and maintain an extrathyroidal reserve of THs that may protect against TH deficiency when TH demand is increased. Thyroid hormones are vital for normal postnatal development. Thus, the postnatal development and growth of tissues responsive to THs has been investigated in TTR null mice. Although the developmental surge in plasma T4 concentrations was evident in 2-week-old TTR null mice, total and free T4 in the plasma were significantly reduced. Characteristics of the developing TTR null mice included delayed suckling-to-weaning transition, delayed onset of growth and retarded longitudinal bone growth. In addition, ileums from newborn TTR null mice displayed disordered cellular structure and contained fewer goblet cells. Although TH homeostasis within the brain of the developing TTR null mice did not appear to be compromised, subtle differences suggested a degree of immaturity in the developing brain, such as higher protein concentrations of cerebrospinal fluid from newborn and 2-week-old TTR null mice than in agematched wild type mice. Collectively, these studies demonstrate the importance of TTR during post-natal development and suggest that the development of the central nervous system is essentially preserved at the expense of peripheral tissues in TTR null mice. Keywords Bone, Brain, Central nervous system, Development, Homeostasis, Thyroid hormones, Transthyretin
J.A. Monk (*) ProScribe Medical Communications (www.proscribe.com.au), 481 Gilbert Rd, Preston VIC 3072, Australia e-mail:
[email protected]
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Abbreviations CNS CSF D1 D2 T3 T4 TBG TH TR TTR
Central nervous system cerebrospinal fluid Type 1 deiodinase Type 2 deiodinase 30 ,3,5-triiodo-L-thyronine 30 ,50 ,3,5-tetraiodo-L-thyronine Thyroxine-binding globulin Thyroid hormone Thyroid hormone receptor Transthyretin
18.1
Introduction
Transthryetin (TTR) is an extracellular protein that binds the thyroid hormones (THs). The distribution of THs to tissues via the blood is essential for TH homeostasis. In humans, albumin and thyroxine-binding globulin (TBG) also bind THs, in addition to TTR, and collectively these proteins are known as the TH distributor proteins (Schreiber and Richardson 1997). The TH distributor proteins ensure the adequate distribution of THs throughout the body, buffer against excess TH uptake into cells and maintain an extrathyroidal reserve of THs that may protect against TH deficiency when TH demand is increased (Mendel et al. 1987; Schreiber and Richardson 1997). Thyroid hormones bound to TH distributor proteins are in equilibrium with unbound THs. By dissociation of TH, the TH distributor proteins sustain the free TH pool in blood and ensure that THs are available to all the cells throughout the body.
18.2
Thyroid Hormones and Development
18.2.1 Thyroid Hormones Transthryetin binds the THs 30 ,50 ,3,5-tetraiodo-L-thyronine (T4), and 30 ,3,5-triiodoL-thyronine (T3). T4 is the predominant TH synthesised in, and secreted from, the thyroid gland (Taurog and Evans 1967) and the predominant TH found in blood (Gross et al. 1950). T3 in blood is secreted by the thyroid gland (Gross and PittRivers 1953), which synthesises T3 de novo and produces T3 by the deiodination of
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T4 (Ishii et al. 1981; van Doorn et al. 1983), and extrathyroidal tissues, which produce T3 by the deiodination of T4 (Pittman et al. 1971). About 80% of T4 secreted into circulation is deiodinated to T3 by tissue specific deiodinases (Larsen et al. 1981). Most, but not all, physiological effects of TH are elicited genomically by T3 binding to specific nuclear receptors known as TH receptors (TR).
18.2.2 Thyroid Hormones in Developing Vertebrates The postnatal development and growth of certain tissues in vertebrates, such as the central nervous system (CNS), bone and the intestine, is explicitly dependent on THs (Yen 2001). Insufficient THs (hypothyroidism) during development can lead to marked intellectual impairment, and arrested growth and delayed bone age or growth of bones in humans (Anderson 2001; Baran 1996; Rovet 1999). These features are most evident in humans who have endemic or myxedematous cretinism (congenital hypothyroidism). The plasma concentrations of T4 rarely fluctuate during the adult life of most vertebrates (Hulbert 2000). In contrast, plasma concentrations of T4 (and T3) rise sharply in developing vertebrates, reaching concentrations greater than those found in adult vertebrates. Plasma concentrations of TH peak at critical developmental stages: during smoulting in salmonid fishes (Specker et al. 1984), during metamorphosis in fish (de Jesus et al. 1991; Miwa and Inui 1987) and amphibians (Mondou and Kaltenbach 1979; Suzuki and Suzuki 1981; Weber et al. 1994); immediately before hatching in birds (Darras et al. 1992; Thommes and Hylka 1977) and reptiles (Shepherdley et al. 2002); during pouch life in marsupials (Buaboocha and Gemmell 1995; Janssens et al. 1990); and either within the first two weeks after birth in altricial mammals, such as mice (Hadj-Sahraoui et al. 2000), or before birth in precocial mammals, such as sheep (Wrutniak et al. 1985). During vertebrate development, the rise in the plasma concentrations of TH is accompanied by the onset of synthesis of an additional TH distributor protein, resulting in an increase in the TH-binding capacity of the blood (Richardson et al. 2005). Those vertebrates that synthesise albumin as the only TH distributor protein during adult life, such as fish, amphibians, reptiles and polyprotodont marsupials, synthesise albumin and TTR during development (Funkenstein et al. 1999; Richardson et al. 2005). Likewise, those species that synthesise albumin and TTR during adult life synthesise albumin, TTR and TBG during development (Richardson et al. 2002; Richardson et al. 2005). The rise in the plasma concentrations of TH is also accompanied by the onset of synthesis of, or the modulation of, deiodinases and TRs in individual tissues (Bianco et al. 2002; O’Shea and Williams 2002). These changes maintain the increased plasma pool of TH, and modify TH levels and the TH response within tissues according to the developmental demands.
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Postnatal Development of Transthyretin Null Mice
18.3.1 Thyroid Hormone Levels in Plasma Thyroid hormone levels were reduced in the plasma of the developing TTR null mice (Table 18.1). Thyroid hormone levels in the plasma from mice increase during development and peak at day 15 after birth before they decline to adult levels (HadjSahraoui et al. 2000). The developmental surge in plasma T4 concentrations was evident in 2-week-old TTR null mice. Plasma levels of total and free T4 were greater in 2-week-old TTR null mice than in adult TTR null mice; however, the concentrations attained were 50% and 46% of those in 2-week-old wild type mice (Table 18.1). Only the total T3 levels in the plasma from 2-week-old TTR null mice were significantly lower than those in 2-week-old wild type mice. As TTR binds a significant proportion of T4 in mouse plasma (Vranckx et al. 1990), reduced plasma levels of total T4 were expected. Given the explicit requirement for THs during development, the postnatal development and growth of tissues responsive to THs may be compromised in TTR null mice as a result of reduced circulating levels of THs.
18.3.2 Postnatal Development of Peripheral Tissues 18.3.2.1
Suckling-to-Weaning Transition
T4 and corticosterone coordinate the changes to the physiological and biochemical processes, such as feeding, digestion and metabolism, that occur during the transition from suckling to weaning (the dietary transition from suckling milk to eating Table 18.1 Plasma concentrations of total and free thyroid hormones (T4 and T3) in 2-week-old (P14) and adult wild type (WT) and transthyretin (TTR) null mice Free T4 (pM) Total T3 (nM) Free T3 (pM) Total T4 (Nm) 71.75 7.83 9.35 1.31 1.07 0.03 1.20 0.07 (n = 4) (n = 6) (n = 4) (n = 4) TTR null 34.23 3.60*** 4.34 0.36*** 0.93 0.04* 1.27 0.08 (n = 4) (n = 6) (n = 4) (n = 4) Adult WT 22.56 1.14 3.21 0.33 1.17 0.10 0.85 0.05 (n = 19) (n = 13) (n = 8) (n = 7) TTR null 15.51 0.74*** 1.19 0.14*** 0.96 0.11 0.66 0.05* (n = 14) (n = 9) (n = 6) (n = 6) Adapted from Monk (2006). Levels of total and free T4, and total and free T3 were measured in plasma by 125I-radioimmunoassay (RIA) (Coat-a-Count kits, Diagnostic Products Corporation, USA). Each sample was assayed in duplicate and the concentrations were averaged to give the total or free TH concentration. Values are mean SEM. Statistical significance between means (one way ANOVA) of the thyroid test examined. Statistical significance is indicated by *p < 0.05, ***p < 0.005 P14
WT
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solids) (Henning 1981). During the first two weeks after birth and before weaning commences, serum levels of insulin and glucose in rats are low (Bla´zquez et al. 1974). While suckling during the first two weeks after birth, pups ingest milk, which is high in fat and low in carbohydrate. During weaning, mammals begin to consume solid food, which is high in carbohydrate and low in fat. As a consequence, insulin and glucose levels in plasma increase, and lipogenesis in the liver and white adipose tissue increases (Girard et al. 1992). Mice are usually fully weaned by 3–4 weeks of age. Coincidentally, the concentration of TTR in the blood from developing rats at weaning (2–4 weeks of age) increases sharply (Thomas and Schreiber 1985). Low blood concentrations of glucose raised the possibility of a delay in the suckling-to-weaning transition in developing TTR null mice. The mean concentration of glucose in the plasma from 2-week-old TTR null mice (11.3 0.61 mM, n = 6) was significantly lower than that in 2-week-old wild type mice (13.3 0.53 mM, n = 6; p < 0.05) (Monk 2006). Hypothyroidism can cause a decrease in glucose concentrations in plasma (Althausen 1949). However, the plasma concentrations of glucose did not differ between adult TTR null mice (20.1 2.16 mM, n = 6) and wild type mice (19.0 0.91 mM, n = 6; n.s.) (Monk 2006), suggesting that the low blood concentrations of glucose in developing TTR null mice may result from a developmental lag in the regulation of glucose levels. Interestingly, the suggested delay in the suckling-to-weaning transition in developing TTR null mice is supported by a significantly (p < 0.05) lower level of malic enzyme mRNA in the liver from 2-week-old TTR null mice, compared with that in the liver from age-matched wild type mice (Monk 2006). Malic enzyme assists in the synthesis of long-chain fatty acids, and the amount of malic enzyme in the liver increases when experimental animals are fed a diet that is high in carbohydrate and low in fat (Goodridge et al. 1996). T3 positively regulates the transcription and stabilises the mRNA precursor of malic enzyme (Song et al. 1988). However, glucose also stimulates the expression and synthesis of malic enzyme (Molero et al. 1993). Thus, in 2-week-old TTR null mice, the lower levels of malic enzyme mRNA may be a result of lower glucose concentrations in the plasma rather than a direct consequence of lower total and free T4 levels. Indeed, similar mRNA levels of type 1 deiodinase (D1) and the TR isoform b1 in the livers from both genotypes suggest that TH function within the liver of 2-week-old TTR null mice is not altered (Monk 2006). However, given that T4 co-ordinates feeding, digestion and metabolism (Henning 1981), the decreased plasma levels of total and free T4 may delay the suckling-to-weaning transition in 2-week-old TTR null mice, resulting in lower plasma concentrations of glucose and, as a consequence, lower levels of malic enzyme mRNA in the liver than those in 2-week-old wild type mice.
18.3.2.2
Onset of Rapid Growth
The blood chemistry of developing TTR null mice suggested that the onset of rapid growth that occurs after two weeks of age in mice may be delayed. The blood
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chemistry of 2-week-old TTR null mice was characterised by lower creatinine concentrations (13.7 3.01 mM, n = 6, vs age-matched wild type mice, 24.3 0.67 mM, n = 6; p < 0.01) and higher plasma urea concentrations (9.60 0.32 mM, n = 6, vs age-matched wild type mice 7.51 0.58 mM, n = 6; p < 0.005) (Monk 2006). Although 2-week-old TTR null mice and wild type mice (7.47 0.15 g, n = 21 vs. 7.13 0.10, n = 21) were of similar body weight (Monk 2006), the decreased plasma concentration of creatinine in 2-week-old TTR null mice suggests reduced skeletal muscle mass. Plasma creatinine concentrations are often lower in mammals with reduced muscle mass. For example, plasma creatinine concentrations in dogs varied according to breed or muscle mass whereas plasma urea concentrations did not (Me´daille et al. 2004). With less muscle mass, the area available for creatine phosphate storage in muscle is reduced; therefore, the conversion of creatine phosphate to creatinine is reduced. Mice experience rapid body growth from 2 to about 5 weeks of age (Fraichard et al. 1997), and plasma urea concentrations decrease during periods of rapid growth when protein anabolism in the liver is favoured (Meyer and Harvey 2004). The lower plasma concentrations of urea in 2-week-old wild type mice, compared with 2-week-old TTR null mice, may be due to increased protein anabolism and the onset of rapid growth. The plasma concentrations of urea in 2-week-old TTR null mice were similar to those in adult wild type and TTR null mice (Monk 2006), suggesting that 2-week-old TTR null mice have not yet entered a period of rapid growth. Despite similar body weights of 2-week-old wild type mice and TTR null mice, the higher plasma concentrations of urea, and lower plasma concentrations of creatinine, in 2-week-old TTR null mice suggest that the onset of rapid growth that occurs after 2 weeks of age may be delayed in TTR null mice.
18.3.2.3
Bone Development
Attributes of hypothyroid juvenile humans who are left untreated include arrested growth, delayed bone age and epiphyseal dysgenesis (Baran 1996; Underwood and Van Wyk 1992). Bones develop and lengthen when chondrocytes (cartilage cells) located in epiphyseal growth plates (the epiphysis is the rounded end of the long bones) mature, proliferate and differentiate in an organised and coordinated manner (Howell and Dean 1992). The activity of chondrocytes is proposed to be highly sensitive to TH concentrations within the growth plate, which are regulated intracellularly by D1 and type 2 deiodinase (D2) (Miura et al. 2002; Shen et al. 2004). The TR isoforms a1, a2, and b1 were localised to reserve zone progenitor chondrocytes and proliferative zone chondrocytes but not hypertrophic zone chondrocytes (Robson et al. 2000; Stevens et al. 2000). T3 was demonstrated to inhibit chondrocyte proliferation, and to stimulate chondrocyte differentiation and terminal differentiation, thus enhancing chondrocyte movement from the proliferative zone into the hypertrophic zone (Robson et al. 2000; Stevens et al. 2000). A crucial period for longitudinal bone growth (the lengthening of the humeri and femur) in mice is from 2 to 4 weeks of age (Fraichard et al. 1997). Longitudinal bone
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growth evident in wild type mice aged between 3 and 4 weeks was absent in TTR null mice (Monk 2006). Femurs were significantly shorter in 3- and 4-week-old TTR null mice than those in age-matched wild type mice, but were of similar length in 10-week-old TTR null and wild type mice (Fig. 18.1). The delay in longitudinal bone growth may be due to a defect in the growth plate dynamics (Monk 2006). Although the normal organisation of chondrocytes was evident, the tibial growth plates in 2-week-old TTR null mice were significantly wider, due to a significantly wider hypertrophic zone, than those in 2-week-old wild type mice. In contrast, the growth plate characteristics of 10-week-old TTR null mice were similar to that of 10-week-old wild type mice (Monk 2006). Given that femur length and tibial growth plate widths are similar in 10-weekold wild type and TTR null mice, the reduced circulating levels of free T4 during development in TTR null mice may have delayed, but not inhibited, linear bone growth. However, the synthesis of D2 in the growth plate suggests that the growth plate is equipped to tolerate fluctuations in circulating TH. A similar growth plate phenotype (increased hypertrophic zone width and delayed bone lengthening) has been reported (Gerber et al. 1999). The inactivation of vascular endothelial growth factor, a protein that mediates angiogenesis, in developing mice resulted in an increase in the hypertrophic zone width, caused by the inhibited resorption of hypertrophic chondrocytes. Vascular endothelial growth factor is essential for the vascular invasion of the growth plate (Gerber et al. 1999), which is also impaired in hypothyroid rats (Lewinson et al. 1989). Whether the reduced circulating levels of
Fig. 18.1 Growth plots of femoral length in developing and adult wild type and transthyretin (TTR) null mice. Differentially stained femurs were dissected from whole skeletons of 3- and 4week-old wild type and TTR null mice, scanned beside a ruler then measured using the Image J 1.31v software. Whole legs from 2- and 10-week-old wild type and TTR null mice were X-rayed, the X-ray film was scanned then the femurs were measured with Image J 1.31v software. Scale bars: 5.0 mm. Data points are mean SEM. Statistical significance is indicated by *p < 0.05, ***p < 0.005. n = 6, except 4-week-old TTR null mice, n = 5, and 10-week-old mice, n = 12
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TH in developing TTR null mice impair the vascular invasion of growth plates, thereby inhibiting apoptosis of hypertrophic chondrocytes and increasing growth plate width, requires further investigation.
18.3.2.4
Maturation of the Ileum
THs are vital for the normal maturation of the small intestine, with development beginning in the duodenum followed by the jejunum then the ileum. THs induce the structural and functional development of the microvillus membrane (Israel et al. 1987) and influence levels of brush border enzymes (Hodin et al. 1992). During the postnatal development in rats, modulation of mRNA levels of the TR isoforms a2 and b1 and elevation of 50 -deiodinase activity ensure that the intestine becomes increasingly responsive to TH (Galton et al. 1991; Hodin et al. 1994). In addition, the synthesis of TTR has been detected in the intestine of foetal humans (Loughna et al. 1995). The villi in the ileum (but not the jejunum or duodenum) from newborn TTR null mice appeared slightly disorganised and contained fewer goblet cells than those from newborn wild type mice (Monk 2006). In TRa/ mice, morphological alterations (reduced diameter, decreased number and size of the villi and significantly reduced number of goblet cells) were detected in the jejunum and ileum but not the duodenum (Fraichard et al. 1997). Although the phenotype of the ileum from newborn TTR null mice was not as striking as that from TRa/ mice, the morphological alteration indicates the requirement of TTR for the distribution of sufficient TH to the intestine.
18.3.3 Postnatal Development of Central Nervous Tissues Thyroid hormones play a pivotal role in orchestrating brain development and differentiation (DeLong 1996). During brain development, the TH network operates to increase and maintain intracellular levels of T3 within the required range. In rats, the brain sequesters a greater amount of circulating T4 from birth to 21 days after birth, a period when THs are critical for brain development, than during adult life (Vigouroux et al. 1979). The majority of T3 in the brain is obtained by the local deiodination of T4 (van Doorn et al. 1985), and the activity of D2 and type 3 deiodinase are modulated during brain development (Kaplan and Yaskoski 1981; Kodding et al. 1986) to ensure that the brain is neither overexposed nor underexposed to T3. In addition, the responsiveness of the brain to T3 is also modulated. mRNA levels of the TR isoform a1 are higher in the cerebral cortex of humans during gestation than adult life whereas mRNA levels of the isoforms a2 and b1 are lower (Chan et al. 2002). Transthyretin may also facilitate TH-related brain development. Transthyretin is the only TH distributor protein synthesised in the epithelial cells of the choroid
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plexus, which is located within the brain and forms the blood–cerebrospinal fluid (CSF) barrier (Dickson et al. 1986; Dickson and Schreiber 1986; Stauder et al. 1986). The secretion of TTR into the CSF facilitates the movement of THs from the blood across the blood–CSF barrier into the CSF (Dickson et al. 1987; Schreiber et al. 1990). Interestingly, in the CSF from human neonates, infants, children and adults, TTR levels were highest in neonates (Larsen and DeLallo 1989). In rats, TTR mRNA levels were increased in the choroid plexus during development and were highest 4 days before birth (Fung et al. 1988), suggesting that TTR synthesis in the choroid plexus is maximal immediately before the spurt in brain growth and the period of TH-related brain development. Data on adult TTR null mice suggest that TTR is required for the adequate uptake of T4 into the brain; the uptake of T4 was decreased and the T4 content in the brain and choroid plexus was 36% and 14% of wild type levels, respectively (Palha et al. 2000; Palha et al. 1997). In addition, the cells that line the ventricles, such as tanycytes, in TTR null mice may be hypothyroid as a result of reduced T4 levels in the CSF in the absence of TTR (Richardson et al. 2007). Thyroid hormones regulate apoptosis of the neural stem cells that reside in the subventricular zone (Lemkine et al. 2005), and in adult TTR null mice, apoptosis of these cells was reduced and was as low as that found in the brains from hypothyroid wild type mice (Richardson et al. 2007). In the light of these results, the possibility of reduced TH levels in the CSF and brains in the absence of TTR and its impact on development must be explored. Despite reduced circulating levels of total and free T4 in 2-week-old TTR null mice, TH homeostasis within the brain of developing TTR null mice does not appear to be compromised (Monk 2006). Although marked reductions in serum T4 concentrations have been shown to increase D2 activity in the cerebral cortex of neonatal rats (Silva and Larsen 1982; van Doorn et al. 1982), D2 mRNA levels in the cerebral cortex from 2-week-old TTR null mice were not altered (Monk 2006). In addition, mRNA levels of T3-responsive genes (Purkinje cell protein 2, myelin basic protein, calbindin and RC3 neurogranin) in the brain did not differ between 2-week-old TTR null mice and wild type mice; however, myelin basic protein mRNA levels were lower in the cerebral cortex of TTR null mice (p = 0.052) (Monk 2006). These results suggested that intracellular T3 levels in the cerebral cortex were normal and indicate a preference for maintenance of TH homeostasis in the CNS at the expense of peripheral tissues. Despite these findings, subtle differences were evident in the brains from developing TTR null mice, compared with age-matched wild type mice, and suggested a degree of immaturity in the developing brain of TTR null mice. The protein concentration in the CSF from newborn (3.92 0.17 mg mL1, n = 5) and 2-week-old (1.03 0.11 mg mL1, n = 4) TTR null mice was higher than those in newborn (2.76 0.13 mg mL1, n = 11; p < 0.01) and 2-week-old (0.555 0.09 mg mL1, n = 8; p < 0.05) wild type mice (Monk 2006). The protein concentration of the CSF decreases during development (Dziegielewska et al. 2000) and can be an indicator for brain maturity. Also, the width of the cerebral cortex appeared to be narrower, the overall size of the brain appeared to be smaller, despite no difference
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in weight, and the lateral ventricles appeared to be larger (ventricle size decreases during development) in the brains of a 2-week-old TTR null mice, compared with age-matched wild type mice (Monk 2006). These apparent differences in the brain require quantitative confirmation and warrant further investigation of CNS tissues in developing TTR null mice.
18.4
Perspectives
Despite reduced circulating levels of total and free T4, the development of the CNS is essentially preserved at the expense of peripheral tissues in TTR null mice. TTR, as part of the TH distributor protein network, may ensure that the temporal progression of physiological and biochemical processes in peripheral tissues that are responsive to TH during periods of increased TH demand, i.e. development. A fatal version of this phenotype has been described for TRa/ mice (Fraichard et al. 1997), which, after 2 weeks of age, exhibit growth arrest and die by 5 weeks of age. Thus, at 2 weeks of age, TTR null mice are at a developmental cusp that initiates their transformation from a pup, which is dependent on its mother, to an independent and adult mouse. Given that TTR null mice survive though to adulthood, further investigation of TTR null mice during this period of life (2 to 5 weeks of age) is required to identify the onset and time course of TH-related developmental events, such as the transition from suckling to weaning and the period of rapid growth.
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de Jesus EG, Hirano T, Inui Y (1991) Changes in cortisol and thyroid hormone concentrations during early development and metamorphosis in the Japanese flounder, Paralichthys olivaceus. Gen Comp Endocrinol 82:369–376 DeLong G (1996) The neuromuscular system and brain in hypothyroidism. In: Braverman L, Utiger R (eds) Werner and Ingbar’s The thyroid. A fundamental and clinical text, 7th edn. Lippincott-Raven, Philadelphia, pp 826–835 Dickson P, Aldred A, Menting J, Marley P, Sawyer W, Schreiber G (1987) Thyroxine transport in choroid plexus. J Biol Chem 262:13907–13915 Dickson PW, Aldred AR, Marley PD, Bannister D, Schreiber G (1986) Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver. J Biol Chem 261:3475–3478 Dickson PW, Schreiber G (1986) High levels of messenger RNA for transthyretin (prealbumin) in human choroid plexus. Neurosci Lett 66:311–315 Dziegielewska KM, Knott GW, Saunders NR (2000) The nature and composition of the internal environment of the developing brain. Cell Mol Neurobiol 20:41–56 Fraichard A, Chassande O, Plateroti M, Roux JP, Trouillas J, Dehay C, Legrand C, Gauthier K, Kedinger M, Malaval L, Rousset B, Samarut J (1997) The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production. EMBO J 16:4412–4420 Fung WP, Thomas T, Dickson PW, Aldred AR, Milland J, Dziadek M, Power B, Hudson P, Schreiber G (1988) Structure and expression of the rat transthyretin (prealbumin) gene. J Biol Chem 263:480–488 Funkenstein B, Perrot V, Brown CL (1999) Cloning of putative piscine (Sparus aurata) transthyretin: developmental expression and tissue distribution. Mol Cell Endocrinol 157:67–73 Galton VA, McCarthy PT, St Germain DL (1991) The ontogeny of iodothyronine deiodinase systems in liver and intestine of the rat. Endocrinology 128:1717–1722 Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N (1999) VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 5:623–628 Girard J, Ferre P, Pegorier JP, Duee PH (1992) Adaptations of glucose and fatty acid metabolism during perinatal period and suckling–weaning transition. Physiol Rev 72:507–562 Goodridge AG, Klautky SA, Fantozzi DA, Baillie RA, Hodnett DW, Chen W, Thurmond DC, Xu G, Roncero C (1996) Nutritional and hormonal regulation of expression of the gene for malic enzyme. Prog Nucleic Acid Res Mol Biol 52:89–122 Gross J, Leblond CP, Franklin AE, Quastel JH (1950) Presence of iodinated amino acids in unhydrolyzed thyroid and plasma. Science 111:605–608 Gross J, Pitt-Rivers R (1953) 3:5:30 -Triiodothyronine. I. Isolation from thyroid gland and synthesis. Biochem J 53:645–650 Hadj-Sahraoui N, Seugnet I, Ghorbel MT, Demeneix B (2000) Hypothyroidism prolongs mitotic activity in the post-natal mouse brain. Neurosci Lett 280:79–82 Henning SJ (1981) Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol 241:G199–G214 Hodin RA, Chamberlain SM, Upton MP (1992) Thyroid hormone differentially regulates rat intestinal brush border enzyme gene expression. Gastroenterology 103:1529–1536 Hodin RA, Meng S, Chamberlain SM (1994) Thyroid hormone responsiveness is developmentally regulated in the rat small intestine: a possible role for the alpha-2 receptor variant. Endocrinology 135:564–568 Howell D, Dean D (1992) The biology, chemistry, and biochemistry of the mammalian growth plate. In: Coe F, Favus M (eds) Disorders of Bone and Mineral Metabolism. Raven Press, New York, pp 313–353 Hulbert AJ (2000) Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc 75:519–631
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Ishii H, Inada M, Tanaka K, Mashio Y, Naito K, Nishikawa M, Imura H (1981) Triiodothyronine generation from thyroxine in human thyroid: enhanced conversion in Graves’ thyroid tissue. J Clin Endocrinol Metab 52:1211–1217 Israel EJ, Pang KY, Harmatz PR, Walker WA (1987) Structural and functional maturation of rat gastrointestinal barrier with thyroxine. Am J Physiol 252:G762–G767 Janssens PA, Grigg JA, Dove H, Hulbert AJ (1990) Thyroid hormones during development of a marsupial, the tammar wallaby, Macropus eugenii. J Endocrinol 127:427–436 Kaplan MM, Yaskoski KA (1981) Maturational patterns of iodothyronine phenolic and tyrosyl ring deiodinase activities in rat cerebrum, cerebellum, and hypothalamus. J Clin Invest 67: 1208–1214 Kodding R, Fuhrmann H, von zur Muhlen A (1986) Investigations on iodothyronine deiodinase activity in the maturing rat brain. Endocrinology 118:1347–1352 Larsen PD, DeLallo L (1989) Cerebrospinal fluid transthyretin in the neonate and bloodcerebrospinal fluid barrier permeability. Ann Neurol 25:628–630 Larsen PR, Silva JE, Kaplan MM (1981) Relationships between circulating and intracellular thyroid hormones: physiological and clinical implications. Endocr Rev 2:87–102 Lemkine GF, Raj A, Alfama G, Turque N, Hassani Z, Alegria-Prevot O, Samarut J, Levi G, Demeneix BA (2005) Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor. FASEB J 19:863–865 Lewinson D, Harel Z, Shenzer P, Silbermann M, Hochberg Z (1989) Effect of thyroid hormone and growth hormone on recovery from hypothyroidism of epiphyseal growth plate cartilage and its adjacent bone. Endocrinology 124:937–945 Loughna S, Bennett P, Moore G (1995) Molecular analysis of the expression of transthyretin in intestine and liver from trisomy 18 fetuses. Hum Genet 95:89–95 Me´daille C, Trumel C, Concordet D, Vergez F, Braun JP (2004) Comparison of plasma/serum urea and creatinine concentrations in the dog: a 5-year retrospective study in a commercial veterinary clinical pathology laboratory. J Vet Med A Physiol Pathol Clin Med 51:119–123 Mendel CM, Weisiger RA, Jones AL, Cavalieri RR (1987) Thyroid hormone-binding proteins in plasma facilitate uniform distribution of thyroxine within tissues: a perfused rat liver study. Endocrinology 120:1742–1749 Meyer D, Harvey J (2004) Veterinary Laboratory Medicine. Interpretation and Diagnosis, 3rd edn. Saunders, St. Louis, MO Miura M, Tanaka K, Komatsu Y, Suda M, Yasoda A, Sakuma Y, Ozasa A, Nakao K (2002) Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate. J Bone Miner Res 17:443–454 Miwa S, Inui Y (1987) Effects of various doses of thyroxine and triiodothyronine on the metamorphosis of flounder (Paralichthys olivaceus). Gen Comp Endocrinol 67:356–363 Molero C, Benito M, Lorenzo M (1993) Regulation of malic enzyme gene expression by nutrients, hormones, and growth factors in fetal hepatocyte primary cultures. J Cell Physiol 155:197–203 Mondou PM, Kaltenbach JC (1979) Thyroxine concentrations in blood serum and pericardial fluid of metamorphosing tadpoles and of adult frogs. Gen Comp Endocrinol 39:343–349 Monk JA (2006) Thyroid hormone homeostasis in developing and adult transthyretin null mice. PhD thesis, The University of Melbourne, Parkville, Australia O’Shea PJ, Williams GR (2002) Insight into the physiological actions of thyroid hormone receptors from genetically modified mice. J Endocrinol 175:553–570 Palha JA, Fernandes R, de Escobar GM, Episkopou V, Gottesman M, Saraiva MJ (2000) Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin-null mouse model. Endocrinology 141:3267–3272 Palha JA, Hays MT, Morreale de Escobar G, Episkopou V, Gottesman ME, Saraiva MJ (1997) Transthyretin is not essential for thyroxine to reach the brain and other tissues in transthyretinnull mice. Am J Physiol 272:E485–E493
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Pittman CS, Chambers JB, Jr., Read VH (1971) The extrathyroidal conversion rate of thyroxine to triiodothyronine in normal man. J Clin Invest 50:1187–1196 Richardson SJ, Aldred AR, Leng SL, Renfree MB, Hulbert AJ, Schreiber G (2002) Developmental profile of thyroid hormone distributor proteins in a marsupial, the tammar wallaby Macropus eugenii. Gen Comp Endocrinol 125:92–103 Richardson SJ, Lemkine GF, Alfama G, Hassani Z, Demeneix BA (2007) Cell division and apoptosis in the adult neural stem cell niche are differentially affected in transthyretin null mice. Neurosci Lett 421:234–238 Richardson SJ, Monk JA, Shepherdley CA, Ebbesson LO, Sin F, Power DM, Frappell PB, Kohrle J, Renfree MB (2005) Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish. Am J Physiol Regul Integr Comp Physiol 288:R1264–R1272 Robson H, Siebler T, Stevens DA, Shalet SM, Williams GR (2000) Thyroid hormone acts directly on growth plate chondrocytes to promote hypertrophic differentiation and inhibit clonal expansion and cell proliferation. Endocrinology 141:3887–3897 Rovet JF (1999) Congenital hypothyroidism: long-term outcome. Thyroid 9:741–748 Schreiber G, Aldred AR, Jaworowski A, Nilsson C, Achen M, Segal M (1990) Thyroxine transport from blood to brain via transthyretin synthesis in choroid plexus. Am J Physiol Regul Integr Comp Physiol 258: R338–R345 Schreiber G, Richardson SJ (1997) The evolution of gene expression, structure and function of transthyretin. Comp Biochem Physiol B Biochem Mol Biol 116:137–160 Shen S, Berry W, Jaques S, Pillai S, Zhu J (2004) Differential expression of iodothyronine deiodinase type 2 in growth plates of chickens divergently selected for incidence of tibial dyschondroplasia. Anim Genet 35:114–118 Shepherdley CA, Daniels CB, Orgeig S, Richardson SJ, Evans BK, Darras VM (2002) Glucocorticoids, thyroid hormones, and iodothyronine deiodinases in embryonic saltwater crocodiles. Am J Physiol Regul Integr Comp Physiol 283:R1155–R1163 Silva JE, Larsen PR (1982) Comparison of iodothyronine 50 -deiodinase and other thyroidhormone-dependent enzyme activities in the cerebral cortex of hypothyroid neonatal rat. Evidence for adaptation to hypothyroidism. J Clin Invest 70:1110–1123 Song MK, Dozin B, Grieco D, Rall JE, Nikodem VM (1988) Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone. J Biol Chem 263: 17970–17974 Specker JL, DiStefano JJ, III, Grau EG, Nishioka RS, Bern HA (1984) Development-associated changes in thyroxine kinetics in juvenile salmon. Endocrinology 115:399–406 Stauder AJ, Dickson PW, Aldred AR, Schreiber G, Mendelsohn FA, Hudson P (1986) Synthesis of transthyretin (pre-albumin) mRNA in choroid plexus epithelial cells, localized by in situ hybridization in rat brain. J Histochem Cytochem 34:949–952 Stevens DA, Hasserjian RP, Robson H, Siebler T, Shalet SM, Williams GR (2000) Thyroid hormones regulate hypertrophic chondrocyte differentiation and expression of parathyroid hormone-related peptide and its receptor during endochondral bone formation. J Bone Miner Res 15:2431–2442 Suzuki S, Suzuki M (1981) Changes in thyroidal and plasma iodine compounds during and after metamorphosis of the bullfrog, Rana catesbeiana. Gen Comp Endocrinol 45:74–81 Taurog A, Evans ES (1967) Extrathyroidal thyroxine formation in completely thyroidectomized rats. Endocrinology 80:915–925 Thomas T, Schreiber G (1985) Acute-phase response of plasma protein synthesis during experimental inflammation in neonatal rats. Inflammation 9:1–7 Thommes RC, Hylka VW (1977) Plasma iodothyronines in the embryonic and immediate posthatch chick. Gen Comp Endocrinol 32:417–422 Underwood L, Van Wyk J (1992) Normal and aberrant growth. In: William’s Textbook of Endocrinology In: Wilson J, Foster D (eds) William’s Textbook of Endocrinology 8th edn. W.B. Saunders Company, Philadelphia, pp 1079–1138
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Chapter 19
TTR Null Mice as a Model to Study the Involvement of TTR in Neurobiology: From Neuropeptide Processing to Nerve Regeneration Carolina Estima Fleming, Ana Filipa Nunes, Ma´rcia Almeida Liz, and Mo´nica Mendes Sousa
Abstract Physiologically, TTR is mainly acknowledged for being the plasma transporter of thyroxine (T4) and retinol. Under pathological conditions, several mutations in TTR are associated with familial amyloid polyneuropathy (FAP), a neurodegenerative disorder characterized by deposition of TTR amyloid fibrils, particularly in the peripheral nervous system (PNS), where it leads to axonal loss and neuronal death. Although it is well established that TTR synthesis occurs in the liver and in the choroid plexus (the sources of TTR in the plasma and cerebrospinal fluid –CSF, respectively), the origin of TTR deposited in the PNS of FAP patients is unknown. Under physiological conditions TTR has access to the nerve both through the blood and CSF. Additionally, a function for TTR in nerve biology could explain its preferential deposition, when mutated, in the PNS. In this respect, several studies using TTR knockout (KO) mice revealed new TTR functions specifically related to the nervous system: (1) the absence of TTR is associated with reduced signs of depressive-like behavior and with memory impairment; (2) TTR participates in sensorimotor performance; (3) TTR regulates neuropeptide maturation and, (4) TTR enhances nerve regeneration. In the following pages, these novel TTR functions related to the nervous system, as well as the use of TTR KO mice as a means to study them, will be discussed. Keywords Nerve regeneration, Neurite outgrowth, Neuropeptide procesing
M.M. Sousa (*) Nerve Regeneration Group, Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, Portugal e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_19, # Springer‐Verlag Berlin Heidelberg 2009
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Introduction
TTR, primarily known as the plasma transporter of thyroxine (T4) and retinol, is mainly synthesized by the liver and choroid plexus of the brain (Gitlin et al. 1975; Aleshire et al. 1983), which are respectively the sources of TTR in the plasma and cerebrospinal fluid (CSF). Several TTR mutations are associated with familial amyloid polyneuropathy (FAP), an autosomal dominant lethal disorder characterized by extracellular deposition of TTR amyloid fibrils, particularly in the peripheral nervous system (PNS), leading to axonal and neuronal loss (Andrade 1952). The mechanisms leading to neurodegeneration in FAP are largely undisclosed. Moreover, the origin of TTR deposited in the PNS of FAP patients is unknown. In this respect, it is possible that TTR might have a role in nerve physiology that would explain why, when mutated, the protein preferentially accumulates in the PNS. Physiologically, two routes of access of TTR to the nerve are possible: (1) through the blood–nerve barrier, which is effective in slowing but not in preventing the entry of proteins into the endoneurium, such that ganglion cells and their axons are probably constantly exposed to serum proteins; and (2) through the CSF–nerve barrier, as peripheral nerve roots contact with CSF, where TTR is present in high levels. A similar reasoning may be applied to the central nervous system (CNS): TTR may access the brain both through the blood–brain barrier and the CSF–brain barrier. As will be detailed below, in addition to FAP, several lines of evidence have recently drawn attention to the importance of TTR in the biology of the nervous system. Still, TTR involvement in the homeostasis of both the PNS and CNS is not yet fully understood. In this respect, TTR KO mice have constituted one of the most valuable tools to link the biology of TTR with that of the CNS and PNS. In the pages that follow, the new evidences placing TTR as a player in neurobiology will be discussed.
19.2
The TTR Knockout Mouse
In 1993, Episkopou et al. generated the TTR KO mouse to investigate the physiological role of TTR in embryonic development and in the adult, specially the role of T4 and retinol, as both are transported through direct or indirect binding to TTR. In general terms, the TTR KO mouse was revealed to be fertile, to have a normal life span, displaying no obvious phenotypic abnormalities postnatally (Episkopou et al. 1993). Since then, several studies have used this strain to address different physiological aspects of the biology of TTR, as summarized and discussed below.
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19.2.1 T4 and Retinol Metabolism in TTR KO Mice: Absence of Major Effects on Thyroid Hormone Function and Retinol Metabolism As expected, plasma from TTR KO mice had decreased levels of both T4 and T3 (Episkopou et al. 1993; Palha et al. 1994); nevertheless, the percentage of free T4 was increased by 50% in their serum which might explain their euthyroid status (Palha 2002). In terms of tissue content of thyroid hormones, whereas both the liver and kidney of TTR KO mice presented no differences in T4 levels when compared to WT mice, their brains showed 30% decreased T4 levels, probably reflecting the absence of TTR from the choroid plexus and CSF (Palha et al. 1997). In fact, although no other T4-binding protein replaces TTR in the CSF of TTR KO mice, no differences were found in the content of T4 in the cortex, cerebellum, and hippocampus (Palha et al. 2000). In conclusion, in spite of being a major T4 carrier in the plasma, apparently, the absence of TTR does not affect thyroid hormone function. In the case of retinol, the retinol-binding protein (RBP) is secreted bound to TTR in a 1:1 molar complex (Monaco et al. 1995), making it reasonable to expect that the absence of TTR would be related to vitamin A deficiency. In fact, TTR KO mice have retinol plasma levels below the level of detection, as well as 3% of the RBP levels found in WT mice (Episkopou et al. 1993). However, mice lacking TTR do not show any symptoms of vitamin A deficiency, such as loss of weight, infections, or eye abnormalities. In agreement with the lack of symptoms of vitamin A deficiency, the total retinol levels in the kidney, liver, testes, and spleen, as well as the levels of RBP in the urine, were not significantly different from WT mice (Wei et al. 1995). Considering the very low levels of plasma retinol, it was surprising that TTR KO mice presented such high levels of retinol in tissues. This finding is probably explained by the fact that in these animals, plasma all-transretinoic acid (the retinol derivative that acts upon transcription factors) is increased, which might be compensating for the low retinol levels (Wei et al. 1995). In conclusion, the above findings suggest that despite their low levels of retinol in the plasma, TTR KO mice present no major defects related to retinol deficiency.
19.2.2 TTR KO Mice as a Tool to Address the Function of TTR in the Nervous System Recently, studies from independent groups using TTR KO mice have enabled a link to be made between TTR and the biology of the nervous system. The detailed phenotypic characterization of this strain comprised a number of behavioral and sensorimotor tests that led to the clear establishment of several impairments caused by the absence of TTR. This primary assessment was followed by both molecular
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studies and research linking TTR to nervous system disease/injury. This body of data will be detailed in the following paragraphs.
19.2.2.1
TTR KO Mice have a Decreased Depressive-like Behavior and a Sensorimotor Impairment
A link between TTR and behavior was first established by Sousa et al. (2004a). In this study, TTR KO mice exhibited increased activity compared to WT animals in the forced swim and locomotor activity tests, which suggested that in the absence of TTR, mice were less prone to develop depressive-like behaviors. In this report, increased levels of norepinephrine in the limbic forebrain of TTR KO mice were shown, although no differences were found in other brain regions. This increase in norepinephrine, a molecule that stimulates brain activity, was correlated with the phenotype observed. Apart from displaying a less depressive-like behavior, TTR KO mice were recently shown to present a sensorimotor impairment (Fleming et al. 2007). This impairment, primarily characterized through SHIRPA (SmithKline Beecham Pharmaceuticals, Harwell MRC Mouse Genome Centre, Imperial College School of Medicine, Royal London Hospital, Phenotype Assessment) (Rogers et al. 1997), progressed with age and included increased limb clasping, poor performance in the vertical pole test, and decreased locomotor activity in aged animals. In relation to locomotor activity, young TTR KO mice were more active than WT littermates, in agreement with previous observations (Sousa et al. 2004a); however, at 12 months of age, this tendency was inverted, probably as a consequence of the motor discoordination of older TTR KO mice (Fig. 19.1). Additionally, in the hot plate test, a standard procedure to measure the nociceptive response to a noxious thermal stimulus, TTR KO mice had an increased latency to react to heat, when compared to WT littermates (Fleming et al. 2007). The observation that TTR KO mice present a sensorimotor impairment suggested a specific function for TTR in nerve physiology. To identify the reasons underlying this phenotype, morphometric and electrophysiological analyses of sciatic nerves were performed but no differences were found between WT and TTR KO littermates (Fleming et al. 2007). Moreover, since the function of the cerebellum is related to the coordination of body movement, the cerebella of both strains were compared. Similar to the sciatic nerve, neither gross anatomical differences nor differences in the structure of the dendritic tree or number of Purkinje cells between the two strains were detected. As such, in spite of the above studies clearly showing that TTR is a player in the normal biology of the nervous system, both the link between TTR and the noradrenergic system, as well as the reason underlying the sensorimotor impairment of TTR KO mice, await identification.
19 TTR Null Mice as a Model to Study the Involvement of TTR in Neurobiology
Fig. 19.1 Limb clasping of a 12-month-old TTR KO mouse
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TTR KO Mice and Age-related Disorders
Besides being a tool to investigate the role of TTR in neurobiology, TTR KO mice have been used to address the role of TTR as a neuroprotective protein in Alzheimer’s disease (AD). Transgenic mice developing AD (Lazarov et al. 2005), maintained in an enriched environment from the time of weaning until the age of six months, resulted in a marked reduction in Ab deposition in the CNS when compared to mice held in standard housing conditions. This reduction in AD pathology was correlated with increased levels of TTR in the brain of mice kept in an enriched environment. In the light of these results, it was hypothesized that the absence of TTR would accelerate amyloid deposition in AD transgenic mice (Choi et al. 2007). In fact, the same authors showed that Ab levels were increased in TTR heterozygous mice crossed with AD transgenics, confirming the initial hypothesis and reinforcing the neuroprotective function of TTR in Ab deposition. More recently, a transgenic mouse model of AD was crossed with TTR KO mice and Ab burden was compared with AD transgenics in a TTR KO background and AD transgenics in a heterozygous TTR background (Wati et al. 2008). Animals in a TTR KO background exhibited decreased levels of Ab deposition, suggesting that the absence of TTR inhibits Ab deposition. This is in clear disagreement with the previous reports that established TTR as being neuroprotective in AD. The reason for these discrepancies might be related to the age of the animals, as well as to the AD mouse model used in the different studies. In order to clarify this issue, WT, heterozygous and TTR KO mice crossed with different AD mouse models should be used. Apart from AD, it has been shown that during aging, decreased levels of TTR are critical to the development of memory impairments (Brouillette and Quirion 2007; Sousa et al. 2007). Not only is a lower TTR gene expression observed in the hippocampus (containing choroid plexus) of aged memoryimpaired rats when compared to aged memory-unimpaired animals (Brouillette and Quirion 2007), but also, during aging, WT mice worsen their performance in spatial reference tasks, which is related to a decline in TTR levels in the CSF (Sousa et al. 2007). Moreover, in the case of TTR KO mice, young adults were seen to display a spatial reference memory impairment when compared to age-matched WT animals (Sousa et al. 2007). As retinoic acid has been implicated in synaptic plasticity, TTR KO mice were injected with this active vitamin A metabolite. Retinoic acid delivery was sufficient to improve their performance in the Morris water maze (Brouillette and Quirion 2007), suggesting that there may still be unanswered questions relating to the impact of TTR in retinol metabolism. Together, the above findings suggest that the absence of TTR accelerates the poorer cognitive performance commonly associated with normal aging and accelerated in AD.
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TTR is Related to Differential Gene Expression in the Nervous System: PAM Upregulation in TTR KO Mice
Given the sensorimotor impairment in the absence of TTR, and to initiate the evaluation of a putative TTR function in the nerve (that could underlie the deposition of mutated TTR in this organ), cDNA microarray analysis of dorsal root ganglia (DRG) from TTR KO mice and mice expressing human TTR (in a TTR KO background) was performed (Nunes et al. 2006). Statistical analysis identified several genes differentially expressed, including some potentially involved in the physiology of the nervous system, namely peptidylglycine a-amidating monooxygenase (PAM) and lipoprotein lipase (LPL), were both upregulated in TTR KO mice (Nunes et al. 2006). The possible links between the increased expression of PAM and LPL and the phenotype displayed by TTR KO mice, together with the insights gained in understanding the function of TTR in the nervous system, are discussed in detail below.
19.3.1 Peptidylglycine a-Amidating Monooxygenase (PAM), a key Neuropeptide Processing Enzyme, is Increased in the Absence of TTR Neuropeptides are stored in secretory granules and are activated by processing enzymes. Approximately 50% of all known neuropeptides are synthesized as biologically inactive glycine-extended precursors that require a carboxy-terminal posttranslational amidation for biological activity. PAM is the only enzyme that C-terminally a-amidates peptides, and is therefore rate-limiting in amidated neuropeptide maturation (Prigge et al. 2000). This integral membrane protein is expressed in a wide variety of cell types, including endocrine, glial and endothelial cells, as well as in many neurons (Prigge et al. 2000). To validate the data obtained by microarrays, immunohistochemistry and semiquantitative RT-PCR were performed, which confirmed that PAM is upregulated both in the CNS and PNS of TTR KO mice (Nunes et al. 2006) (Fig. 19.2). Moreover, in vitro, absence of TTR led to increased PAM expression in neuronal and neuronal-like cell cultures, as cells grown in the absence of TTR displayed increased PAM expression when compared to cells grown with this protein (Nunes et al. 2006). Addition of TTR to KO serum was able to diminish PAM synthesis, demonstrating that TTR itself is responsible for differential PAM expression. In summary, the above results show that TTR is involved in the homeostasis of the nervous system, namely by regulating neuropeptide maturation through the downregulation of PAM expression.
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Fig. 19.2 PAM immunohistochemistry in the sciatic nerve (upper panels) and spinal cord (lower panels) of WT (left) and TTR KO mice (right)
19.3.2 Increased PAM levels in the Absence of TTR are not Related to an Effect on PAM mRNA Stability It is possible that the effect of TTR on PAM gene expression might be exerted by its major ligands, retinol, and/or T4. Hypothyroidism increases PAM expression by enhancing the stability of PAM mRNA in the cytoplasm (Fraboulet et al. 1996); however, although TTR is the major T4 carrier in the CSF, TTR KOs are euthyroid (Episkopou et al. 1993), suggesting that the effect of TTR on PAM expression is unlikely to result from impaired thyroid hormone homeostasis. Retinol, the precursor of active retinoic acid, has a wide influence on gene expression and could be a possible mechanism for the regulation of PAM gene expression. However, despite the low plasma retinol levels, TTR KOs lack symptoms of vitamin A deficiency (Episkopou et al. 1993), suggesting that the effect of TTR on PAM expression probably does not result from impaired retinoic acid metabolism. The most commonly described mechanism for the regulation of PAM gene expression involves the alteration of PAM mRNA stability. Thyroid hormones, estrogens and b-adrenergic agonists are examples of molecules affecting PAM
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mRNA half-life. Likewise, TTR could regulate PAM levels by altering its mRNA stability in spite of influencing its transcriptional rate. To verify this hypothesis, PAM mRNA half-life was determined in primary cortical neurons grown either in the presence or absence of TTR (Nunes et al. unpublished observations). Actinomycin D was added to the culture medium to block de novo transcription and PAM mRNA decay was analyzed by RT-PCR at different time points after actinomycin D treatment. For all time points, a similar decay of PAM mRNA levels was observed with time in both conditions. This result excludes the possible involvement of TTR in PAM mRNA stability as the putative mechanism for regulation of PAM gene expression. It also suggests that the increased PAM levels observed in the absence of TTR, both in vivo and in vitro, are the result of an increased transcriptional rate. As such, presently, the mechanism underlying PAM regulation by TTR is still not understood and should be the subject of further investigation.
19.3.3 TTR KOs are a New Mouse Model for Increased Neuropeptide Y (NPY) Following the identification of PAM as a molecule overexpressed in the absence of TTR, the biological implications of this overexpression were assessed in TTR KO mice. As described below, the data obtained showed that TTR KOs are a novel model for increased NPY levels, further reinforcing the role of this protein in the physiology of the nervous system.
19.3.3.1
Amidated Neuropeptide Y is Increased in TTR KO Mice
To evaluate whether PAM overexpression in TTR KOs has biological consequences resulting from increased PAM activity, amidated NPY content in TTR KO and WT mice was compared (Nunes et al. 2006). NPY is the major neuropeptide present in the mammalian central and peripheral nervous system (Pedrazzini et al. 2003) and its activation requires C-terminal a-amidation by PAM. As the CSF neurotransmitter content reflects CNS synaptic events, NPY levels in the CSF of TTR KO and WT mice were compared; as expected given their increased PAM expression, TTR KOs presented approximately twofold increased NPY levels in the CSF. As the hippocampus is one of the most abundant NPY sources, its NPY levels were also determined; hippocampus from TTR KO mice presented fivefold increased NPY. Similarly, in the PNS, TTR KOs displayed two- and threefold increased NPY in the dorsal root ganglia (DRG) and sciatic nerve, respectively. To exclude the variation of NPY as a consequence of differential expression, semiquantitative NPY RT-PCR analysis of different nervous system regions was performed. In all tested tissues, no statistical differences were found in NPY mRNA between strains, supporting the hypothesis that increased NPY in TTR KO mice results from increased processing and activation by PAM (Nunes et al. 2006).
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Lipoprotein Lipase, a Gene Activated by NPY, is Increased in the Absence of TTR
One of the most relevant NPY effects is its action on energy balance by increasing energy intake and decreasing its expenditure. This effect is achieved by augmenting lipid storage in white adipose tissue (WAT) through the increase of the synthesis and activity of LPL and the decrease of brown adipose tissue thermogenesis (Billington et al. 1994; Kotz et al. 1998). It is noteworthy that in the microarray analysis referred to above, upregulation of LPL mRNA in TTR KOs was revealed, consistent with the increased PAM mRNA expression and NPY levels in these animals. As expected, WAT from TTR KOs displayed more than twofold increased LPL activity. Next, Nunes et al. (2006) inquired into whether a similar effect might be observed in the nervous system; for that, the expression and activity of LPL in the CNS and PNS were analyzed. Similar to the findings in the WAT, increased LPL was reported in the brain, spinal cord and DRG from TTR KO mice. The balance between energy production and dissipation is a function of temperature regulation and any imbalance is manifested as changes in body temperature. Since NPY has been shown to induce hypothermia (Esteban et al. 1989), the core body temperature of WT and TTR KO mice was measured (Nunes et al. 2006). Although in young mice no statistical difference was found, 12-month-old TTR KOs presented a lower core body temperature than WTs, reflecting decreased energy expenditure, possibly as a consequence of increased NPY levels. To evaluate whether increased NPY levels in TTR KO mice result in additional biological consequences, and to verify whether, as has been reported, NPY preferentially enhances carbohydrate ingestion (Pedrazzini et al. 2003), TTR KO and WT mice were compared for regular chow intake and preference for a high-carbohydrate diet (Nunes et al. 2006). Although no differences in average body weight or average regular chow intake were detected, TTR KO mice presented increased intake and preference for the high carbohydrate diet, as expected from their increased NPY levels. It is noteworthy that in other mouse and rat models of NPY overexpression, the systematic lack of any major effect in energy homeostasis as well as unaltered body weight and food intake are observed (Herzog 2003). In this respect, except for the increased carbohydrate consumption and preference, TTR KO mice behave similarly, showing no difference in these parameters.
19.3.3.3
NPY Overexpression in the Context of the Behavioral Phenotype of TTR KO Mice
A growing body of data implicates NPY in depression, by demonstrating that this neuropetide is an antidepressant neurotransmitter (Heilig 2004). As such, the fact that in TTR KO mice NPY is increasedcorrelates well with the fact that TTR KOs have a decreased depressive-like behavior compared with WT animals (Sousa et al. 2004a). In that report, increased norepinephrine levels in the absence of TTR were
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suggested to reveal the role of TTR involvement in depression (Sousa et al. 2004). However, it is possible that NPY might be responsible, at least in part, for the behavioral phenotype of TTR KO mice and this issue should be the subject of additional analysis.
19.3.3.4
The Use of TTR KO Mice as a NPY Overexpressor Model
Thus far, only two transgenic mouse models for NPY overexpression have been described: in the brain-restricted model (using the neuronal promoter Thy1), a minor 1.15 increase of NPY levels was found (Inui et al. 1998), whereas in the second model (using the endogenous NPY promoter), fivefold increased brain NPY mRNA levels were reported (Thiele et al. 1998). These reports nevertheless, lack the description of NPY levels in the PNS and in the CSF, thus not allowing direct comparison with the TTR KOs. It is however, noteworthy that in a rat model of hippocampal NPY overexpression (Thorsell et al. 2000), NPY is increased by approximately 30% in the hippocampus, whereas in the TTR KO mice reported by Nunes et al. (2006), a higher increase is observed: approximately 80% in the hippocampus, 40% in the CSF, 50% in the DRG and 70% in the sciatic nerve. One should however, bear in mind that it is highly likely that changes in PAM gene expression lead to changes in levels of other amidated neuropeptides. Supporting this hypothesis, increased levels of substance P, another major amidated neuropeptide, were found in the PNS and plasma of TTR KO mice (Nunes et al. 2006). The physiological consequences of this observation, as well as the possible increase of other amidated neuropeptides, should be further investigated. Although one should not discard the hypothesis that increased levels of other amidated neuropeptides may produce some complexity, TTR KO mice not only display increased NPY levels when compared with other NPY overexpressor models, but also present an accompanying NPY overexpressor phenotype, including decreased energy expenditure, decreased depressive-like behavior and increased carbohydrate consumption and preference, most of which are not commonly observed in other NPY overexpressor models. In summary, TTR KO mice display increased amidated NPY levels, without augmented NPY gene expression, and present a NPY overexpressor phenotype; as such, these animals offer a unique model to study both peripheral and central actions of NPY, as well as a new animal model for increased NPY levels (Nunes et al. 2006).
19.4
TTR Enhances Nerve Regeneration
Besides the microarray analysis, nerve crush was performed in WT and TTR KO mice, to further unravel whether TTR has a role in nerve physiology, given the possibility that upon injury, the consequences arising from the absence of TTR might
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be exacerbated. The data obtained, as discussed below, led to the important finding that TTR enhances nerve regeneration, a process of the utmost importance in biological systems. Although the PNS has a regeneration permissive environment, since axons sprout in adult peripheral nerves after injury, target innervation is often incomplete, resulting in a disappointing functional recovery. The fact that TTR enhances nerve regeneration represents an additional demonstration that this protein participates in nerve biology. Furthermore, the assignment of a TTR function in nerve biology and repair, may explain its preferential deposition, when mutated, in the PNS of FAP patients.
19.4.1 After Sciatic Nerve Crush, Lack of TTR is Related to a Delayed Regeneration Capacity As referred to above, and given the possibility that following nerve injury the consequences arising from the absence of TTR might be exacerbated, nerve crush was performed in WT and TTR KO mice and regeneration was assessed 15 and 30 days post-crush, functionally and by morphometric analysis of the distal nerve stumps (Fleming et al. 2007). At the functional level, in vivo nerve conduction velocity was determined and locomotor activity was assessed. TTR KO mice presented a statistically significant lower nerve conduction velocity, when compared to WT littermates, which was the first evidence pointing towards a decreased regenerative capacity in the absence of TTR. Moreover, despite the higher locomotor activity prior crush, TTR KO mice presented a decreased locomotor activity following nerve injury, further suggesting a slower regeneration. To assess whether the decreased functional performance of TTR KO mice correlated with neuropathological findings, nerve regeneration, was scored by morphometry. After 15 days of regeneration, the total number of myelinated fibers of TTR KO mice was approximately 20% lower than in WT littermates. Moreover, following 30 days of regeneration, TTR KOs showed an approximately 40% decreased density of unmyelinated fibers when compared to WT animals. To further ascertain that TTR increases nerve regeneration, Thy1.2-TTR mice, human TTR transgenic mice with TTR expression both in sensory and motor neurons, resulting in the presence of TTR in the sciatic nerve (Sousa et al. 2004b), were backcrossed to the TTR KO background, so that the resulting mouse strain (Thy1.2-TTRxTTR KO mice) does not expresses endogenous mouse TTR but expresses human TTR in neurons (Fleming et al. 2007). When Thy1.2-TTRxTTR KO mice were submitted to nerve crush, neuronal TTR expression in a TTR KO background led to an accelerated regenerative capacity when compared to both WT and TTR KO mice. After 15 days of regeneration, Thy1.2TTRxTTR KO mice presented an approximately 50% increase both in the number of myelinated fibers and in the density of unmyelinated fibers, when compared to TTR KO mice.
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The functional and morphometric analysis presented above demonstrates that TTR enhances and accelerates nerve regeneration which, following injury, may be crucial for timely target innervation and for regaining of functional capacity.
19.4.2 Assessment of the Cellular Mechanism Through Which TTR Enhances Nerve Regeneration The mechanism whereby the absence of TTR is responsible for delayed regeneration, should shed light on the involvement of TTR in nerve physiology and regeneration, as well as on FAP pathology. Again, the possibility that the effect of TTR in nerve regeneration , might be exerted by its major ligands, retinol and T4 cannot be excluded. However, as referred to previously for PAM gene expression, although TTR is the major T4 carrier in the CSF, TTR KO mice are euthyroid (Episkopou et al. 1993), suggesting that TTR effect on nerve regeneration is unlikely to result from impaired thyroid hormone homeostasis. In the case of retinol, despite low plasma retinol levels, TTR KO mice do not show any symptoms of vitamin A deficiency (Episkopou et al. 1993), suggesting that TTR effect does not result from impaired retinoic acid metabolism. In the following pages, the efforts to unravel the cellular mechanisms through which TTR enhances nerve regeneration, are described. Given the data presented here, it is unlikely that TTR exerts its effect in neurite outgrowth through its major ligands: not only was TTR directly responsible for rescuing the decreased intrinsic ability to grow neurites of TTR KO DRG neurons cultivated in a T4- and retinol-free medium (Fleming et al. 2007), but also, I84S TTR, a TTR mutant with very low affinity for both T4 and RBP (Berni et al. 1994; Refetoff et al. 1986), behaved similarly to WT TTR and was able to rescue the phenotype of cells grown in TTR KO serum.
19.4.2.1
The Absence of TTR does not Affect Neuronal Survival Following Nerve Crush
To further understand the delayed regeneration of TTR KO mice, neuronal survival was assessed after nerve crush (Fleming et al. 2007). As the number of fibers in the proximal nerve stump reflects the number of DRG neurons and motorneurons that survived injury, regardless of axonal growth occurring distally, the proximal nerve segments of WT and TTR KO mice were evaluated by morphometry 30 days after the crush. No differences were found in the density of myelinated or unmyelinated fibers between strains, demonstrating that the role of TTR in nerve regeneration is unrelated to neuronal survival/death. To further validate this result, the density of L4–6 DRG neurons was determined 30 days after the nerve crush; again, no significant differences between strains were found, reinforcing the conclusion that
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Fig. 19.3 Neurite outgrowth of DRG neurons from WT (right) and TTR KO (left) mice. Arrows highlight neurites
the effect of TTR in nerve regeneration is not associated with neuronal survival/ death (Fleming et al. 2007).
19.4.2.2
TTR Increases Neurite Outgrowth
The possibility that TTR elicits neurite outgrowth and extension was next assessed as these are essential requirements for axonal regeneration to occur (Fleming et al. 2007). PC12 cells exposed to TTR KO serum displayed a 20% and a 30% decrease, respectively, in the neurite number per cell and in the length of the longest neurite, when compared to cells grown with WT serum. To establish whether absence of TTR was itself responsible for decreased neurite number and size, TTR KO serum was supplemented with WT TTR; addition of TTR was able to totally rescue the phenotype observed in the absence of this protein. To determine whether TTR KO sensory neurons already display an intrinsic decreased neurite outgrowth, as a consequence of their physiological TTR-free environment, DRG neurons from WT and TTR KO mice were grown in the absence of TTR, and their neurite number and size were compared (Fleming et al. 2007). Similar to PC12 cells, DRG neurons from TTR KO mice presented a 25% and a 15% decreased neurite number and length of the longest neurite, respectively, when compared to WT DRG neurons (Fig. 19.3). Again, addition of TTR to the culture medium was able to rescue the phenotype of TTR KO DRG neurons, demonstrating that this protein is directly responsible for the differential neurite outgrowth, which can account for the decreased regenerative capacity of TTR KO mice. Summing up, the above findings demonstrate that TTR enhances nerve regeneration, affecting primarily neurons. Future work should now unravel the molecular mechanism underlying this finding.
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The Proteolytic Activity of TTR may Participate in the Biology of the Nervous System
In the plasma, a small TTR fraction is carried in high-density lipoproteins (HDL), through binding to apolipoprotein A-I (apoA-I) (Sousa et al. 2000). Besides being a transporter, TTR is able to cleave the C-terminus of apoA-I, being a novel cryptic protease (Liz et al. 2004, 2005). The relevance of apoA-I cleavage by TTR in lipoprotein metabolism was determined. Upon TTR cleavage, HDL displayed a reduced capacity to promote cholesterol efflux and cleaved apoA-I displays increased amyloidogenicity (Liz et al. 2007). Several features including an optimum pH for activity of approximately 7, inhibition by serine protease inhibitors and cleavage in apoA-I after a Phe residue, strongly suggest that TTR is a serine protease (Liz et al. 2004). However, the catalytic mechanism of TTR remains to be solved. The cryptic nature of TTR proteolytic activity derives not only from the fact that it lacks canonical structural protease determinants, but also because its physiological function is apparently unrelated to proteolysis (Liz et al. 2005). As it is possible that apoA-I may not be the major TTR substrate, and given the phenotypes of TTR KO mice discussed in the previous paragraphs (which can be related to the absence of TTR proteolytic activity and not to the absence of the protein itself), upcoming studies should concentrate on the analysis of the physiological relevance of TTR proteolytic activity in the nervous system. In this respect, it is worth mentioning that very recently, when the nature of the TTR/Ab interaction was further investigated, TTR was found to be able to cleave Ab in multiple positions (Costa et al. 2008) with some of the Ab peptides generated displaying lower amyloidogenic potential than the full length counterpart. Moreover, TTR was also able to degrade aggregated forms of Ab. These results confirmed the hypothesis that the proteolytic activity of TTR has an impact on the biology of the nervous system. Additional putative substrates in the CNS and/or CNS are now waiting to be disclosed.
19.6
Conclusions
Although in recent years a growing amount of data has revealed that TTR is an important protein in the biology of the nervous system, a number of questions were raised while others remain unanswered, specially those related to the mechanism through which TTR exerts its action in the CNS and PNS. In respect to the importance of TTR in the brain, the pattern of TTR gene expression both throughout evolution and during embryonic development is noteworthy. The conservation of TTR gene expression in the choroid plexus from reptiles to mammals led to the hypothesis that the expression of this gene first arose in the brain of reptiles (Schreiber et al. 2002). Additionally, during human embryonic development, TTR is first expressed in the tela choroidea, the precursor
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of the choroid plexus, followed by expression in the liver (Harms et al. 1991; Richardson et al. 1994). This blueprint of TTR gene expression in the choroid plexus, conserved throughout evolution and starting early in embryonic development, points to a pivotal role for TTR in the nervous system. It is also noteworthy that given the recent reports assigning novel functions for TTR in the biology of the nervous system, namely its involvement in behavior (Sousa et al. 2004a), memory (Brouillette and Quirion 2007), neuropeptide processing (Nunes et al. 2006), and nerve regeneration, (Fleming et al. 2007), the development of gene therapies for FAP aiming at silencing/reducing TTR production should be envisaged with caution. As most FAP patients are heterozygous, gene therapy efforts should be directed to specifically suppress the mutated TTR allele. In the near future, one could expect that the links between TTR biology and the biology of the nervous system will be rapidly strengthened and deepened.
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Gitlin D, Gitlin JD (1975) Fetal and neonatal development of human plasma proteins. In: Putnam FW (ed) The plasma proteins, Vol II, 2nd edn. Academic Press, New York Harms PJ, Tu GF, Richardson SJ, Aldred AR, Jaworowski A, Schreiber G (1991) Transthyretin (prealbumin) gene expression in choroid plexus is strongly conserved during evolution of vertebrates. Comp Biochem Physiol B 99:239–249 Heilig M (2004) The NPY system in stress, anxiety and depression. Neuropeptides 38:213–224 Herzog H (2003) Neuropeptide Y and energy homeostasis: insights from Y receptor knockout models. Eur J Pharmacol 480:21–29 Inui A, Okita M, Nakajima M, Momose K, Ueno N, Teranishi A, Miura M, Hirosue Y, Sano K, Sato M, Watanabe M, Sakai T, Watanabe T, Ishida K, Silver J, Baba S, Kasuga M (1998) Anxiety-like behavior in transgenic mice with brain expression of neuropeptide Y. Proc Assoc Am Physicians 110:171–182 Kotz CM, Briggs JE, Grace MK, Levine AS, Billington CJ (1998) Divergence of the feeding and thermogenic pathways influenced by NPY in the hypothalamic PVN of the rat. Am J Physiol 275:R471–R477 Lazarov O, Robinson J, Tang YP, Hairston IS, Korade-Mirnics Z, Lee VM, Hersh LB, Sapolsky RM, Mirnics K, Sisodia SS (2005) Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice. Cell 120:701–713 Liz MA, Sousa MM (2005) Deciphering cryptic proteases. Cell Mol Life Sci 62:989–1002 Monaco HL, Mancia F, Rizzi M, Coda A (1995) Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268:1039–1041 Liz MA, Faro CJ, Saraiva MJ, Sousa MM (2004) Transthyretin, a new cryptic protease. J Biol Chem 279:21431–21438 Liz MA, Sousa MM (2005) Deciphering cryptic proteases. Cell Mol Life Sci 62:989–1002 Liz MA, Gomes CM, Saraiva MJ, Sousa MM (2007) ApoA-I cleaved by transthyretin has reduced ability to promote cholesterol efflux and increased amyloidogenicity. J Lipid Res 48:2385–2395 Nunes AF, Saraiva MJ, Sousa MM (2006) Transthyretin knockouts are a new mouse model for increased neuropeptide Y. FASEB J 20:166–168 Palha JA (2002) Transthyretin as a thyroid hormone carrier: function revisited. Clin Chem Lab Med 40:1292–1300 Palha JA, Episkopou V, Maeda S, Shimada K, Gottesman ME, Saraiva MJM (1994) Thyroid hormone metabolism in a transthyretin-null mouse strain. J Biol Chem 269:33135–33139 Palha JA, Hays MT, Morreale de Escobar G, Episkopou V, Gottesman ME, Saraiva MJ (1997) Transthyretin is not essential for thyroxine to reach the brain and other tissues in transthyretinnull mice. Am J Physiol 272:E485–E493 Palha JA, Fernandes R, de Escobar GM, Episkopou V, Gottesman M, Saraiva MJ (2000) Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin-null mouse model. Endocrinology 141:3267–3272 Pedrazzini T, Pralong F, Grouzmann E (2003) Neuropeptide Y: the universal soldier. Cell Mol Life Sci 60:350–377 Prigge ST, Mains RE, Eipper BA, Amzel LM (2000) New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell Mol Life Sci 57:1236–1259 Refetoff S, Dwulet FE, Benson MD (1986) Reduced affinity for thyroxine in two of three structural thyroxine-binding prealbumin variants associated with familial amyloidotic polyneuropathy. J Clin Endocrinol Metab 63:1432–1437 Richardson SJ, Bradley AJ, Duan W, Wettenhall RE, Harms PJ, Babon JJ, Southwell BR, Nicol S, Donnellan S, Schreiber G (1994) Evolution of marsupial and other vertebrate thyroxinebinding plasma proteins. Am J Physiol 266:R1359–R1370 Rogers DC, Fisher EM, Brown SD, Peters J, Hunter AJ, Martin JE (1997) Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment. Mamm Genome 8:711–713
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Schreiber G (2002) The evolution of transthyretin synthesis in the choroid plexus. Clin Chem Lab Med 40:1200–1210 Sousa JC, Grandela C, Fernandez-Ruiz J, de Miguel R, de Sousa L, Magalhaes AI, Saraiva MJ, Sousa N, Palha JA (2004a) Transthyretin is involved in depression-like behaviour and exploratory activity. J Neurochem 88:1052–1058 Sousa JC, Marques F, Dias-Ferreira E, Cerqueira JJ, Sousa N, Palha JA (2007) Transthyretin influences spatial reference memory. Neurobiol Learn Mem 88:381–385 Sousa MM, Berglund L, Saraiva MJ (2000) Transthyretin in high density lipoproteins: association with apolipoprotein A-I. J Lipid Res 41:58–65 Sousa MM, Monteiro F, Saraiva MJ (2004b) Lack of amyloid deposition in the peripheral nervous system of transgenic mice expressing human mutant transthyretin in neurons. FENS Abstracts 2:A159.14. Thiele TE, Marsh DJ, Ste Marie L, Bernstein IL, Palmiter RD (1998) Ethanol consumption and resistance are inversely related to neuropeptide Y levels. Nature 396:366–369 Thorsell A, Michalkiewicz M, Dumont Y, Quirion R, Caberlotto L, Rimondini R, Mathe AA, Heilig M (2000) Behavioral insensitivity to restraint stress, absent fear suppression of behavior and impaired spatial learning in transgenic rats with hippocampal neuropeptide Y overexpression. Proc Natl Acad Sci USA 97:12852–12857 Wati H, Kawarabayashi T, Matsubara E, Kasai A, Hirasawa T, Kubota T, Harigaya Y, Shoji M, Maeda S (2008) Transthyretin Accelerates Vascular Abeta Deposition in a Mouse Model of Alzheimer’s Disease. Brain Pathol. 19:48–57 Wei S, Episkopou V, Piantedosi R, Maeda S, Shimada K, Gottesman ME, Blaner WS (1995) Studies on the metabolism of retinol and retinol-binding protein in transthyretin-deficient mice produced by homologous recombination. J Biol Chem 270:866–870
Chapter 20
Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease Yves Ingenbleek
Abstract Transthyretin (TTR) is a 55-kDa protein secreted mainly by the choroid plexus and the liver. Whereas its intracerebral production appears as a stable secretory process allowing even distribution of intrathecal thyroid hormones, its hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly. Both morbid conditions are governed by distinct pathogenic mechanisms leading to the reduction in size of lean body mass (LBM). The liver production of TTR integrates the dietary and stressful components of any disease spectrum, explaining why it is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequalled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyperhomocysteinemic states, acquired metabolic disorders currently ascribed to dietary restriction in water-soluble vitamins. Sulfur (S)-deficiency is proposed as an additional causal factor in the sizeable proportion of hyperhomocysteinemic patients characterized by adequate vitamin intake but experiencing varying degrees of nitrogen (N)-depletion. Owing to the fact that N and S coexist in plant and animal tissues within tightly related concentrations, decreasing LBM as an effect of dietary shortage and/or excessive hypercatabolic losses induces proportionate S-losses. Regardless of water-soluble vitamin status, elevation of homocysteine plasma levels is negatively correlated with LBM reduction and declining TTR plasma levels. These findings occur as the result of impaired cystathionine-b-synthase activity, an enzyme initiating the transsulfuration pathway and whose suppression promotes the upstream accumulation and remethylation of homocysteine molecules. Under conditions of N- and S-deficiencies, the maintenance of methionine homeostasis indicates high metabolic priority.
Y. Ingenbleek Laboratory of Nutrition, University Louis Pasteur Strasbourg e-mail:
[email protected]
S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_20, # Springer‐Verlag Berlin Heidelberg 2009
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Keywords Body composition, Protein components, Biomarkers, Lean body mass, Transthyretin, Nutritional status, Protein malnutrition, Stressful disorders, Elderly persons, Homocysteine, Alzheimer’s disease
20.1
Introduction
The homotetrameric transthyretin (TTR) molecule (55 kDa) was first identified in cerebrospinal fluid (CSF) (Kabat et al. 1942) and later in human serum (Schultze et al. 1956). The initial name of tryptophan (Trp)-rich prealbumin (PA) arose from the unusual Trp content of this indispensable amino acid (IAA) and from the observation that the protein was migrating in front of the serum-albumin (ALB) peak in the then currently used electrophoretic systems. Besides thyroxine-binding globulin (TBG) and ALB, PA was rapidly recognized as one of the three specific binding-proteins (BPs) involved in transporting thyroid hormones (Ingbar 1958), hence the thyroxine-binding prealbumin (TBPA) appellation given at that time. The further discovery that TBPA was also carrying the bulk of circulating retinol through the small retinol-binding protein (RBP, 21 kDa) (Kanai et al. 1968) lent credence to the more physiologically informative TTR denomination, emphasizing the dual conveying role played by the BP in biological fluids (Goodman et al. 1981). The aim of the present chapter is to review the most recent findings collected on TTR in health and disease, focusing particularly on stressful disorders and hyperhomocysteinemia.
20.2
Body Composition Studies
The first attempts to evaluate the protein components of the human body were grounded on anthropometric criteria (Gurney and Jelliffe 1973; Keys et al. 1950) or direct tissue analyses of diseased persons (Clarys et al. 1984; Mitsipoulos et al. 1998). Subsequent more dynamic approaches relied on nitrogen (N) balance studies that have now lost much of their previous importance in nutritional knowledge. N balance surveys indeed depend not only on the level of protein intake but also upon energy supply (Young et al. 1992). Individuals submitted to N-restricted regimens are basically able to maintain N homeostasis until very late in the starvation processes (Young et al. 1992). Moreover, N balance only provides an overall estimate of N gains and losses but fails to identify the tissue sites and specific interorgan fluxes involved (Kopple 1987). The most recent analytical tools employ elaborate physico-chemical techniques using stable or labeled isotopes, prompt-g neutron activation, computerized tomography, dual photon absorptiometry, bioelectric impedance analysis (BIA), magnetic
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resonance imaging (MRI) and dual X-ray absorptiometry (DEXA). These sophisticated methods have been successfully applied for the appraisal of fat-free mass (FFM) (Lukaski et al. 1985), total body N (TBN) (Cohn et al. 1981; Ellis et al. 1982), lean body mass (LBM) (Segal et al. 1988) and skeletal muscle (SM) mass (Janssen et al. 2000). Schematically, the human body may be divided into two major compartments, namely fat mass (FM) and FFM that is obtained by substracting FM from body weight (BW). The fat cell mass sequesters about 80% of the total body lipids, is poorly hydrated and contains only small quantities of lean tissues and nonfat constituents. FFM comprises the sizeable part of lean tissues and minor mineral compounds among which are Ca, P, Na, and Cl pools totaling about 1.7 kg or 2.5% of BW in a healthy man weighing 70 kg (Forbes 1996). Subtraction of mineral mass from FFM provides LBM, a composite aggregation of organs and tissues with specific functional properties. LBM is thus nearly but not strictly equivalent to FFM. With extracellular mineral content subtracted, LBM accounts for most of total body proteins (TBP) and of TBN assuming a mean 6.25 ratio between protein and N content (International Commission on Radiation Protection 1975). SM accounts for 45% of TBN whereas the remaining 55% is in nonmuscle lean tissues (Cohn et al. 1981). The LBM of the reference man contains 98% of total body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass) with a relatively higher sequestration of K in SM and of S in the outermost skin layer, hair and nails. The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g) (Forbes 1996). The average hydration level of LBM in healthy subjects of all age is 73% with the proportion of the intracellular/extracellular fluid spaces being 4:3. Table 20.1 collects findings from several research groups (Battezzatti et al. 2007; Cohn et al. 1981; Ellis et al. 1982; Janssen et al. 2000) showing the main protein components of the body in healthy men and women. Whatever the analytical methodology utilized, all selected parameters demonstrate a clear-cut gender dimorphism, whether referring to absolute values or body weight or body height ratios. The data also reveal that all parameters are maintained in the form of stabilized plateau levels throughout sexual maturity. LBM represents about two thirds of BW in healthy subjects whereas SM, its main ponderal component, amounts to 37–40% (Cohn et al. Table 20.1 Body composition studies showing the main protein components in healthy adult humans TBNb,c (kg) TBPd (kg) SMe (kg) LBMa,c (kg) TTRf (mg L1) BWa,b (kg) M 76–79 1.8–1.9 11.2–11.8 33 54–62 290–320 F 60 1.2–1.4 7.5–8.7 21 36–39 250–280 a Body weight (BW) and lean body mass (LBM) measured by Battezzatti et al. (2007) using BIA and DEXA technologies b BW and TBN measured by Ellis et al. (1982) using prompt g-neutron activation c TBN and LBM measured by Cohn et al. (1981) using prompt g-neutron activation d Total body protein (TBP) is the result of TBN multiplied by 6.25 (International Commission on Radiation Protection 1975) e Skeletal muscle (SM) mass measured by Janssen et al. (2000) using MRI f Data from Bienvenu et al. (1996) using immunoturbidimetry
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1981). SM is of particular relevance in nutritional studies due to its capacity to serve as a major reservoir of amino acids (AAs) and as a dispenser of gluconeogenic substrates. An indirect estimate of SM size consists in the measurement of urinary creatinine, end-product of the nonenzymatic hydrolysis of phosphocreatine which is limited to muscle cells (Boorsook and Dubnoff 1947). Creatininuria has daily fluctuations dependent on intake. Following appropriate dietary control and provided that 24-h urinary samples are properly collected, both variables maintain constant ratios stabilized around 1 g creatininuria per 18.6 kg SM (Heymsfield et al. 1983). On average, SM mass is 36% heavier in men than women (Janssen et al. 2000) and this is reflected by the sexual difference in mean creatininuria values being around 23 mg kg1 BW in males versus 16 mg kg1 BW in females. The information on the body composition of children is scarcer than that on adult subjects, but the topic is currently of increasing interest. Starting from the reference fetus (Ziegler et al. 1976), a growing body of studies have utilized the abovedescribed analytical tools to evaluate body characteristics in childhood (Fomon et al. 1982; Fors et al. 2002; Houtkooper et al. 1992; Kim et al. 2006; Lohman and Going 2006; Okasora et al. 1999). There exists an overall consensus among workers that from birth until the onset of puberty children undergo continuing growth associated with similar age-related alterations in the chemical composition of the body. Referring to BW, the percentage of total body water decreases whereas the proportion of bone mineral content, FM, FFM, LBM and SM increase steadily without significant differences in body composition between prepubertal children. The development of enlarged LBM and SM sizes in adolescent boys and the increased FM in adolescent girls is attributed to sex hormones and differentiated responsiveness to growth factors stimulating anabolic processes (Mauras 2001; Veldhuis et al. 2005). During ageing, all the protein components of the human body depicted in Table 20.1 decrease regularly (Baumgartner et al. 1995; Forbes and Reina 1973; Fuller et al. 1996). This shrinking tendency is especially well documented for SM (Baumgartner et al. 1999; Evans and Campbell 1993; Gallagher et al. 1997; Janssen et al. 2000; Lukaski 1997) whose absolute amount is preserved until the end of the fifth decade, consistent with studies showing unmodified muscle structure (Lexell et al. 1986), intracellular K content (Kehayias et al. 1997) and working capacity (Hurley 1995). TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline with an accelerated decrease after 65 years (Cohn et al. 1983; Ellis et al. 1982). SM undergoes a 15% reduction in size per decade (Janssen et al. 2000), an involutive process indirectly revealed by the progressive lowering of creatininuria values after 55 years (Rowe et al. 1976; Tzankoff and Norris 1977). The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women (Gallagher et al. 1997; Hansen et al. 1999) decreasing strength and functional capacity (Evans and Campbell 1993). The downward SM slope may be somewhat prevented by physical training (Evans 1991) or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation (Visser et al. 2002) or in critically ill patients whose muscle mass undergoes proteolysis and contractile dysfunction (Mitch and Goldberg 1996; Zoico and Roubenoff 2002).
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Besides cytokine-induced SM involution, the multifactorial mechanisms usually involved in the occurrence of sarcopenia are changes in dietary intake, disuse atrophy, downregulation of androgen secretory patterns and gradual cellular refractoriness to trophic and anabolic stimuli (Baumgartner et al. 1999; Dutta and Hadley 1995).
20.3
Transthyretin in Health and Protein-Depleted States
The serial measurement of plasma TTR in healthy children shows that BP values are low in the neonatal period and rise linearly with superimposable concentrations in both sexes during infant growth (Bienvenu et al. 1996; Malvy et al. 1992; Vahlquist et al. 1975) consistent with superimposable N accretion and protein synthesis rates (Young et al. 1975). Sexual differences occur at the onset of adolescence with higher values recorded in male teenagers. Gender dimorphism is attributed to the impact of androgenic hormones known to exert direct stimulatory effects on liver TTR synthesis (Braverman and Ingbar 1967) and to anabolic events associated with postpubertal alterations (Mauras 2001; Veldhuis et al. 2005). TTR concentrations stabilize thereafter in both sexes (Bienvenu et al. 1996; Sachs and Bernstein 1986). Starting from the sixties, TTR values progressively decline showing steeper slopes in elderly males (Bienvenu et al. 1996). The lowering trend seems to be initiated by the attenuation of androgen influences and trophic stimuli with increasing age (Gallagher et al. 1997). The normal human TTR trajectory from birth to death has been well documented by scientists belonging to the Foundation for Blood Research [FBR]. The immunoturbidimetric measurement of TTR (Ledue et al. 1987) was done on blood samples from nearly 70,000 healthy US citizens and the values are given in Table 20.2, fully strengthening the validity of preliminary data previously published in an FBR position monograph (Bienvenu et al. 1996). The data unambiguously demonstrate the striking sex- and age-dependent similarities between the evolutionary patterns of TTR and of those of N-components comprised in Table 20.1. The plasma concentration of any circulating protein is determined by the balance between its rate of synthesis and catabolism or leakage into the intravascular space. Following FAO/WHO/UNU recommendations for healthy adults, the safe level of protein intake turns around 0.75 g k1 day1 (FAO/WHO/United Nations University 1985). This amount sustains normal growth and maintains the plasma concentrations of most biological parameters. Nevertheless, recent studies have shown that under these circumstances, TTR plasma level and pool size remain constant because its synthetic and catabolic rates are both downregulated concomitantly (Afolabi et al. 2004). The data confirm previous turnover surveys suggesting that the FAO/WHO/UNU guidelines are marginally inadequate to maintain the metabolic reserve capacities required to mount optimal responses to stress (Young and Marchini 1990). TTR is the first plasma protein to decline in response to marginal protein restricion, thus working as an early signal warning that adaptive mechanisms maintaining homeostasis are undergoing decompensation.
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Table 20.2 Plasma transthyretin values by age and sex in healthy humans (n = 68,720) Age Group Females (n = 47,881) Males (n = 20,839) (years) Under 1 1–2 3–4 5–6 7–8 9–10 11–12 13–14 15–16 17–18 19–20 21–30 31–40 41–50 51–60 61–70 71–80 81–90 91–100
n
Age
Mean
s.d.
n
Age
Mean
s.d.
9 77 104 122 127 189 278 446 615 564 566 4,124 8,898 11,446 8,467 5,677 4,341 1,705 126
0.6 2.1 4.0 6.0 8.0 10.1 12.0 14.1 16.0 18.0 20.0 26.8 36.4 46.0 55.6 65.9 75.7 84.6 93.6
182.1 177.1 177.3 184.6 196.1 208.3 228.9 243.8 257.4 259.9 265.0 265.2 263.4 265.2 274.5 273.2 268.0 257.7 247.9
79.6 47.7 44.6 41.3 43.5 40.6 47.8 48.4 46.5 49.6 54.2 54.3 52.4 53.5 55.3 59.1 61.6 61.9 71.8
22 76 103 126 138 141 168 176 255 212 184 1,340 3,328 4,707 4,067 2,858 2,189 703 46
0.7 1.9 4.0 6.0 8.1 10.1 12.1 14.0 16.1 18.0 20.0 26.8 36.5 46.0 55.7 65.8 75.5 84.4 93.0
172.7 179.8 174.2 178.6 190.2 211.0 221.9 255.2 272.8 282.2 285.3 299.6 309.5 308.3 301.8 290.0 277.6 262.1 236.3
44.4 44.5 36.0 41.8 41.3 45.7 47.3 44.9 52.4 50.1 50.8 52.1 58.8 60.5 61.1 61.2 58.5 60.6 76.4
TTR was proposed as a marker of protein nutritional status following a clinical investigation undertaken in 1972 on protein-energy malnourished (PEM) Senegalese children (Ingenbleek et al. 1972). By comparison with ALB and transferrin (TF) plasma values, TTR revealed a much higher degree of sensitivity to changes in protein status that has been attributed to its shorter biological half-life (2 days) and to its unusual Trp richness (Ingenbleek et al. 1972, 1975a). Transcription of the TTR gene in the liver is directed by CCAAT/enhancer binding protein (C/EBP) bound to hepatocyte nuclear factor 1 (HNF1) under the control of several other HNFs (Costa et al. 1989). The mechanism responsible for the suppressed TTR synthesis in PEM-states is a restricted AA and energy supply working as limiting factors (Ingenbleek and Young 2002). Exprimental studies on rat liver models have shown direct transcriptional (Strauss et al. 1994) and translational (Kimball 2002) regulatory controls exerted by dietary protein, leading to the drop of liver TTRmRNA levels and correspondingly diminished secretion of the mature BP in the bloodstream. LBM may be schematically subdivided into a visceral pool (liver, intestine, thymoleucocytic tissue) characterized by high metabolic reactiveness to any alteration in nutritional status, a structural pool (SM, skin, soluble collagen) made up of slowly turning over proteins and a nonexchangeable pool (connective tissues, tendons and cytoskeleton) poorly sensitive to dietary and stress-induced influences (Ingenbleek and Young 2002). The fractional synthesis and renewal rates
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of the liver and of the gut mucosa are about 10- to 20-times more rapid than that of SM mass, explaining why the small-sized visceral pool makes an absolute contribution to the daily turnover of body proteins at least equal to that defining the structural pool (Ingenbleek and Young 2002). Fasting or dietary restriction causes early and graded responses in visceral tissues (Gofferje and Kozlik 1977; Large et al. 1980; Smale et al. 1980) as opposed to the delayed reactivity disclosed by SM (Lennmarken et al. 1986). In the declared stage of PEM, multivariate analyses have shown that the predictive ability of outcome offered by TTR is independent of that provided by ALB and TF (Brasseur et al. 1994). The consequences and severity of PEM on the three main visceral tissues appear to be well correlated with declining TTR concentrations as illustrated by the extent of liver steatosis (Ingenbleek et al. 1972), the flattening of gut endothelial mucosa (McMillan et al. 2001) and the functional impairment of defence capacities (Moulias et al. 1985). During PEM, all tissue components listed in Table 20.1, including those belonging to the structural compartment, show significant reductions in protein content. Nevertheless, creatininuria is poorly informative of the magnitude of muscle proteolysis in early PEM stages as more than 10% SM loss is required before the catabolite undergoes a significant decrease (Shenkin et al. 1996). In contrast, sarcopenia found in longlasting malnutrition is associated with concomitant lowering of creatininuria values (Arroyave et al. 1961), implying gradual exhaustion of gluconeogenic compounds and AAs of survival value with ominous prognosis (Briend et al. 1989; Heymsfield et al. 1982). It is also worth mentioning that TTR values show normal Gaussian distributions in healthy neonates (Warner and Jang 1999) and in preadolescent children (Ingenbleek 1977) providing therefore a substrate for epidemiological surveys on protein nutritional status between population groups (Reifen et al. 2003). The rapidly turning over TTR protein is highly responsive to any change in protein flux and energy supply, being clearly situated on the cutting edge of the equipoise. It is beyond the scope of this chapter to go over all clinical conditions shown to benefit from using TTR as a biomarker since a recent review has dealt with these aspects (Ingenbleek 2008). Rather, we would like to pinpoint some salient examples of TTR usefulness for assessing nutritional adequacy in preterm infants and healthy neonates (Lee et al. 2001; Nissim et al. 1983), for monitoring the best N:energy intake of infant formulas (Kashyap et al. 1990) and for correcting the balance between nutrient classes in uncontrolled diabetes (Kobbah et al. 1988), weight reducing programs (Gofferje and Kozlik 1977) and inherited diseases of AA metabolism (Acosta et al. 2005; Arnold et al. 2002). It is well known that (sub) clinical PEM exists in many adult (Bistrian et al. 1976; Devoto et al. 2006) and elderly (Constans et al. 1992; Polge et al. 1997; Sandman et al. 1987; Sergi et al. 2006) hospitalized subjects. Serial measurement of TTR is helpful for grading the severity of chronic disorders and for alerting physicians as to the validity of therapeutic strategies (Bernstein and Pleban 1996; Devoto et al. 2006). Plasma TTR values correlate with TBN fluctuations in kidney patients (Mushnick et al. 2003) and with FFM index in elderly noninfected persons (Sergi et al. 2006). PEM is a common finding in Alzheimer’s disease (AD) and multiple-infarct dementia (MID). TTR participates directly to the maintenance of memory and
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normal cognitive processes during the ageing process (Brouillette and Quirion 2007; Sousa et al. 2007), potentially by acting on the retinoid and thyroid hormone signalling pathways. Moreover, TTR may bind amyloid b-peptide in vitro, preventing its transformation into toxic amyloid neurofibrils and amyloid plaques (Schwarzman et al. 1994). This protective activity is likely to be reduced in AD patients due to their low plasma (Sandman et al. 1987) and CSF (Riisøen 1988) TTR concentrations. Moreover, TTR works as a limiting factor for plasma retinol transport (Ingenbleek et al. 1975b). Retinol may be converted firstly into retinal and thereafter into physiologically active all-trans- and 13-cis- retinoic acids (RAs), end-products of a two-step oxidation procedure regulated by retinaldehyde dehydrogenase enzymes (Blomhoff and Blomhoff 2006; Kim et al. 1992). Retinol is the rate-limiting determinant of both RA derivative concentrations (Fex et al. 1996), implying that any fluctuation in TTR protein status might entail corresponding alterations in cellular bioavailability of retinoid compounds. The intracellular activities exerted by retinoids are mediated by a large variety of membrane, cytosolic and nuclear receptors (Blomhoff and Blomhoff 2006; Napoli 1999). During normal ageing, the concentration of retinoids declines in cerebral tissues (Goodman and Pardee 2003), a regression likely to be more pronounced in malnourished AD patients (Ingenbleek 2008; Sandman et al. 1987). In animal models, depletion of RAs causes the deposition of amyloid b-peptides, favoring the formation of amyloid plaques (Corcoran et al. 2004). Aged animals are characterized by the downregulation of RA receptor expression (Etchamendy et al. 2001). Exogenous administration of RAs corrects the involutive process (Etchamendy et al. 2001) and maintains the activity of proteins involved in the control of amyloidogenic pathways (Husson et al. 2006). In addition, retinol may disaggregate preformed amyloid b-fibrils more effectively than do RAs (Ono et al. 2004), showing that retinol and RAs work in concert to prevent the cerebral damage of elderly people. These recent data have nutritional implications that have been tentatively described elsewhere (Ingenbleek 2008), emphasising that optimal protein nutritional status, assessed by TTR and retinol concentrations stabilized within normal ranges, offers optimal protection against neurodeterioration risk in AD and MID patients.
20.4
Transthyretin in Acute and Chronic Stressful Disorders
Inflammatory disorders of any cause are initiated by activated leukocytes releasing a shower of cytokines working as autocrine, paracrine, and endocrine molecules (Bienvenu et al. 2000). Experimental studies (Heinrich et al. 1990) and clinical investigations (Banks et al. 1995) have shown that interleukin-6 (IL-6) is a key mediator possessing a nuclear factor (NF) displaying a high degree of homology with C/EBP-HNF1 which competes for the same DNA response element of the IL-6 gene (Isshiki et al. 1991). IL-6 is not expressed under healthy conditions, explaining why the acute phase proteins (APPs) are kept at baseline plasma levels. Under acute stressful conditions, cytokines abruptly suppress the hepatic production of visceral
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proteins (Murakami et al. 1988) whereas protein turnover is strongly stimulated as a result of both augmented tissue proteolysis (mainly in the muscle mass) and enhanced specific tissue protein synthesis (mainly in the liver and at the site of injury). Muscle protein breakdown (Mitch and Goldberg 1996; Zoico and Roubenoff 2002) releases AAs which are preferentially driven into the hepatic precursor pool and utilized for the production of APPs with C-reactive protein (CRP) as an emblematic representative (Deodhar 1989) and of other defence and repair molecules (Hasselgren et al. 1988; Long et al. 1977). The rate of protein degradation generally exceeds the rate at which AAs are used for protein synthesis (Arnold et al. 1993; Tomkins et al. 1983) yielding a net negative balance associated with increased output of urea, ammonia, creatinine, 3-methylhistidine and other minor N catabolites in the urine (Beisel 1975; Long et al. 1981). Both visceral and metabolic N pools participate in the acute responses of the stressed body but the balance within and between these pools is determined by the nature and severity of the insult. The gap between synthesis and breakdown widens as the stress becomes more severe, resulting in increased catabolic and oxidative processes and urinary N losses. As a result, all protein components listed in Table 20.1 undergo varying degrees of size reduction that may be quantified by noninvasive tissue analyses or indirectly by the measurement of urinary N losses. In very severe cases of injury in adults, N output may rise to as high as 40 g N1 day1 or about 250 g N1week1, which corresponds to a loss of about 1.5 kg proteins or 8 kg LBM. The data are consistent with findings showing that TBP approximates 20% of LBM in healthy adults (Veldhuis et al. 2005). The peak of N urinary excretion culminates within 3–5 days after the initiation of acute injury (Cuthbertson 1942; Kasper et al. 1975). This coincides with the nadir recorded for the negative N balance and for TTR values (Ingenbleek and Young 2002; Kasper et al. 1975). When stressful condition subsides, provided that appropriate nutritional support is offered, both N balance and TTR levels improve and return to the physiological range within a couple of days. In contrast, inadequate dietary management (Hoover et al. 1980) or multiple injuries, severe sepsis and metabolic complications (Finn et al. 1996) result in persistent N losses with subnormal TTR plasma concentrations (Devakonda et al. 2008). The evolutionary patterns of N urinary output and of plasma TTR thus appear as mirror images of each other, which suggests that the BP reflects the depletion of LBM in both acute and chronic disease processes. Despite etiopathogenic mechanisms distinct from those characterizing PEM, TTR concentrations appear to reflect the loss or gain of LBM in stressful conditions, working as an excellent predictive tool of later outcome (Holland et al. 2001; Robinson et al. 2003; Sreedhara et al. 1996). Investigations on kidney patients have defined a plasma TTR threshold of 110 mg L1 as indicative of weak survival likelihood (Perez Valdivieso et al. 2008), consistent with the cut-off line of 100 mg L1 described in critically ill (Devakonda et al. 2008) and cancer patients (Geisler et al. 2007; Ho et al. 2003). The onset of injury and the surge of proinflammatory cytokines also stimulate the overproduction of counterregulatory hormones (glucocorticoids, catecholamines, glucagon and growth hormone GH) opposing the hypoglycemic and anabolic effects
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of oversecreted insulin. Taken together, these adaptive hormonal responses create a dichotomous stage characterized by insulin refractoriness and overall downregulation of synthetic processes in healthy tissues contrasting with upregulated activities in hepatic and inflamed territories (Ingenbleek and Bernstein 1999a, b). The IL-6induced suppressed liver production of TTR, but also of other BPs such as RBP, TBG and corticosteroid-binding globulin (CBG) amplifies the hormonal climate generated by the stress response (Ingenbleek and Bernstein 1999a, b). Following the law of mass action (Mendel 1989), the drop in plasma concentrations of these BPs releases significantly augmented amounts of ligands in physiologically active form. As a result, injured regions are transitorily flooded with waves of BP-derived thyroid, retinoid and steroid compounds fine-tuning the defence and repair mechanisms primarily initiated by proinflammatory cytokines (Ingenbleek and Bernstein 1999a, b). In the course of stress, transient elevation of thyroxine, retinol and cortisol concentrations in the urinary output attest to the fact that unmetabolized hormonal fractions undergo kidney leakage. The data imply that: (1) TTR, RBP, TBG, and CBG actively participate in the development of adaptive stress responses and should no longer be regarded as negative APPs and (2) the capacity of the stressed body to mount adequate responses is significantly impaired in the case of preexisting PEM because the amount of ligands released in free form is proportionate to the decrement recorded between pre and post-stress BP levels. The complexity and multifaceted aspects of these inflammatory events have been tentatively collected under the denomination of Nutritionally Dependent Adaptive Dichotomy (NDAD) (Ingenbleek and Bernstein 1999a, b). Theoretically, each protein component shown in Table 20.1 might become a candidate to represent the magnitude of TBN depletion and to predict the resulting outcome of diseased patients in the course of stress. Creatininuria (Arroyave et al. 1961), creatinine index (Desmeules et al. 2004; Terrier et al. 2008) or sarcopenia (Briend et al. 1989; Heymsfield et al. 1982) are used as excellent predictors of morbidity and likely mortality in epidemiological surveys and stressful disorders of medium or long-lasting duration. However, owing to their poor initial reactivity, these parameters fail to identify the early metabolic alterations of stress injury. Moreover, cautious interpretation is required here due to the fact that creatininuria hardly reaches 10% of all nitrogenous catabolites excreted in the urinary output (Ingenbleek and Young 2002), far less than urea, which results from deamination/ transamination reactions occurring in all body tissues. Finally, these SM parameters mainly reflect the downsizing of the structural compartment and miss the slower involutive processes affecting the visceral compartment. These distinct evolutionary patterns are illustrated by TBN and TBK that are both decreasing but not at the same rates (Cohn et al. 1983; Ellis et al. 1982). TBN displays a blunt slope which reflects more closely the slowly moving changes affecting the visceral compartment whereas TBK appears as a more dynamic and more rapidly turning over indicator (Cohn et al. 1983; Ellis et al. 1982). This implies that assessing sarcopenia and related parameters underrate the visceral life supporting component which is relatively spared until late in the disease process (Cohn et al. 1983; Ellis et al. 1982). In other words, the visceral compartment, which is the first to react also
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shows the longest resistance to the metabolic changes associated with the stress response, implying that the decline in SM mass, taken alone, might well underestimate the survival potential of the threatened body. Critically ill patients monitored for 21 days in intensive care units showed a 15% reduction in TBP whereas TBK was already reduced by 20% (Wood et al. 1984). In protracted debilitating disorders with terminal sarcopenia, the depletion of body cell mass did not exceed 45% of baseline values (Kotler et al. 1989). Measurement of LBM appears, therefore, as a more balanced approach since its encompasses both visceral and structural evolutionary patterns. LBM shrinking may be the consequence of either dietary restriction reducing protein syntheses to levels compatible with survival or that of cytokine-induced tissue proteolysis exceeding protein synthesis and resulting in a net body negative N balance. The size of LBM in turn determines plasma TTR concentrations whose liver production similarly depends on both dietary provision and inflammatory conditions. In animal cancer models, reduced TBN pools were correlated with decreasing plasma TTR values and provided the same predictive ability (Enrione et al. 1987). In kidney patients, LBM is proposed as an excellent predictor of outcome (Desmeules et al. 2004) working in the same direction as TTR plasma levels (Chertow et al. 2005; Holland et al. 2001; Terrier et al. 2008). Kidney patients with diabetic nephropathy may lose around 4 g albuminuria per day and deplete their LBM compartment by about 3.4 kg after a year (Pupim et al. 2005). Such loss of active metabolic tissue, which corresponds to about 7% of LBM in healthy individuals, is accompanied by an accelerated mortality rate (Pupim et al. 2005). In contrast, a recent prospective and randomized survey has shown that high N intake, supposed to preserve LBM reserves, reduces significantly the mortality rate of kidney patients and is positively correlated with the alterations of TTR plasma concentrations appearing as the sole predictor of final outcome (Cano et al. 2007). Taken together, these findings bolster the concept that the fluctuations of LBM integrate both nutritional and inflammatory facets of any disease spectrum and determine the level of liver TTR production rate. In the light of these recent integrative aspects, the long-lasting debate as to whether TTR should be regarded as a PEM marker or as a phlogistic indicator (Johnson 1999) now appears obsolete. It must nevertheless be kept in mind that nutritional and inflammatory factors working in varying proportions may similarly affect the body economy, making it hard to assess the role played by each component. The same downregulation of LBM values may indeed be determined by a severe PEM state associated with minor inflammatory burden or by a major stressful disorder affecting patients standing in relatively good nutritional status. Hence, the requisite to set up a comprehensive formula including all aspects of the disease paradigm. Several attempts have been made in that direction based on assembling biometric, hematological or immune parameters.The so-called prognostic inflammatory and nutritional index (PINI) has been beneficially applied in more than 100 clinical investigations covering most medical disciplines. The PINI scoring formula is a simple biochemical quotient aggregating two markers of nutritional (ALB and TTR) and of inflammatory (CRP and AGP or a1-acid
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glycoprotein, orosomucoid) states, providing a grading of the severity of any disease process (Ingenbleek and Carpentier 1985). PINI ¼
AGPðmg=LÞ CRPðmg=LÞ ALBðg=LÞ TTRðmg=LÞ
A number of recently promoted analytical tools such as surface-enhanced laser desorption/ionization (SELDI) or matrix-assisted laser desorption/ionization (MALDI) combined with time of flight-mass spectrometry (TOF-MS) can be used to the same end. The advent of these sophisticated and costly proteomic fingerprinting methods analyses the protein content of cells, tissues and body fluids such as plasma, urine, and CSF. The diagnostic and therapeutic usefulness of these approaches has been described in a recent review (Schweigert 2007). The proteomic detecting technology has highlighted that the native TTR monomer may coexist with four additional TTR variants having slightly higher molecular mass due to the disulfide S-S binding of small S-containing compounds (sulfonate, cysteine, cysteinylglycine, and glutathione) on cysteine 10 of the normal TTR amino acid sequence (Schweigert 2007). It is noteworthy that most SELDI or MALDI workers interested in defining protein nutritional status have chosen TTR as a biomarker, showing that there exists a large consensus considering the BP as the most reliable indicator of protein depletion in most morbid circumstances.
20.5
The Transthyretin-Homocysteine Saga
Total homocysteine (tHcy) is a S-containing AA not found in customary diets but endogenously produced in the body of mammals by the enzymatic transmethylation of methionine (Met), one of the eight IAAs supplied by staplefoods. Figure 20.1 shows that tHcy may either serve as precursor substrate for the synthesis of new Met molecules along the remethylation (RM) pathway or undergo irreversible kidney leakage through a cascade of derivatives defining the transsulfuration (TS) pathway. Hcy is thus situated at the crossroad of RM and TS pathways that are regulated by three water-soluble vitamins (pyridoxine, B6; folates, B9; cobalamins, B12). 5-Methyl-tetrahydrofolate (5-CH3-THF) operates as a donor of the methyl group in the RM process whereas cobalamins and pyridoxine work as cofactors of both enzymes governing the Hcy-metabolizing machinery, Met-synthase (EC 2.1.1.13) and cystathionine-b-synthase (CbS, EC 4.2.1.22), respectively (Fig. 20.1). Experimental studies have clarified the mechanisms whereby S-containing compounds distribute between both competing pathways so as to maintain Met homeostasis (Finkelstein and Martin 1984, 1986). It is also known how deficient intake of water-soluble vitamins may lead to hyperhomocysteinemic status likely to favor the development of atherothrombogenic events (Hankey and Eikelboom 1999; Welsch and Loscalzo 1998). Dietary deprivation of 5-CH3-THF (Kang et al. 1987) or of
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Fig. 20.1 Schematic configuration of the methionine (Met)-homocysteine (Hcy) cycle. Met supplied by dietary proteins undergoes transmethylation process to release Hcy and CH3. The latter compound is taken up by acceptor molecules (creatine, hormones, neurotransmitters, phospholipids) whereas the former may be remethylated to Met along the remethylation (RM) pathway or irreversibly degraded throughout the transsulfuration (TS) cascade. Both RM and TS pathways stand in competition under the control of 3 watersoluble vitamins so as to maintain Met homeostasis. 5-Methyl-tetrahydrofolate (5-CH3-THF, vit B9) is the donor of the CH3 group required for the remethylating process whereas cobalamins (vit B12) and pyridoxine (vit B6) operate as cofactors of Met-synthase and CbS activities, respectively. 1 Methionine-synthetase (Met-synthase), 2 cystathionine-b-synthase (CbS), Cysta cystathionine, Cys cysteine, Tau taurine, SO42 sulfaturia
cobalamins (Stabler et al. 1990) can depress Met-synthase activity, favoring the downstream accumulation of tHcy in biological fluids. Pyridoxine shortage may inhibit the activity of CbS, the rate-limiting step initiating the TS cascade, thereby promoting the upstream sequestration of tHcy (Ubbink et al. 1996). Serial measurement of tHcy shows that the low values recorded during the neonatal period increase linearly, without sexual difference, in preadolescent children but display gender dimorphism at puberty (Must et al. 2003; Rauh et al. 2001; Reddy 1997) and throughout sexual maturity (Battezzatti et al. 2007; Jacques et al. 1999). These tHcy temporal patterns from birth until the end of adulthood show striking similarities with LBM and TTR values (Battezzatti et al. 2007; Bienvenu et al. 1996; Dierkes et al. 2001). Thereafter, normal tHcy concentrations tend to rise (Bates et al. 1997; McCaddon et al. 1998), clearly diverging from declining TTR and LBM profiles.
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This age-related distinction occurs independently of water-soluble vitamin status (Strassburg et al. 2004) and appears to be genetically determined, explaining that normal elderly people constitute a target group incuring increased risk for tHcydependent thrombovascular disorders. A tentative explanation may be proposed in light of comparable data reported for plasma leptin, an adipokine (16 kDa) mainly secreted by white adipocytes and displaying a gender difference positively correlated with FM stores during sexual maturity. As expected, normal leptin values are higher in healthy adult female subjects characterized by a larger fat cell mass but reveal paradoxical elevation in elderly male subjects despite the decrease in total body fat. Deficient androgenic secretion could be involved as exogenous testosterone normalizes leptin values (Jockenho¨vel et al. 1997). Moreover, the only determinant of leptin levels is the androgen/estrogen ratio, indicating a major influence of sex steroids on leptin production (Jockenho¨vel et al. 1997). Because estradiol status is involved in tHcy variance (Dierkes et al. 2001) and sustains complex relationships with both androgens and GH (Giannoulis et al. 2006; Leung et al. 2004), a rearrangement of the sex hormone/trophic balance observed during normal adulthood could well explain the disrupted tHcy profile registered during normal ageing. The interactions of sex steroids with tHcy apparently operate as an effector of the hypothalamic–pituitary–gonadal–LBM axis. Regardless of vitamin-B status, this normal evolution toward rising tHcy levels may be accelerated in elderly patients suffering low grade disease activity documented by supranormal plasma APPs (CRP, a1-antichymotrypsin) and cytokine (IL-1, IL-6, tumor-necrosis factor a) concentrations (Bates et al. 1997; Gori et al. 2005; Visser et al. 2002; Wikby et al. 2006). The fact that significant positive correlations are found between tHcy and plasma urea (Koehler et al. 1996) and plasma creatinine (Bates et al. 1997; Koehler et al. 1996; Pancharuniti et al. 1994) indicates that both visceral and muscular tissues undergo proteolytic degradation throughout the course of rampant inflammatory burden. Summing up, the data indicate that in healthy individuals, tHcy plasma concentrations maintain positive correlations with LBM and TTR from birth until the end of adulthood. Starting from the onset of normal old age, tHcy values become disconnected from LBM control and reveal diverging trends with TTR values. Of utmost importance is the finding that, contrary to all protein components figuring in Table 20.1 which are downregulated in protein-depleted states, tHcy values are upregulated. A rationale explaining this unexpected converting process is proposed below. Hyperhomocysteinemia is an acquired clinical entity characterized by mild or moderate elevation in tHcy blood values found in apparently healthy individuals (McCully 1969). This distinct morbid condition appears as a public health problem of increasing importance in the general population, being regarded as an independent and graded risk factor for vascular pathogenesis unrelated to hypercholesterolemia, arterial hypertension, diabetes and smoking (Hankey and Eikelboom 1999; Welsch and Loscalzo 1998). There exists overall agreement that dietary deficiency in watersoluble vitamin(s) operates as a primary causal factor. Nevertheless, preventive and/or therapeutic vitamin supplementation trials have disclosed significant differences in their degree of potency. Folates are usually regarded as the most
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important regulatory nutrient in restoring tHcy plasma values, whereas pyridoxine appears as the weaker determinant. Cobalamin deficiency occupies an intermediary position affecting more particularly vegan subjects. Studies grounded on stepwise multiple regression analysis have concluded that the two main watersoluble vitamins account for only 28% of tHcy variance (Pancharuniti et al. 1994), whereas vitamins B6, B9, and B12, taken together, did not account for more than 30–40% of variance (Lussier-Cacan et al. 1996). Moreover, a number of hyperhomocysteinemic conditions are not responsive to folate and pyridoxine supplementation (BosyWestphal et al. 2001). This situation prompted us to search for other causal factors which might fill the gap between the public health data and the vitamin triad deficiencies currently incriminated. We suggest that links established between erratically dispersed observations have generated the increasingly documented proposal that S – the forgotten element – plays central roles in nutritional epidemiology (Ingenbleek and Young 2004). Aminoacidemia studies performed in PEM children (Antener et al. 1981; Holt et al. 1963), adult patients (Smith et al. 1974) and elderly subjects (Polge et al. 1997) have reported that the concentrations of plasma IAAs invariably display lowering trends as the morbid condition worsens. The depressed tendency is especially pronounced in the case of tryptophan (Antener et al. 1981) and for the so-called branched-chain AAs (BCAAs, isoleucine, leucine, valine) the decreases in which are regarded as a salient PEM feature following the direction outlined by TTR (Ingenbleek et al. 1986). Met constitutes a notable exception to the abovedescribed evolutionary profiles, showing unusual stability in chronically proteindepleted states. Maintenance of normal methioninemia is associated with supranormal tHcy blood values in PEM adults (Ingenbleek et al. 1986) and increased tHcy leakage in the urinary output of PEM children (Antener et al. 1981). In contrast, most plasma and urinary S-containing compounds produced along the TS pathway downstream to CbSconverting step (Fig. 20.1) display significantly diminished values. This is notably the case for cystathionine (Ingenbleek et al. 1986), glutathione (Jackson 1986), taurine (Gray et al. 1994) and sulfaturia (Ittyerah 1969). Such distorted patterns are reminiscent of abnormalities defining homocystinuria, an inborn disease of Met metabolism characterized by CbS refractoriness to pyridoxine stimuli (Mudd et al. 1985), thereby promoting the upstream retention of tHcy in biological fluids. It was hypothesized more than 20 years ago (Ingenbleek et al. 1986) that PEM is apparently able to similarly depress CbS activity, suggesting that the enzyme is a N-status sensitive step working as a bidirectional lockgate, overstimulated by high Met intake (Finkelstein and Martin 1986) and downregulated under N-deprivation conditions (Ingenbleek et al. 2002). Confirmation that N dietary deprivation may inhibit CbS activity was recently provided by experimental studies in rats (Okawa et al. 2006). As a result, the tHcy precursor pool is enlarged in biological fluids, boosting Met remethylation processes along the RM pathway, consistent with studies showing overstimulation of Met-synthase activity in conditions of protein restriction (Storch et al. 1990). In other words, high tHcy plasma concentrations observed in PEM states are the dark side of adaptive mechanisms for maintaining Met homeostasis. The uncommon steadiness of this IAA in such an unfavorable
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environment is in keeping with the unique role played by Met in the preservation of N body stores (Fuller et al. 1989; Owens and Bergen 1983), in the production of Scontaining molecules of survival importance (biotine, coenzyme A, glutathione, taurine, thiamine) and in the myriad of molecular, structural, functional, and metabolic properties exerted by S-compounds that are described in detail elsewhere (Ingenbleek and Young 2004). Increased homocystinuria in PEM patients indicates that varying proportions of unmetabolized Hcy molecules undergo renal leakage. The Met-deficiency theory clearly challenges the traditional water-oluble vitamin concept, explaining much of the remaining variance and providing a unifying concept for the nutritional factors involved in the maintenance of Met homeostasis. The classical interpretation that strict vegans, who consume plenty of folates in their diet (Gregory 1995) and manifest nevertheless higher tHcy plasma concentrations than omnivorous counterparts (Hung et al. 2002; Krajcovicova´-Kudla´kova´ et al. 2000), needs to be revisited. On the basis of hematological and biochemical criteria, cobalamin deficiency is one of the most prevalent vitamin-deficiencies wordwide (Stabler and Allen 2004), being often incriminated as deficient in vegan subjects (Hung et al. 2002; Krajcovicova´-Kudla´kova´ et al. 2000). It seems, however, likely that its true causal impact on rising tHcy values is substantially overestimated in most studies owing to the modest contribution played by cobalamins on tHcy variance analyses. In contrast, there exists a growing body of converging data indicating that the role played by the protein component is largely underscored in vegan studies. It is worth recalling that S is the main intracellular anion coexisting with N within a constant mean S:N ratio (1:14.5) in animal tissues and dietary products of animal origin (Ingenbleek 2006). The mean S:N ratio found in plant items ranges from 1:20 to 1:35, a proportion that does not optimally meet human tissue requirements (Ingenbleek 2006), paving the way for borderline S and N deficiencies. A recent Taiwanese investigation on hyperhomocysteinemic nuns consuming traditional vegetarian regimens consisting of mainly rice, soy products, vegetables and fruits with few or no dairy items illustrates such clinical misinterpretation (Hung et al. 2002). The authors reported that folates and cobalamins, taken together, accounted for only 28.6% of tHcy variance in the vegetarian cohort whereas pyridoxine was inoperative (Hung et al. 2002). The daily vegetable N and Met intakes were situated highly significantly (p < 0.001) below the recommended allowances for humans (FAO/WHO/United Nations University 1985), causing a stage of unrecognized PEM documented by significantly depressed BCAA plasma concentrations. Met levels escaped the overall decline in IAAs levels, emphasizing that efficient homeostatic mechanisms operate at the expense of an acquired hyperhomocysteinemic state. No difference in ALB values was recorded between omnivorous and vegan subjects, in accordance with the poor responsiveness of that plasma protein to marginal alterations in protein nutritional status (Ingenbleek et al. 1975a). The diagnosis of subclinical PEM was missed because the authors ignored the exquisitely sensitive TTR detecting power. A proper PEM identification would have allowed the authors to confirm the previously described TTR–tHcy relationship (Fig. 20.2) that was established in Western Africa from comparable field studies involving country dwellers living on plant products. Figure 20.2 indicates that
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Fig. 20.2 Measurement of TTR and tHcy concentrations in control subjects (C) and subclinically malnourished goitrous patients recruited in three cohorts of 20 adult individuals representing the stages I, II, and III of thyroid swelling following WHO criteria. TTR and tHcy values are expressed as mean standard deviation (horizontal and vertical bars). In the C group, male (M) and female (F) data are shown separately, indicating that both TTR and tHcy manifest gender dimorphism. The data reveal that declining nutritional status, as assessed by TTR values, is negatively correlated with rising tHcy concentrations
plotting TTR against tHcy values yields a negative correlation linking deterioration of protein nutrition and hyperhomocysteinemia, regardless of water-soluble vitamin status. In terms of tHcy-induced atherothrombogenic risks, future studies carried on vegan subjects should assess the benefits generated by low lipid energy and high fiber intake and the health hazards created by chronic N insufficiency and permanently disordered IAA nurture. Excessive urinary N and S leakage accompanying any disease process may cause the same deleterious effects as those resulting from dietary shortage of N and S nutrients. The concept that acute or chronic stressful conditions may exert similar inhibitory effects on CbS activity and thereby promote hyperhomocysteinemic states is founded on previous studies showing that hypercatabolic states are characterized by increased urinary N and S losses maintaining tightly correlated depletion rates (Cuthbertson 1931; Ingenbleek and Young 2004; Sherman and Hawk 1900) which reflect the S:N ratio found in tissues undergoing cytokineinduced proteolysis. This has been documented in coronary infarction (Turgan et al. 1999) and in acute pancreatitis (Yuzbasioglu et al. 2008) where tHcy elevation evolves too rapidly to allow for a nutritional vitamin B-deficit explanation. As a matter of fact, tHcy is regarded as a stable plasma parameter (Garg et al. 1997) and the two investigations cited (Turgan et al. 1999; Yuzbasioglu et al. 2008) indeed report unaltered folate and cobalamin plasma concentrations. We assume that the elevated tHcy values culminate with the topmost level reached by N- and
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S-catabolites in the urinary output and coincide with the nadir recorded for TTR values. There exists also a number of chronic stressful disorders characterized by high tHcy values and for which a valid causal explanation is still lacking. Patients affected by morbid obesity who are submitted to severe BW reduction programs (Gallistl et al. 2001) or undergo bariatric surgery often develop secondary hyperhomocysteinemic status as early as six months (Sheu et al. 2001) or one year (Borson-Chazot et al. 1999) after gastroplasty. The available data strongly suggest that the BW reduction observed postoperatively is the result of an expected FM loss entailing significant alleviation of all conventional cardiovascular risk factors (Borson-Chazot et al. 1999; Sheu et al. 2001) but associated with undiagnosed LBM wastage causally related to imbalanced regimen and/or intestinal malabsorptive syndromes. Gastroplasty for morbid obesity may cause body composition changes (Strauss et al. 2003) and varying degrees of protein-depleted states including severe PEM (Faintuch et al. 2004). In multiple linear regression analysis performed on obese patients, LBM was indeed the sole independent variable negatively correlated with rising tHcy blood values (Gallistl et al. 2001). The same relationship has been documented in patients with types I and II diabetes mellitus who reveal tHcy plasma levels elevated in proportion to the severity of their nephropathy (Chico et al. 1998). Univariate correlations and multiple regression analyses point to the level of albuminuria as the strongest independent parameter associated with high tHcy values (Chico et al. 1998). Cirrhotic patients who are submitted to orthotopic liver transplantation maintain postsurgical elevation of creatinine and fibrinogen plasma concentrations that are positively correlated with tHcy values (Bosy-Westphal et al. 2001), indicating the persistence of low grade muscle proteolysis. There is no doubt that the routine measurement of both TTR and tHcy will clarify many aspects of these intricate disease conditions and increase the interest of many future nutritional studies.
20.6
Concluding Perspectives
The clinical usefulness of TTR as a nutritional biomarker, described in the early seventies (Ingenbleek et al. 1972) has been substantially disregarded by the scientific community for nearly four decades. This long-lasting reluctance expressed by many investigators is largely due to the fact that protein malnutrition and stressful disorders of various causes have combined inhibitory effects on hepatic TTR synthesis. Declining TTR plasma concentrations may result from either dietary protein and energy restrictions (Strauss et al. 1994) or from cytokine-induced transcriptional blockade (Murakami et al. 1988) of its hepatic synthesis. The proposed marker was therefore seen as having high sensitivity but poor specificity. Recent advances in protein metabolism settle the controversy by throwing further light on the relationships between TTR and the N-components of body composition. Among all parameters listed in Table 20.1, SM and derived compounds constitute excellent predictive tools of outcome in long-lasting disorders. However, their
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clinical reliability is limited by their poor reactivity during the initial stages of stress and by the fact that muscle tissue proteolysis underestimates the whole body capacity to surmount the survival crisis. LBM status appears as a most reliable indicator of malnutrition and inflammation since its fluctuations encompass both morbid conditions and represent a more balanced assessment of visceral and structural tissue reserves. The developmental patterns of LBM and TTR exhibit striking similarities. Both parameters rise from birth to puberty, manifest gender dimorphism during full sexual maturity then decrease during ageing. Uncomplicated PEM primarily affects both visceral and structural pools of LBM with distinct kinetics, reducing protein synthesis to levels compatible with prolonged survival. In acute or chronic stressful disorders, LBM undergoes muscle proteolysis exceeding the upregulation of protein syntheses in liver and injured areas, yielding a net body negative N balance. These adaptive responses are well identified by the measurement of TTR plasma concentrations which therefore appear as a plasma marker for LBM fluctuations. Attenuation of stress and/or introduction of nutritional rehabilitation restores both LBM and TTR to normal values following parallel slopes. TTR fulfills, therefore, a unique position in assessing actual protein nutritional status, monitoring the efficacy of dietetic support and predicting the patient’s outcome (Bernstein and Pleban 1996). TTR is a simple, rapid and inexpensive biochemical micromethod allowing the correct follow-up of any diseased person. TTR is regarded as the gold standard protein marker for PEM states (Bernstein et al. 1995; Ingenbleek and Young 1994) with the additional advantages of reducing duration of hospitalization and the risk of undesirable complications and mortality. When TTR screening is performed in the first hours of hospitalization and followed by appropriate nutritional support, significant reductions in costs ensue (Bernstein and Ingenbleek 2002; Potter and Luxton 2002). Met is an S-containing IAA that plays roles of survival importance in body economy and whose essentiality is closely correlated with N metabolism. Met benefits from high homeostasis priority that is tightly regulated by the dietary intake of N and water-soluble vitamins normally supplied by the regular consumption of well-balanced omnivorous regimens. In the case of restricted N and S intakes, as seen in vegetarianism (Hung et al. 2002; Krajcovicova´-Kudla´kova´ et al. 2000), anorexia nervosa (Moyano et al. 1998) or subclinical PEM (Ingenbleek et al. 2002), Met homeostasis is maintained at the expense of impaired CbS activity, allowing the upstream retention of enlarged tHcy pools in body fluids and the activation of remethylating tHcy ! Met processes. The same compensatory mechanisms may take place in the course of any acute stressful condition exhausting LBM reserves through excessive N and S losses. Regardless of water-soluble vitamin status, the sudden rise of plasma tHcy in acutely ill patients (Turgan et al. 1999; Yuzbasioglu et al. 2008) is more likely to occur in subjects living on borderline TBN and TBS status. We assume that the acquired biochemical anomaly is largely underrated in clinical practice and should also develop in many diseased conditions characterized by massive hypercatabolic losses such as in typhoid fever or malaria.
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In chronically ill patients, high tHcy plasma values may arise from the loss of body proteins through the kidneys (Chico et al. 1998) or through the intestinal and bronchial mucosas as predicted for protein-losing enteropathy or cystic fibrosis. Hyperhomocysteinemic states may also result from long-lasting evolutive diseases such as myeloproliferative leukemias, auto-immune disorders or neurodegenerative processes often characterized by alternating bouts of inflammation and remission. Every clinical relapse should correspond to a stage of hypercatabolic resurgence with transiently increased tHcy plasma levels coinciding with stepwise deterioration of LBM resources and of TTR values. Taken together, the data show that the serial measurement of TTR should be an integral part of any preventive or therapeutic program since its omission may result in misleading conclusions. Complementing the assessment of water-soluble vitamin status, the measurement of TTR allows to set up an unifying concept for the main nutritional factors involved in the control of Met homeostasis.
Acknowledgments The tremendously high amount of TTR data that was generously provided by Robert Ritchie, Thomas B. Ledue, and Dwight E. Smith (Foundation for Blood Research, Scarborough, Maine 04074, USA) and collected in Table 20.2 of this chapter is gratefully acknowledged.
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Index
A
E
Age-related diseases, 316 Allantoin, 96–98, 110, 111 Alzheimer’s disease (AD), 286–289 Amphibian, 28, 29, 33, 35–39, 49, 52 Antibody therapy, 222
Endocrine disruptors, 159–167 Eutherian, 24, 26, 28–30, 33–39 Exons, 46, 47, 49, 50, 52, 53, 55
B Behavioral disorders, 286–289, 314 Binding pockets, 5, 10, 11, 13, 16 Bone, 299, 302, 303 Brain, 304–306
F Familial amyloidotic polyneuropathy (FAP), 2, 14, 16, 17, 174, 176, 179–181, 191–199, 215–231 Fibril disruption, 221 Fish (seabream), 69, 103, 146–151, 153 Flavones, 10 Flavonoids, 160–164
C Caenorhabditis elegans, 111, 117, 118, 120 Cardiomyopathy, 174–180, 183–185, 191, 241, 245–247, 249, 251, 252 Cerebrospinal fluid (CSF), 45, 305 Chimera, 124, 129–134, 137 Choroid plexus, 24, 28, 31–32, 35–36, 39 Clinical symptoms, 216, 224, 227 Codons, 47, 49, 52, 53 Cooperativity, 206 Crystal space group and packing, 5 Crystal structure, 3, 4, 7, 8, 11, 14, 78, 81–84, 87, 88, 91, 96, 101–106, 129–136 Cys-10, 203–207, 210, 211
G Gene disruption, 268, 272, 273, 282 Gene therapy, 216, 227–230 Gut, 304
H Haplotype, 176, 178, 183 Health-related states, 333 Heat-shock proteins (hsp), 195, 196 His-31, 201–212 Homocysteine, 340–346 5-Hydroxyisourate hydrolase (HIUase), 65–71, 77–92, 96–107
D Deiodinase, 61 Development, 298–306
I Introns, 46, 47, 49, 52, 53, 55
359
360
Index
L
S
Liver, 26–34, 36, 39 Liver transplantation, 239–254
Salmonella dublin, 80–92 Selenoprotein, 61 Ser-46, 207, 208, 211 Serum amyloid P component (SAP), 270–275 Species, 3, 5, 11, 16 Stress disorders, 330, 336–340, 346 Structure activity, 2
M Marsupial, 23–40, 49, 52 Microheterogeneity, 201–212 Monotreme, 24, 33–35, 39 Mutations, 174, 176–183
N Negative cooperativity, 2, 8–10, 12, 17 Nerve regeneration, 321–324, 326 Neurodegeneration, 192, 195, 196 Neuropathy, 240, 243–245 Neuropeptide Y (NPY), 319–321 Non-steroidal anti-inflammatory drugs (NSAID), 162–164, 224–225
O Open reading frame (ORF), 47 Operon, 89, 91 Organ complications, 245–249
P Pathogens, 191–199 Peptidylglycine alpha-amidating monooxygenase (PAM), 317–321, 323 Peripheral nervous system (PNS), 191, 192, 196, 199 Peripheral neuropathy, 243–244 Periplasm, 82, 91, 92 Plasma exchange, 217 Protein Data Bank (PDB), 3, 5, 16 Protein-depletion, 333–336, 342, 346
T Teleost, 144, 146, 147, 150, 151, 154 Thyroid hormone distributor proteins (THDPs), 26–28, 30–34, 36, 39, 60, 61, 64 Thyroxine-binding globulin (TBG), 2, 26–28, 30, 31, 33 Transgenic mouse models, 267–272, 274, 277 Transthyretin-like protein (TLP), 60, 65, 66, 68–71, 77–92, 97, 111, 112, 117–121 Transthyretin (TTR)-null mice, 281–290, 297–306, 311–326 Transthyretin-related proteins (TRP), 109–119, 121 Treatment, 215–218, 222, 224, 225, 227, 228, 230, 231
U Ureide, 96, 109
V Vitamin A, 123, 144–146, 150–152, 154
R
W
Retinol-binding protein (RBP), 28, 29, 32, 35, 62, 123–138, 143–154, 285, 286
Wallaby, 207–210 Weaning, 300–301, 306