Current Topics in Cellular Regulation, Volume 34 (Current Topics in Cellular Regulation)

CURRENT TOPICS IN

Cellular Regulation Volume 34

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CURRENT TOPICS IN

Cellular Regulation edited by Earl R. Stadtman

National Institutes of Health Bethesda, Maryland

P. Boon Chock

National Institutes of Health Bethesda, Maryland

Volume 34

ACADEMIC PRESS San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. © Copyright © 1996 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc.

A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NWl 7DX International Standard Serial Number: 0070-2137 International Standard Book Number: 0-12-152834-0 PRINTED IN THE UNITED STATES OF AMERICA 96 97 98 99 00 01 EB 9 8 7 6 5

4

3 2 1

Contents

Regulatory Features of Multicatalytic and 26S Proteases LAURA HOFFMAN AND M A R T I N RECHSTEINER

I. II. III. IV. V. VI.

Introduction Structural and Enzymatic Properties of MCP Structural and Enzymatic Properties of 26S Protease Regulation of MCP and 26S Protease Regulation by MCP and 26S Proteases Summary References

1 2 5 8 22 26 26

Calponin STEVEN J. WINDER AND MICHAEL P. WALSH

I. II. III. IV. V.

Introduction Biochemical Properties of Calponin Functional Properties of Calponin Structure-Function Relations Calponin and Caldesmon References

33 36 43 57 58 59

Type III Cyclic Nucleotide Phosphodiesterases and Insulin Action VINCENT C . MANGANIELLO, MASATO TAIRA, TETSURO KONO, EVA DEGERMAN, AND P E R BELFRAGE

I. Introduction II. Cyclic Nucleotide PDE Gene Families III. Type III cGMP-Inhibited Cyclic Nucleotide Phosphodiesterases References

63 64 70 91

VI

CONTENTS

Mammalian Aminoacyl-tRNA Synthetases DAVID C. H . YANG

I. II. III. rV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XrV. XV. XVI. XVEI. XVIII. XIX. XX.

Introduction Classification of Mammalian Aminoacyl-tRNA Synthetases General Structure of the RS Complex Dissociation and Organization of the Synthetase Complex Primary Structures of Mammalian Synthetases Distinct Characteristics of N-Terminal Extensions in Mammalian Aminoacyl-tRNA Synthetases Functional Significance of Synthetase Complex Aspartyl-tRNA Synthetase Arginyl-tRNA Synthetase Lysyl-tRNA Synthetase GluPro-tRNA Synthetase Valyl-tRNA Synthetase Complex Tryptophanyl-tRNA Synthetase Seryl-tRNA Synthetase Threonyl-tRNA Synthetase Correlation of the Classifications of Amino Acids and Mammalian Aminoacyl-tRNA Synthetases Autoantibodies to Mammalian Synthetases Aminoacyl-tRNA Synthetases as Multifunctional Proteins Organization of Synthetases and the Protein Biosynthetic Machinery Prospects References

101 102 102 105 107 108 Ill 112 118 119 120 122 124 125 126 126 127 128 129 129 131

Regulation of Interaction between Signaling Protein Chey and Flageller Motor during Bacterial Chemotaxis RiNA B A R A K AND M I C H A E L E I S E N B A C H

I. II. III. IV.

Introduction Proteins That Participate in CheY-Switch Interaction Regulation of the CheY-Switch Interaction Concluding Remarks References

137 140 143 153 153

Chemical Biology of Nitric Oxide: Regulation and Protective and Toxic Mechanisms DAVID A. WINK, INGEBORG HANBAUER, MATTHEW B . GRISHAM, FRANCOISE LAVAL, RAYMOND W . N I M S , JACQUES LAVAL, J O H N COOK, ROBERTO PACELLI, JAMES LIEBMANN, MURALI KRISHNA, PETER C . FORD, AND JAMES R . MITCHELL

I. Introduction

159

CONTENTS II. III. rV. V. VI.

Chemical Aspects of Nitric Oxide Biochemical Targets for Nitric Oxide Extracellular and Intracellular Metabolism of Nitric Oxide Nitric Oxide and Oxidative Stress Conclusions: Direct versus Indirect Effects of Nitric Oxide on Biological Systems References

Vll 161 167 172 175 182 183

Nutritional and Hormonal Regulation of Glutathione Homeostasis CARLA G. TAYLOR, LAURA E . NAGY, AND TAMMY M . BRAY I. Introduction II. Glutathione Synthesis and Interorgan Homeostasis III. Regulation of Tissue Glutathione Concentration by Diet and Nutritional Status IV. Regulation of Glutathione by Hormones V. Glutathione in the Vicious Cycle of Disease, Infection, and Malnutrition References

189 191 192 195 200 204

Protein Folding and Association: In Vitro Studies for Self-Organization and Targeting in the Cell RAINER JAENICKE I. II. III. IV. V. VI.

INDEX

Introduction Hierarchies of Structure, Stability, and Folding Mechanism of Folding and Association Cellular Aspects Practical Aspects Conclusions References

209 212 218 257 293 297 301 315

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CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Regulatory Features of Multicatalytic and 26S Proteases LAURA HOFFMAN MARTIN RECHSTEINER Department of Biochemistry University of Utah School of Medicine Salt Lake City, Utah 84132

I. Introduction Intracellular proteolysis serves as an important regulatory mechanism (see Refs. 1-6 for reviews). Specific enzymes are subject to rapid degradation in response to changing nutritional conditions as seen, for example, during glucose repression in yeast (7) or polyaminestimulated degradation of ornithine decarboxylase (8). A host of eukaryotic transcription factors are naturally short-lived proteins (1). Moreover, cell cycle progression requires destruction of cyclins, the polypeptide activators of cell division cycle 2 (cdc2) kinase (9-11). The number of proteases involved in removing such rapidly degraded proteins is less clear. In fact, surprisingly few endoproteases have been localized to the nucleus and C5^osol of eukaryotic cells. Whereas there is an abundance of lysosomal cathepsins (12) and secreted proteases (13), only a handful of C3rtoplasmic endoproteases have been identified. These include the calpains (14), a 70-kDa metalloprotease (15), proline endopeptidase (16), a recently described interleukin 1(3 processing enzyme (17,18), and two large degradative enzymes, the multicatalytic protease (MCP), and the 26S ATP/ubiquitin-dependent protease (19,20). In this chapter, we focus on regulatory aspects of the latter two proteolytic complexes. The MCP and 26S complexes provide rich possibilities for regulation. Besides traditional mechanisms, such as control of enzyme levels or phosphorylation, potential combinatorial associations between families of MCP subunits and perhaps even larger families of ATPase subunits could generate a variety of specific proteolytic complexes. When the reported activators and inhibitors of MCP are considered, the regulatory capabilities are substantial (see Fig. 1). Furthermore, the nuclear/ cytoplasmic distribution of the two proteases varies with development and growth state. Changes in the locations of the proteases could well affect their access to substrates. Despite these possibilities, there are 1

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

LAURA HOFFMAN AND MARTIN RECHSTEINER

activated

MCP

FIG. 1. Schematic representation of the association between MCP and various regulatory complexes. MCP is shown as the central cylinder that can interact with several other protein complexes. The ATPase complex (AC) combines with MCP in an ATPdependent reaction to form the 26S protease. When the regulator (REG) interacts with MCP, the resulting MCP complex is activated for hydrolysis of small fluorogenic peptides. A second MCP complex with increased peptidase activity has an additional subunit with an apparent Mr of 160,000. Latent MCP may contain an additional protein of ~30 kDa that inhibits MCP's activity. Evidence supporting these interactions is presented in the text. Finally, a 15S ATPase complex is composed of a protein homologous to the S4 subfamily of ATPase (AC) subunits. It could conceivably interact with MCP although there are no data supporting this hypothesis.

only a few well-documented examples of regulation of MCP or the 26S enzyme. Consequently, several topics are covered in this chapter because they illustrate conceivable regulatory strategies, not because it has been demonstrated that such mechanisms operate within cells.

11. Structural and Enzymatic Properties of MCP During the past few years considerable progress has been made regarding the structural and functional aspects of the multicatalytic protease, and recent publications are available on the primary structures of MCP subunits, their arrangements in the proteolytic complex, and on various proteolytic activities of MCP (19-23). Although this chapter focuses on the regulatory aspects of MCP, a brief structural review should prove useful. Because the multicataljrtic protease is a large (700 kDa), complex (>10 subunits), potentially dangerous (broad

MULTICATALYTIC AND 26S PROTEASES

3

cleavage specificity), and ubiquitous protease, its existence in the cytoplasm has been somewhat of an enigma. However, studies have indicated that MCP is, in fact, a well-regulated and versatile enzyme. It has been identified as a component of several multisubunit complexes with varying specific activities toward fluorogenic peptides and varying abilities to degrade polypeptide substrates. Although there are two reports that an MCP-like protease may be present in prokaryotes (24,25), MCP proper appears to be restricted to eukaryotes and the archea. In these organisms, the multicatalytic protease is a cylindrical structure composed of four stacked rings each containing six or seven subunits (21). The archaebacterial Thermoplasma enzyme consists of only two subunits, termed a and p, whose sequences are related (26). Immunoelectron microscopy studies on the Thermoplasma enzjrme demonstrate that the j8 subunits, which may provide the enzjrme's active sites, are located in the inner two rings of the cylinder; the a subunits, which may serve regulatory rather than proteolytic functions, form rings at each end of the cylinder (27). Two-dimensional gels reveal more than a dozen MCP subunits in eukaryotes (see Fig. 2). Genes for 10 distinct MCP subunits have been cloned from a variety of organisms, including yeast (28), Drosophila (29), Xenopus (30), and mammals (31,32). The deduced sequences of eukaryotic MCP subunits, now classified as a or j8 according to their similarity to the Thermoplasma subunits, are unlike those of any previously identified proteases. MCP subunits are, however, related to each other and thus seem to represent a new family of proteases (22). There is little doubt that the eukaryotic multicatalytic protease contains several distinct proteolytic activities. Early studies demonstrated a very broad cleavage specificity for MCP, e.g., the enzyme cleaves peptides after basic, acidic, and hydrophobic residues (33). Protease inhibitors provided the key observation implicating distinct active sites in peptide bond breakage. Orlowski and Wilk (34) found that certain compounds inhibited cleavage of one peptide while actually stimulating hydrolysis of another. The existence of multiple genes encoding a and j8 subunits is consistent with the presence of distinct protease active sites in MCP. It also opens the possibility that MCP activities might be regulated by altering the subunit composition within the MCP particle. The catalytic mechanism employed by MCP has not been determined. Data on inhibition of MCPs isolated from various organisms are compatible with peptide bond cleavage being mediated by either serine or cysteine residues (see Table I). Analyses of MCP subunit sequences have not resolved the issue. Histidine, a key residue in the catalytic triad of serine proteases, is not conserved among MCP subunits, nor

r.

LAURA HOFFMAN AND MARTIN RECHSTEINER P*^

7 I

6

I

5

I

MW ^^^

4

I

I

7

I

6

I

5

4 I

ATPase complex

66-

45 29-

•

24 -

Regulator

26S prdlease •< 66 -

\< 4529-

U^ >. ' >•

— 0

24 -

FIG. 2. 2D PAGE patterns of subunits from MCP, AC, 26S protease, and regulator. Proteins in the various complexes were separated in the first dimension by isoelectric focusing (pH 10-3) and in the second dimension by denaturing SDS-PAGE, and the gels were subsequently silver stained. Three major MCP subxinits are denoted by open arrowheads; MCP proteins are also found in the 26S protease, but not in the ATPase complex or regulator. The ATPase complex subunits between 30 and 110 kDa are shown, although resolution of the 100- and 110-kDa proteins was not obtained. Four of the AC proteins are denoted by closed arrowheads for orientation; the same set of proteins are also found in the 26S protease. The 30-kDa regulator protein typically resolves as a protein with a p/ of ~5.5 (open circle). Occasionally, an additional protein with a higher p/ can also be resolved. The region where regulator would migrate is circled on the AC and 26S protease gels. The regulator protein is clearly absent from the ATPase and 26S protease complexes.

are there patterns of histidine and cysteine characteristic of sulfhydryl proteases. At present, it seems hkely that MCP will prove to be an at3rpical serine protease (22). The enzyme is shown in Fig. 1 as a hollow cylinder, a structure much like that of microtubules. It is not clear whether the particle contains an aqueous central channel, nor is it clear where the protease active sites are located. However, it is attractive to

MULTICATALYTIC AND 26S PROTEASES TABLE I INHIBITORS OF SUC-LEU-LEU-VAL-TYR-MCA CLEAVAGE BY M C P

SoureeofMCP Rabbit reticuloc5d:e°'' Compound Chymostatin Antipain Pepstatin Leupeptin PMSF^ TPCK^ NEM'' EDTA EGTA Calpain inhibitor I Calpain inhibitor II

Sea urchin sperm^

Spinach*^

Cone. ifjiM)

Inhib.

(%)

Cone. ifiM)

Inhib.

Cone. ifiM)

Inhib.

250 250 250 250 2500 250 2500

83 0 STIM 20 0 5 85

100 100 10 100 2000 100 1000

99 23 STIM 63 61 7 98

100 100 100 5000 100 500 5000 1000

92 6 0 57 23 0 95 0

—

—

—

1000 250

4 87

— — —

250

65

—

(%)

— — — —

— —

(%)

Thermc)plasma^ Cone. ifiM) 16 18 18 25

— —

Inhib.

(%) 0 0 0 11

— —

— —

4000 5000 5000 26

0 97 100 100

—

25

73

Note. Cone., eoneentration; Inhib., inhibition. " Hough et al. (41). *Dubiele^a/. (100). 'Inahaietal. (185). ^Ozaki etai (186). ^ Dahlmann e^ a/. (187). ^ PMSF, phenylmethylsulfonyl fluoride. ^ TPCK, tosyl-phenylalanine ehloromethyl ketone. '' NEM, N-ethylmaleimide.

imagine that the active sites Hne a central canal. This would prevent indiscriminate proteolysis of cellular proteins by MCP.

III. Structural and Enzymatic Properties of 26S Protease A major advance in our understanding of intracellular proteolysis was the demonstration that ubiquitin (Ub) targets proteins for destruction. In two classic 1980 papers, Hershko and colleagues (35,36) showed that a 8.5-kDa protein, later identified as Ub (37), was required for ATP-dependent degradation of bovine serum albumin and RNase in rabbit reticulocyte lysate. They also found that Ub was covalently attached to the protein substrates. Based on these findings, they proposed that Ub marks proteins for destruction. Others have suggested

b

LAURA HOFFMAN AND MARTIN RECHSTEINER

that Ub has proteolytic activity (38) or that it stimulates proteolysis by inactivating an endogenous protease inhibitor (39). However, most who study Ub-mediated proteolysis would agree with some version of the pathway presented in Fig. 3. In this figure, the carboxyl terminus of Ub is shown to be activated by an enzyme (El), transferred as a high-energy thiol ester to Ub carrier proteins (E2s), and subsequently deposited in monomeric form on amino groups of histones (H2A) or as poly (Ub) chains on proteolytic substrates (S). The latter reaction often requires the participation of a ubiquitin protein ligase (E3). The marked substrates are then shown to be degraded and Ub is recycled. Experimental support for the scheme in Fig. 3 is considerable, and it has been reviewed on several occasions during the past few years (1-4).

l^

S peptides

FIG. 3. Schematic representation of the ubiquitin-mediated proteolytic pathway. Starting at the top of the diagram, the carboxyl terminus of ubiquitin (Ub) is shown to be activated by the E l enzyme and transferred as a reactive thiol ester to one of several small E2 carrier proteins. Ub is then conjugated directly to lysine-119 on histone H2A or to lysine amino groups on proteolytic substrates (S) by a Ub-ligase enzyme (E3). Monoubiquitinated proteins, such as H2A, are apparently not targeted for degradation. For other proteins though, Lys-48 of ubiquitin is used as a target site for building ubiquitin "chains." These polyubiquitinated proteins are substrates for degradation by the 26S protease (P) which hydrolyzes the substrate in an ATP-dependent reaction, generating small peptide products and recycled ubiquitin molecules.

MULTICATALYTIC AND 26S PROTEASES

7

A major feature of the marking hypothesis is the existence of a protease that specifically degrades ubiquitinated proteins. In 1986, Hough et al. (40) identified an enz3rme that degraded Ub-lysozyme conjugates in an ATP-dependent reaction; a year later they reported its purification (41). The 26S enzyme contains at least 20 different polypeptides (see Fig. 2), including a subset with molecular weights and isoelectric points characteristic of MCP subunits. Similarities in subunit composition led to the proposal that MCP subunits were integral parts of the 26S enzyme (42); the larger (>42 kDa) polypeptides were proposed to confer ATP dependence as well as Ub recognition on the 26S protease. Based on electron microscopy images obtained in 1970 (43) and subunit stoichiometries, Hough et aL (42) proposed a specific arrangement for MCP and other subunits in the 26S complex (see Fig. 1). Two lines of evidence support the shared subunit h5rpothesis and the idea that higher molecular weight polypeptides confer energy dependence. Whereas Hough et al. (41) isolated Ub-conjugate degrading activity as a 26S enzyme, Hershko and colleagues found that mixing three smaller components, termed CFl, CF2, and CF3, was required for degradation of Ub-lysozyme (44). Subsequent studies demonstrated that CF3 is the multicatalytic protease (45-47). Armon et al. (48) also showed that an NTPase activity is generated upon assembly of the CFs; this result has been confirmed (49). Recent cDNA cloning provides a second observation supporting the idea that the larger 26S subunits confer energy dependence. Dubiel et al. (50) reported the sequence for a 51-kDa polypeptide, subunit 4, from human red cell 26S protease. Subunit 4 belongs to a novel eukaryotic ATPase family that includes yeast CDC48p and yeast PASlp, Chinese hamster NSF, Xenopus p97, and four proteins—TBPl, TBP7, MSSl, and SUGl—very closely related to subunit 4 (50). The latter four proteins together with S4 constitute a subfamily of proteins, each about 440 residues, that contains one nucleotide binding site. The other members of the ATPase family are roughly twice as long and contain two ATP consensus sites per chain. Functionally, we believe that the S4 subfamily ATPase subunits serve to present substrates to the proteolytic core provided by MCP (see Ref. 22 and below for further discussion). In addition, two ATP-dependent proteases from Escherichia coli, Lon and Clp, are almost certainly members of this family (22). Given the structural and functional parallels between the 26S protease, Clp, and Lon, we proposed that the three enzymes are evolutionarily related (22). Structually, 26S and Clp appear to be closer relatives. Both are

8

LAURA HOFFMAN AND MARTIN RECHSTEINER

composed of hexameric rings of protease subunits (ClpP and MCP) that associate with separate ATPase subunits (ClpA and S4-Hke proteins). However, from an enzymatic perspective, the 26S enzyme is more Hke Lon. Both 26S and Lon can utiHze ATP, CTP, GTP, or UTP for proteolysis. In addition, both enzymes exhibit high affinity for nucleotides; the A'ni for proteolysis is about 30 fiM ATP (51). By contrast, Clp is specific for ATP and much higher levels of the nucleotide (K^n -1000 /xM) are needed to support protein breakdown (52). In this regard, the ATPase activity of ClpA resembles that of Xenopus p97. The latter may also be specific for ATP; it requires high levels of nucleotide for hydrolysis (53) and, interestingly, it also forms a hexamer (see Fig. 1 and below). IV. Regulation of MCP and 26S Protease A. Regulation of Protease Levels The concentration of MCP varies markedly among tissues. Values range from more than 2% of the total protein in rat thymus and testes (54) to as little as 0.01% of human lymphocyte proteins (55). Typically, however, MCP is a reasonably abundant cellular constituent present at about 0.5% of soluble proteins or roughly one MCP for every two ribosomes (see Table II). TABLE II LEVELS OF MCP

IN TISSUES OR CELL TYPES

MCP levels Tissue or cell type Human lymphocytes MOLT-10 HL60 Human renal cells Rat muscle Rat testis Rat th3mius Rat muscle Rat liver Rat kidney Sea urchin egg

jjLg per milligram cell protein 0.135 1.36 3.94 -7.0 3.27 23.6 28.5 1.1 9.4 2.0

—

Molecules per cell 1.2 5.8 1.7 2.9

X W X 10^ X W X W

— —

Ref.

55 56 54

1.2 X 10^

—

4 X 10^ 8.5 X W 2 X 10«

" For lymphocytes we assumed 1 mg protein = 10^ cells. For all other cell types we assumed 1 mg = 2 x 10^ cells.

57 58

MULTICATALYTIC AND 26S PROTEASES

9

The steady-state concentration of an enzyme is determined by its rates of synthesis and degradation (59). Three studies indicate that MCP is a relatively stable enzyme. It is now widely accepted that certain forms of "prosomes" are equivalent to MCP (6,22). Hence, some examples are drawn from the prosomes literature. Akhayat et al. (60) could not detect synthesis of prosomes (MCP) in developing sea urchins. Because the quantity of prosomes was unchanged after 48 hr of development, Akhayat et al, (60) concluded that MCP is metabolically stable. Hendil (61) examined MCP turnover in human HeLa cells and found that all MCP subunits exhibited half-lives of ~5 days. He also observed that MCP synthesis was not induced by heat shock or cell crowding. Tanaka and Ichihara (62) reported a significantly longer half-life for rat liver MCP. They found that MCP was roughly 1% of soluble liver proteins, and its apparent half-life was 12-15 days. If MCP generally proves to be as stable as it is in sea urchin, HeLa cells, or rat liver, then differential synthesis must account for the several hundred-fold range in concentrations shown in Table II. Several groups have demonstrated differential accumulation of MCP in developing tissues. Using antibodies against 28- and 35-kDa subunits of MCP, Klein et al (63) observed large differences in immunofluorescent staining ofDrosophila embryonic tissues. MCP was shown to be particularly concentrated in cells undergoing morphogenetic movements anterior and posterior of the cephalic furrow. The authors also reported transient accumulation of MCP in pole cells. This observation suggests that MCP subunits can be rapidly degraded in some tissues, although one cannot rule out masking of epitopes as an explanation for reduced staining in pole cells from older embryos. Tanaka and colleagues examined the synthesis of MCP subunits in human hematopoietic and renal tumor cells (64). Having established that the levels of MCP subunits and their mRNAs were much higher in malignant human hematopoietic cells (65), they compared the expression of MCP subunits in normal peripheral T lymphoc3rtes to MCP expression in human leukemia cells (66). Rapid synthesis of both MCP subunits and the higher-molecular-weight proteins characteristic of the 26S protease was observed after treating normal T lymphocytes with mitogens. By contrast, when various leukemic cell lines were induced to differentiate, there was reduced expression of mRNAs that encode MCP subunits and reduced synthesis of MCP proteins. Because the intracellular levels of MCP did not change markedly after either treatment, the authors proposed the existence of two pools of MCP—a larger metabolically stable pool and a small pool of rapidly degraded MCP subunits. However, the data presented are consistent with a

10

LAURA HOFFMAN AND MARTIN RECHSTEINER

single pool of MCP subunits. Since the specific activities of radiolabeled proteins were not measured, the extent to which absolute amounts of MCP protein should have increased cannot be determined. B. Expression of Specific MCP Subunits

As noted in the Introduction, the existence of multiple MCP subunits and numerous 26S ATPase subunits would allow eukaryotic cells, in principle, to generate large numbers of specific proteases by combinatorial association to these components. Do eukaryotic cells mix and match subunits? The answer is not clear, although three studies suggest that this may be a regulatory strategy. Haass and Hoetzel (67) examined Drosophila MCP by twodimensional PAGE and found changes during development. A relatively simple subunit pattern present in early embryos and Schneiders S3 cells became increasingly more complex in older embryos and adult flies. The authors proposed that the new protein species arose by posttranslational modification, but they recognized that synthesis of distinct subunits could account for the more complicated patterns at later developmental stages. Similarly, Ahn et al. (68) reported significant changes in the levels of five MCP subunits in developing chick muscle; the intensity of three species increased while that of two subunits declined. These investigators concluded that some subunits, at least, are under developmental control. A great deal of excitement has been generated by the possible role of MCP in antigen presentation (see Refs. 69 and 70 for reviews). MCP and/or the 26S protease are candidates for generating the peptides that bind major histocompatibility complex (MHC) class I receptors. Although circumstantial, there is a reasonable body of evidence supporting this idea. First, genes for MCP-like subunits, Ring 10 and Ring 12, are located in the MHC complex (71-73). Second, y-interferon (yIFN) stimulates production of many components in the antigen presentation pathway. In fact, expression of two MCP-like subunits whose genes are located in the major histocompatibility locus provides particularly strong evidence for tissue-specific expression of unique MCP subunits. Yang et al. (74) found that five new polypeptides were present in MCP complexes immunopurified from HeLa cells treated with yIFN. The use of human lymphoblastoid cell lines deleted for Ring 12 allowed these investigators to identify a specifically induced protein, subunit b, as the Ring 12 gene product. Using ammonium sulfate fractionation to separate MCP from 26S protease, Yang et al. provided evidence that y-IFN-induced MCP subunits partitioned uniquely between the two enzymes. That is, of five induced subunits, two were

MULTICATALYTIC AND 26S PROTEASES

11

found in the 26S enzyme and three in MCP. Finally, cell fractionation studies indicated that MCP particles isolated from microsomes were enriched in several y-IFN-induced subunits. This is consistent with an overall presentation scheme in which MCP generates peptides that enter microsomes by specific peptide transporters, also encoded in the MHC, and then subsequently bind class I receptors for movement to the cell surface. C. Regulation by Posttranslational Modification 1. PHOSPHORYLATION

Phosphorylation is, without a doubt, the major reaction by which eukaryotes regulate biochemical processes (75). Therefore, it is, surprising that so few examples of MCP or 26S protease regulation by protein kinases can be found. The only evidence that MCP subunits are phosphorylated in vivo was provided by Haass and Kloetzel (67) who showed that growing Drosophila S3 cells in medium containing ^^P-labeled phosphate resulted in labeled MCP subunits. Based on sequence analysis of MCP subunits, Haass et al. (76) and Tanaka et al. (77) proposed that a tyrosine in a src consensus site was the phosphorylated residue. However, this proposal has not yet been directly confirmed. Pereira and Wilk (78) reported that 27- and 28-kDa subunits of bovine pituitary MCP were major substrates for a copuripfying cAMPdependent kinase. Two additional subunits (31 and 24 kDa) were phosphorylated to a lesser extent. Unfortunately, evidence was not presented demonstrating that phosphorylation affected the proteolytic activity of MCP. Hough et al. (79) observed ^^P-labeled phosphate incorporation into at least two subunits (110 and 62 kDa) of the 26S ATPase complex. On pharmacological grounds, the responsible protein kinase appeared to be casein kinase II, Ferrell et al. (unpublished observations, 1988) extended these studies by showing that serine and, to a lesser degree, threonine are the phosphate-accepting residues. Moreover, the extent of phosphorylation is markedly affected by two inhibitors of Ub-conjugate degradation, hemin and aurintricarboxylic acid (see Fig. 4). 2. GLYCOSYLATION

Whether MCP subunits are covalently modified by sugars is a controversial subject. Using lectin blotting procedures, Schmid and colleagues reported the presence of glucosyl-, mannosyl- andN-acetylgalactosaminyl residues in plant MCP (80,81). Rivett and Sweeney (82) also claim that three subunits of rat liver MCP bind concanavalin. On the other

12

LAURA HOFFMAN AND MARTIN RECHSTEINER

B

A P2

Pi

^ '^ if O

45 — 3629—

£

-•

<

PS

2 c O 0) »o i: <

^^^k^

• ^

^ PT

X

origin

24 — 20 —

-f-

pH 1.9

250Vx6h

(-)

FIG. 4. Phosphorylation of 26S protease subunits. (A) Rabbit reticulocyte 26S protease was purified to apparent homogeneity and incubated with 20 /JLM y-^^P-ATP in the presence or absence of hemin and aurintricarboxylic acid (ATA), both of which are inhibitors of conjugate degradation. The extent of phosphorylation was then determined by separation of subunits on SDS-PAGE gels and subsequent autoradiography. ATA severely depresses phosphorylation, whereas hemin promotes it. Pi and P2 represent different pools of 26S protease obtained from hydroxylapatite. (B) The 26S protease was incubated with 32PO4-ATP, and its subunits were separated on SDS-PAGE gels. The 110kDa subunit was excised from the gel and digested with pronase, and the phosphorylated amino acids were separated by electrophoresis. The resulting autoradiogram shows that serine is the predominant phosphorylated residue with minor labeling of threonine.

hand, Haass and Kloetzel (67) and Kaltoft et al. (83) did not find carbohydrate in Drosophila or human MCPs, respectively. 3. PROTEOLYTIC PROCESSING

Conversion of inactive precursors to active enzymes by peptide bond cleavage is a recurrent theme in the field of proteolysis. At the physiological level, it is the central control step in blood clotting (84), and it plays a key role in activating digestive enzymes (85) as well as in regulating blood pressure (86). The mechanism also operates at the cellular level. Some lysosomal cathepsins are activated by removal of prepro regions (87), as are processing enzmes in the secretory pathway (88). The same holds true for cytoplasmic proteases. Calpain is activated by cleavage of the 80-kDa heavy chain (89) and there is evidence

MULTICATALYTIC AND 26S PROTEASES

13

that the interleukin lj8 protease is activated by hydrolysis of an internal peptide bond (17,18). In this context, it is perhaps not surprising to find that j3 subunits of MCP are also processed. This has been shown clearly by studies on the archaebacterial enzyme from Thermoplasma. Zwickl et al. (90) expressed T. acidophilum a and /3 subunits in E. coli and found that 8 residues were removed from the N terminus of the f3 chain. This was apparently an MCP-mediated reaction since it was dependent on coexpression of the a subunit. Studies by Lilley et al. (19) on rat liver MCP indicate that removal of N-terminal extensions from /3 subunits occurs generally. Direct peptide sequencing revealed that numerous rat MCP (3 chains began at a threonine about 10-20 residues into the sequences deduced from cDNAs. As a further parallel between the 26S and Clp proteases, Maurizi and colleagues have shown that ClpP is also missing 14 residues from its N terminus (92). In all these cases, processing is thought to activate precursor subunits thereby ensuring that protease activity is confined to the assembled particle. Two studies employing Western blot analyses suggest that additional processing reactions may occur. Kreutzer-Schmid and Schmid (93) probed HeLa nuclear and cytoplasmic extracts with a monoclonal antibody to a prosomal 27-kDa protein. Surprisingly, the major immunoreactive species was a 38-kDa nuclear protein. On digestion with V8 protease, the 38-kDa protein produced a 27-kDa species and several smaller peptides with Mr values similar to those obtained from the 27-kDa prosomal protein. Since p27 has been identified as a member of the MCP a-subunit family (94), these studies raise the possibility that an MCP a subunit is produced as a 38-kDa precursor. Weitman and Etlinger (95) have also obtained a monoclonal antibody that reacts with a 32-kDa protein associated with latent MCP particles. The same monoclonal recognizes a 28-kDa protein in activated MCP particles and a 41-kDa protein in unpurified preparations of MCP. These authors also suggest that the 41-kDa protein may be a precursor to the 32/28-kDa species. There are several reports of self-digestion by MCP. Tanaka and Ichihara (96) found that rat liver MCP subunits disappeared on addition of high levels of urea, presumably by autocatalytic cleavages. Lee et al. (97) observed proteolytic degradation of certain subunits in active as opposed to latent forms of human MCP. Yu et al. (98) observed a more limited degradation following dialysis of bovine pituitary MCP against low ionic strength Tris buffers. It is doubtful that any of these manipulations reflect physiological control mechanisms. They do, how-

14

LAURA HOFFMAN AND MARTIN RECHSTEINER

ever, demonstrate that a large proteolytic particle can be activated or destroyed by self-cleavage reactions. D. Regulation of MCP by Associated Proteins

In Fig. 1, MCP is shown associating with a variety of other protein complexes that affect its activity. Only the ATPase complex (AC) has been demonstrated to influence substrate selection by conferring the ability to degrade Ub conjugates on MCP. It seems likely, however, that other proteins in the diagram will serve similar roles. Consequently, all are considered potential agents for regulating the stability of cellular proteins. 1. ACTIVATORS

Two protein complexes that activate peptide hydrolysis by MCP have been purified and characterized. One consists of a presumed hexamer of 30-kDa subunits (99,100). The other is a multisubunit complex containing at least 10 proteins (101). Because this more complicated protein complex is a central component of the 26S protease, it is considered first. Starting with non-ATP-depleted rabbit reticulocyte lysate, Hough et al. (41) isolated a single 26S proteolytic complex capable of degrading Ub-lysozyme conjugates. By contrast, Hershko and colleagues (44) observed breakdown of Ub-lysozyme conjugates only on combining three factors obtained from ATP-depleted lysate. The three factors, termed CFl, CF2, and CF3, had molecular masses of approximately 600, 250, and 650 kDa, respectively. When combined in the presence of Mg2+-ATP, the three factors disappeared, and a large (>1000 kDa) ATP-dependent protease formed. Ganoth et al. (44) concluded that CFl, CF2, and CF3 combine to form the 26S protease. The properties of CF3 were similar in many ways to those of the multicatalytic protease and, as noted, subsequent studies confirmed that CF3 was, indeed, MCP (45-47). Two groups have characterized complexes that either correspond to CFl or to CFl plus CF2. Hoffman et al. (101) discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26S protease (e.g., proteins with molecular masses between 30 and 110 kDa). Incubation of this particle with MCP and Mg^^-ATP resulted in its association with MCP, significant stimulation of peptide hydrolysis by MCP, and generation of a protease capable of degrading Ub-lysozyme conjugates. Based on the sedimentation characteristics of the protein complex, and its ability to form the 26S protease when combined with MCP, Hoffman

MULTICATALYTIC AND 26S PROTEASES

15

et al, suggested that it corresponds to CFl and CF2. A 51-kDa polypeptide in the particle belongs to a family of putative ATPases (50), and the protein complex has been found to exhibit ATPase activity (L. Hoffman et al, manuscript in preparation). For this reason, we now substitute the term, ATPase complex (AC), for a rather inelegant descriptor, "the ball," used previously. Udvardy (102) has also purified and characterized a multiprotein complex, the /x, particle, from Drosophila oocytes. Like the reticulocyte ATPase complex, the /x particle does not possess protease activity. However, in the presence of ATP, it combines with Drosphila MCP to form a 26S protease capable of degrading Ub-yolk protein conjugates. In contrast to the results of Hoffman et al. (101), a third component is required for assembly of /x and MCP, but this component is not incorporated into the 26S protease. Udvardy suggests that CF2 may not be incorporated into the 26S enzyme. In this scheme, the /x particle would be equivalent to CFl, MCP would equal CF3, and the unincorporated assembly factor would be CF2. If, on the other hand, the /x particle corresponds to CFl and CF2, then a fourth factor is needed to generate the Drosophila 26S protease. Several groups have identified a smaller protein complex that stimulates peptide hydrolysis by MCP. Yukawa et al, (103) described a factor from platelets that enhanced MCP's ch)rmotrypsin- and trypsin-like activities; ATP was not required for activation. More complete descriptions of this factor were published in 1992, when three groups characterized a ~200- to 300-kDa protein complex that activiates hydrolysis of certain fluorogenic peptides (99,100,104). In two cases, the activator or regulator was purified sufficiently to identify its subunit composition. Chu-Ping et al (99) reported that bovine red cell MCP activator has a native molecular weight of —180,000 and is composed of a single 28-kDa subunit. The activator, which is presumably a hexamer, stimulated three distinct peptidase activities by increasing Kiax and reducing K^. The activator did not stimulate hydrolysis of proteins. Dubiel et aL (100) obtained similar results for human red blood cell regulator. They found that the regulator sedimented at U S and was composed of two closely related 30-kDa subunits. When added to MCP, the regulator stimulated hydrolysis of two fluorogenic peptides by almost 60-fold, whereas hydrolysis of two other peptides was stimulated only 3- to 10-fold. The human regulator did not stimulate hydrolysis of Ub-lysozyme conjugates, bovine serine albumin, or lysozyme. Using glycerol gradients, native gels, and two-dimensional PAGE, this group presented evidence that activation results from the reversible association of

16

LAURA HOFFMAN AND MARTIN RECHSTEINER

regulator and MCP. That is, neither component appears to be permanently affected by activation. Cloning of the cDNA encoding one of the two 30-kDa subunits revealed that it is a 249-residue protein (Realini et ai, manuscript in preparation, 1993). Like the ATPase subunits, the MCP regulator sequence possesses a stretch of amino acids strongly predicted to form coiled coils. The potential significance of coiled coil structural motifs is discussed below. Figure 1 depicts a hexamer of p97 subunits associating with MCP to form a complex similar to the 26S protease. Although the diagrammed reaction between p97 and MCP is entirely hypothetical, it is included in the figure because of some evident parallels between p97 and members of the S4-like subfamily of putative ATPases. Before discussing those parallels, a brief review of p97 is in order. In 1976, White and Ralston (105) extracted red blood cell membranes in 0.1 mM EDTA and obtained a soluble Mg^^-ATPase. The enzyme appeared to be specific for ATP in that CTP or GTP were not hydrolyzed. Later studies on the purified red cell ATPase showed that it has a native molecular weight of about 500,000 and is composed of a single 100-kDa subunit (106). The K^r, of the enzyme for ATP is 1 mM, and it is inhibited by AT-ethylmaleimide, Cd^^, Zn^^, andp-chloromercuribenzoate. In 1990, Peters et al. (53) described a 15S ATPase present in extracts ofXenopus laevis oocytes. Their enzyme displays sixfold radial symmetry and is composed of a single subunit with an apparent Mr of 97,000 (e.g., p97). Peters et al. (53) prepared antibodies to p97 and demonstrated that it was present in a wide variety of organisms and tissues. They also obtained the sequence of p97 from cDNA clones and found that it was closely related to two proteins involved in secretion, NSF, or its yeast equivalent SeclSp (107,108). Subsequent studies showed that p97 is even more closely related to the protein encoded by a yeast cell cycle mutant, cdc48 (109). Regarding Fig. 1, the evidence that p97 may assemble with MCP is as follows: The putative S4-like ATPases are members of a family of larger proteins that contain two candidate ATP-binding sites. Within this extended family are two proteins, cdc48p and p97, that bear close resemblance to the S4 subfamily. CDC48p and p97 have predicted coiled coil regions spaced relative to one of their ATP-binding sites at positions equivalent to proteins in the S4 subfamily. Like S4, the larger proteins have conserved cysteine residues C-terminal to the ATPbinding site. Moreover, the sixfold radial symmetry of p97 (53) suggests that it might well interact with the six a subunits at each end of MCP. Thus, there is reason to suspect that p97 will be shown to associate with MCP.

MULTICATALYTIC AND 26S PROTEASES 2.

17

INHIBITORS

Proteins that inhibit MCP activity have also been reported. Almost a decade ago, Speiser and Etlinger (39) proposed that ATP stimulates proteolysis in reticulocyte extracts by repressing an endogenous protease inhibitor. Three years later, Murakami and Etlinger (110) purified a hexameric complex of 40-kDa subunits that inhibited both calpain and MCP. Because heating destroyed inhibitor activity against MCP, but not against calpain, the authors concluded that different domains on the inhibitor interacted with each protease. Two papers have implicated the 40-kDa inhibitor in 26S protease function. Li and Etlinger (111) report that a ubiquitinated derivative of the 40-kDa inhibitor is a component of the 26S protease. However, this claim should be viewed with extreme caution since the subunit pattern of their "26S protease" bears no resemblance to the polypeptide pattern of the 26S protease reported by four independent groups (41,45,47,112). DriscoU et al. (113) claim that a 250-kDa ATP-stabiHzed inhibitor of MCP is a 26S protease component. They propose that the 250-kDa native complex, which is composed of 40-kDa subunits, corresponds to the CF2 component identified by Hershko and colleagues. As previously mentioned, Udvardy (102) questioned the idea that CF2 is incorporated into the 26S protease. Clearly, further experimentation will be required to assess the importance of inhibitors in 26S protease function. In 1991, Etlinger and colleagues (114) reported the isolation of yet another MCP inhibitor from human erythrocytes. This factor is apparently a tetramer since its native molecular mass is 200 kDa, and it is composed of a single 50-kDa subunit. A specific monoclonal antibody and peptide sequencing distinguished this inhibitor from the 40-kDa inhibitor. Li et al. (114) suggested that the 50-kDa inhibitor plays a role in Ub-mediated proteolysis. Finally, DeMartino and colleagues (115) have purified an inhibitor that forms multimers under nondenaturing conditions. It appears to be composed of a single, self-associating 31-kDa polypeptide. The protein inhibits both the three distinct catalytic activities of MCP and the enzyme's ability to degrade casein, lysoz3nne, and bovine serum albumin. These authors also suggest that this inhibitor may play a role in ATP/Ub-mediated proteolysis. Whereas there seems to be little doubt that protein complexes exist which can stimulate or inhibit peptide bond hydrolysis by MCP, certain key questions remain unanswered. Do the inhibitors represent bona fide regulatory molecules or substrates? Are the activators subcompo-

18

LAURA HOFFMAN AND MARTIN RECHSTEINER

nents of larger protein assemblies (e.g., ATPase complexes) or independent regulators? We believe it is premature to assign specific roles to the various activators and inhibitors. 3. ACTIVATION AND INHIBITION BY SMALL MOLECULES

There are reports that MCP can be activated or inhibited by fatty acids (116), detergents (116-119), sulfated lactosylceramides (120), inorganic ions (121,122), polylysine (117,123), and heating (124). Some of these findings might reflect relevant physiological regulatory mechanisms, e.g., palmitylation or myristoylation of proteins could conceivably target them for destruction by MCP (125). However, in our view the observed effects of the various small molecules do not invoke plausible control mechanisms. Hence, they are not covered further in this essay. E. Subcellular Distribution of MCP and 26S Proteases Experiments performed a decade ago convincingly demonstrated that a Xenopus 22S cylinder particle, now known to be MCP, is present in both nucleus and cytoplasm. Kleinschmidt et al. (126) analyzed extracts from manually dissected oocyte germinal vesicles and cytoplasms on 2D PAGE gels. They found identical MCP subunits in each compartment. In a companion paper, Hiigle et al. (127) obtained antibodies to a 30-kDa subunit from Xenopus MCP and examined the distribution of the enzyme in various tissues by immunofluorescence microscopy. MCP was enriched in the nuclei of all cells examined, which included liver, muscle, a Xenopus culture cell line, and ovarian tissues. In liver and muscle a characteristic punctate or speckled pattern was observed within nuclei; nucleoli and heterochromatin were not significantly stained. During mitosis of Xenopus A^ cells, MCP was dispersed throughout the cell, but apparently was not present on metaphase chromosomes. There have been a number of additional studies on the location of MCP in the intervening 10 years (see Table III for a summary). Despite the fact that a wide variety of tissues and species have been examined, certain themes emerge. MCP is largely C3rtoplasmic in zygotes and early embryos, although it becomes increasingly nuclear as development proceeds. The nuclear/cytoplasmic distribution of MCP can vary among cells within a specific tissue. The enzyme is, however, more heavily concentrated in nuclei of dividing or cancerous cells. In some nondividing cells, e.g., Drosophila salivary glands, the protease is apparently absent from the nucleus. Immimofluorescence staining often reveals a speckled or clustered pattern for MCP in both nucleus and cytoplasm.

Cell type Sea urchin 2-cell embryo Sea urchin blastula Dmsophila salivary gland M o u e 3T3 Human HeLa Avian erythroblasts Newt embryo Preblastula Postblastula Rat liver Human renal cancer cells Normal human kidney cells Ascidian embryo Rat kangaroo cells HeLa Monkey kidney cells Rat liver Human El3 2 culture cells Ovarian granulosa cells

Technique

IF” or Western blot MAb’ to 27-kDa subunit

IF IF, MAb 27 kDA IF, MAb 27 kDa IF, MAbs 27 kDa, 28 kDa, 29 kDa, 31 kDa IF MAb IF, Pc‘ to r a t MCP IMHCd MAb + PC antibodies IF, MAb to subunit with pZ 6.3 IF MAbs 27 kDa, 29 kDa, 31 kDa IF, MAbs 27 kDa, 29 kDa. 31 kDa PC to rat liver MCP EM,’ collodial gold rat liver MCP

IF with PC to r a t muscle or

IF, Immunofluorescence. * MAb, monoclonal antibody. PC, polyclonal antibodies. IMHC, immunohistochemistry. EM, electromicroscopy.

TABLE 111 SUBCELLULAR LOCALIZATION OF MCP SUBUNITS

++ +

+

++

+++

++

+

+

++

Nucleus

+++

++ +++

++

++ ++ +

+++

+++ +++

+++ ++ ++

Cytoplasm

Speckled Distributed on intermediate filame Speckled

“Speckled” distnbution

Location

+

+++ ++

+ + -

-

+ -

++

++

+

++

Speckled or clustered distribution d relative to spindle

No mention of clustering

Specific locations relative to mitotic

Large differences in stain intensity

++

20

LAURA HOFFMAN AND MARTIN RECHSTEINER

However, a clustered distribution of MCP was not reported in a recent electron microscopic study (137). These generalizations hold for almost all studies except those reported by Scherrer and colleagues (129-131,135,136). This group consistently finds "prosome" antigens associated with intermediate filaments. Presumably their antibodies identify MCP, although a clear connection between prosomal antigens and MCP subunits has not been documented by these investigators. Moreover, the fixation procedures used by Scherrer and colleagues are not traditional. In contrast to most protocols, cell membranes are disrupted by detergent prior to fixation. For these reasons, the reported association between prosomal proteins and intermediate filaments may not apply to MCP. Two members of the S4-like subfamily of ATPases have also been localized predominantly in the nucleus. Nelbock et al. (139) produced polyclonal rabbit antibodies to TBPl and found the protein mainly in the nucleus of COS cells. Similar results were obtained with TBP7, a homolog of TBPl (140). In addition, two other members of the subfamily, MSSl and SUGl (141,142), have apparent effects on transcription so they are also likely to be located in the nucleus. It has been proposed that TBPl, MSSl, and SUGl are ATP-dependent transcription factors (141). Although this possibility cannot be eliminated, the observed effects on gene expression can be explained by proteolytic mechanisms (50), and it is likely that all members of the S4-like subfamily are components of the 26S protease. In fact, MSSl has recently been identified as subunit 7 of the human 26S enzyme (143). Other components of the Ub-mediated proteolytic pathway are also concentrated in the nucleus. Cook and Chock (144) report that the Ubactivating enzyme, El, is largely nuclear. The yeast cdc34 gene product, a ubiquitin-carrier protein, is also localized in nuclei (145). Finally, there is evidence that ubiquitin conjugates may also be enriched in nuclei. After microinjecting ^^^I-labeled ubiquitin into HeLa cells, Carlson and Rechsteiner (146) found, as expected, Ub-H2A histone conjugates were exclusively nuclear. They also observed an abundance of larger conjugates in the nuclear fraction. Likewise, Beers et al. (147) found numerous high-molecular-weight conjugates in plant cell nuclei. Finally, immunolocalization of Ub conjugates in rat cardiomyocytes revealed a "speckled" nuclear pattern (148). Current evidence indicates that the multicatalytic protease, the 26S protease, the ubiquitin activating enzyme, and the Ub carrier proteins are often enriched in the nucleus. Moreover, high-molecular-weight Ub conjugates, the presumed substrates for the 26S protease, are also prominent within nuclei. This raises the possibility that a large portion

MULTICATALYTIC AND 26S PROTEASES

21

of selective intracellular proteolysis occurs within the nuclear compartment. There is, furthermore, a voluminous literature on regulated nuclear entry of transcription factors, such as N F - K B and dorsal (149152). Thus, one can imagine that certain proteins are stable in the cytoplasm and rapidly degraded after entry into the nucleus. Compartment-specific degradation would provide an effective mechanism for controlling the metabolic stability of proteins. F. Multicatalytic Protease and 26S Activities during Development, during Cell Cycle, and after Physiological Stress

Changes in MCP or 26S activities must be viewed with some caution for a variety of reasons. First, a number of activators and inhibitors of MCP have been described (see above). Hence, one cannot know whether apparent changes in protease activity reflect the enzjones proper or changes in regulatory factors. Of course, the latter possibility is still physiologically relevant. Second, the multicatalytic protease is a notoriously "sticky" particle, and one must determine that copurifying activities are integral components of the protease complex. Likewise, the 26S protease can be fragile so apparent changes may reflect handling. Readers should view the following section with these caveats in mind. Chung, Tanaka, and colleagues have measured MCP peptidase activities during chick development (68). In embryonic muscle, they observed 2- or 3-fold decreases in MCFs chymotrypsin- and trypsin-like activities between embryonic Days 8 and 20. By contrast, there was a 4-fold increase in polylysine-stimulated casein degradation over the same period. Chymotryptic activity was relatively stable in developing chick brain and liver, but trypsin-like activity, assayed with the fluorogenic peptide Cbz-ARR-MNA, increased 30-fold in liver between Days 11 and 14. As previously mentioned, these investigators found changes in the relative proportion of five MCP subunits in developing muscle. Two groups have reported decreased MCP and 26S protease activity in maturing erj^hroid cells. Using density purified rabbit reticulocytes and mature red cells, Di Cola et al, (153) observed coordinate threefold decreases in several MCP peptidase activities. This was paralleled by twofold lower degradation of Ub conjugates by the 26S protease. Tsukahara et al. (154) similarly observed a decrease in the 26S protease on dimethyl sulfoxide-induced differentiation of murine erythroleukemia cells. There is a single report that the 26S protease is activated at specific points in the cell cycle. Kawahara et al. (155) measured peptide hydrolysis in developing ascidian embryos and found two peaks of chymotryptic activity corresponding to prophase and metaphase of the third cleavage

22

LAURA HOFFMAN AND MARTIN RECHSTEINER

stage. On the other hand, Mahaffey et al. (iinpubUshed observations, 1993) observed no changes in peptide hydrolysis or Ub-conjugate degradation during the first two cleavage cycles in Xenopus egg extract. Finally, two papers describe changes in MCP activity following physiological stress. Kuehn et al. (156) examined MCP peptidase activities in muscles of fasting rats. Whereas the amount of MCP protein was unchanged over the 3 days of starvation, cleavage of the chymotryptic substrate, Suc-Leu-Leu-Val-Tyr-MCA, fell threefold. This decrease was not observed in testis or thymus of the fasted animals. On the other hand, Medina et al, (157) found enhanced ATP-dependent proteolysis in fasted rat muscle. Based on changes in poly(Ub) transcripts and levels of Ub conjugates, they attributed this increase to the Ubdependent pathway.

V. Regulation by MCP and 26S Proteases Interest in the regulation of multicatalytic and 26S proteases stems, in large part, from mounting evidence that they, in turn, exert important controls on other metabolic pathways. Almost two decades ago, Schimke (59) pointed out that regulatory proteins would be metabolically labile because rapid changes in their concentration demand a short half-life. The list of rapidly degraded intracellular proteins grows daily and now includes transcription factors, oncoproteins, protein kinase-associated subunits, and key metabolic enzymes (158). Given the expanding numbers of short-lived proteins, a major task is to identify the proteases responsible for their destruction. Some proteins, at least, appear to be substrates for the 26S enzyme. A. Natural Substrates for MCP and the 26S Protease In Fig. 1, MCP is associating with various factors thought to affect its activity. It is not clear whether the central core particle, MCP, is able to degrade intact proteins. In fact, we suspect that the regulator/ MCP complex may simply be a very efficient peptidase rather than an endoproteinase. At the same time, MCP is known to be a central component of the 26S enzyme (22), so discussions of 26S substrates include, perforce, substrates for MCP. There are, however, several papers that implicate MCP alone in the destruction of oxidized proteins. Thus, before considering substrates of the larger 26S enzyme, the potential role of MCP in removing oxidized proteins from cells is reviewed. Although Goldberg and Boches (159) claimed that oxidized red cell proteins are degraded by an ATP-dependent process, the same labora-

MULTICATALYTIC AND 26S PROTEASES

23

tory arrived at different conclusions in several later studies. Fagan et al. (160) reported that hemoglobin oxidized by nitrite or phenylhydrazine was rapidly degraded, and this process was not inhibited by ATP depletion. Likewise, Davies and Goldberg (161) observed that proteins damaged by oxygen radicals are degraded by red cell extracts lacking nucleoside triphosphates. Two studies identify MCP as the responsible enzyme. Pacifici et al. (162) found that 70-80% of the degradative activity against oxidatively modified proteins was exhibited by a 670kDa proteinase complex, which they called M.O.P. Since the subunit pattern of M.O.P. is virtually identical to the SDS-PAGE profile for MCP, the two enzymes are presumably the same. Sacchetta et al. (163) obtained similar results using phenylhydrazine-denatured hemoglobin as substrate. These investigators found that MCP was unable to hydrolyze native Hb. It did, however, produce peptides from denatured globin. Interestingly, free amino acids were not final products of the reaction. In contrast to these studies, Fagan and Waxman (164) have recently concluded that red cell MCP is not responsible for degrading oxidantdamaged hemoglobin in crude red cell extracts. Using a combination of protease inhibitors and antibodies, these investigators present reasonably convincing evidence that insulinase, a 100-kDa metalloprotease, is responsible for most ATP-independent proteolysis of oxidized hemoglobin. Thus, isolated MCP may be capable of degrading oxidized hemoglobin, but it does not appear to be the principal protease doing so in crude red cell extracts. Several studies by Rivett implicate rat liver MCP in the degradation of oxidized proteins. Using oxidized glutamine S5nithetase (glutamateammonia ligase) as a substrate, she identified four rat liver proteases that degrade the inactive bacterial enzyme (165). Two of the enz3rmes were calpains, one was cathepsin D, and one was a large, ~300-kDa, protease with an alkaline pH optimum. Subsequent purification of the larger enzyme identified it as MCP, and improved gel filtration showed its molecular weight to be 650,000 (166). As with the studies using hemoglobin, it is difficult to know the fraction of oxidized proteins degraded by MCP within rat liver cells. To date, the 26S protease is the only enzyme known to degrade proteins conjugated to Ub. It seems reasonable, therefore, to consider proteins whose degradation is mediated by Ub as 26S substrates. A variety of intracellular proteins are thought to be degraded by Ubmediated pathways (see Table IV). However, as noted in an earlier review (1), the evidence implicating Ub in the destruction of natural cellular constituents remains largely circumstantial. Except for detailed studies on artificial Ub-j8-galactosidase substrates, the evidence

24

LAURA HOFFMAN AND MARTIN RECHSTEINER TABLE IV NATURAL SUBSTRATES OF 26S PROTEASE

Protein

Ref.

Protein

Ref.

Phytochrome MATa2 repressor Cyclin p53 Myc, Fos Cytochrome P450 2E1

174 175 11,184 176 177 178

PDGF receptor Mos Cytochrome P450 3A Retinoblastoma Ribonucleotide reductase subunit M l Ornithine decarboxylase

179 180 181 182 183 167

is mainly a correlation between the appearance of Ub-conjugates to a specific protein and the protein's rapid disappearance. Because the proteins listed in Table IV are present at such low concentrations within cells, it has not been possible to isolate Ub-conjugated derivatives and directly assay their degradation by the 26S protease. No doubt, additional proteins will join those in Table IV, but newer approaches will be required to obtain conclusive evidence that any of the proteins are degraded by Ub-mediated (26S) pathways. Murakami et aL (167) have reported that ornithine decarboxylase (ODC), an extremely short-lived enzyme, is degraded directly by the 26S protease. Rapid proteolysis of ODC required the addition of antizyme which forms a noncovalent complex with ODC (168). However, ubiquitination was not required. This demonstration that the 26S protease can directly degrade certain proteins is consistent with several previous observations. Bercovich et al. (169) showed that immunoprecipitation of the Ub-activating enzyme from reticulocyte lysates did not prevent ODC degradation. Likewise, thermal inactivation of E l in the mutant cell line, ts85, did not inhibit ODC turnover (170). B. Postulated Molecular Mechanisms for Target Selection

It seems clear that the 26S protease is a versatile enzyme capable of degrading both ubiquitinated and nonconjugated proteins. Exactly how the enzyme recognizes substrates has not been elucidated. Presumably, a subunit(s) in the ATPase complex can recognize ubiquitin or poly(lJb) chains. However, repeated attempts to identify a Ub-binding subunit using photoaflfinity cross-linking approaches have not yet proved successful (Ustrell et aL, unpublished observations, 1993). Hershko and colleagues have identified two isopeptidases (171,172). One of the enzymes acts on poly(Ub) chains, but it is not associated with the 26S complex (171). The other ubiquitin C-terminal hydrolase

MULTICATALYTIC AND 26S PROTEASES

25

activity is associated with the 26S complex and, interestingly, it requires ATP, CTP, or GTP to hydrolyze ubiquitin-lysozyme isopeptide bonds (172). This isopeptidase activity is tightly coupled to proteolysis, and these investigators propose that the enzyme releases Ub from substrate amino groups in the final stages of proteolysis. It is well estabUshed that intertwined a hehces (e.g., leucine zippers or coiled coils) can play important roles in protein-protein associations. Lupas et al (173) have devised an algorithm for predicting the occurrence of coiled coil regions in proteins. Application of this algorithm to the various proteolytic components shown in Fig. 1 produced some intriguing patterns. Potential coiled coil regions are present in MCP subunits, in the U S regulator subunit, and in all members of the S4-like subfamily of putative ATPases (see Fig. 5A). Conceivably, these regions promote binding of regulator or ATPase subunits to MCP. However, based on sequence variability in the S4-like subfamily, we have suggested that the coiled coil domains on the ATPase subunits at least, serve to bind substrates of the 26S enzyme (22). This helix-shuffle hypothesis can account for the rapid degradation of those unassembled

100 F

Regulator

100

200

300

Residue number FIG. 5. Coiled coils as possible substrate recognition motifs. (A) The sequences of three proteins (regulator, subunit 9 of human MCP, and subunit 4 of the 26S protease) were analyzed for potential coiled coil motifs by the algorithm of Lupas et al (173). Regions with high coiled coil probabilities are shown in the diagram. (B) Coiled coils may be used to target proteins for proteolytic degradation as in the hypothetical scheme shown. A mechanism for proteolytic substrate recognition by the 26S protease is proposed by which unpaired a helices of Fos, which normally dimerize with similar regions on Jun, could dimerize with a helices present in ATPase subunits of the 26S protease. This interaction with S4 rather than Jun would result in degradation of Fos.

26

LAURA HOFFMAN AND MARTIN RECHSTEINER

proteins that possess leucine zippers as shown schematically in Fig. 5B. Whether correct or not, the idea has the virtue of being readily testable by site-directed mutagenesis.

VI. Summary It should be clear from the foregoing accounts that our understanding of MCP and 26S regulation is still rudimentary. Moreover, we have only recently identified about a dozen natural substrates of these two proteases. Those outside the field may view the situation with some dismay. Those who study the MCP and 26S enzymes are provided with rich opportunities to address fundamental questions of protein catabolism and metabolic regulation. NOTE ADDED IN PROOF. There have been several significant advances in the two years that have elapsed between the submission and publication of this manuscript. While it is not possible to bring the entire manuscript up to date, several references must be added. Cloning of the 30-kDa activating protein (188) and description of Ub-conjugate degradation in the Xenopus cell cycle system (189) were referred to here as unpublished. Also, the Ub-conjugate binding subunit of the 26S protease has been identified (190), activation of MCP by two regulatory complexes has been further detailed (191), and a protein-protein interaction hjrpothesis for subunits and substrates has been proposed (192). Finally, we now refer to the multisubunit complex which combines with MCP to form the 26S protease as the Regulatory Complex (RC).

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

REFERENCES Rechsteiner, M. (1991). Cell 66, 615-618. Finley, D., and Chau, V. (1991). Anna. Rev. Cell Biol. 7, 25-69. Hershko, A., and Ciehanover, A. (1992). Annu. Rev. Biochem. 61, 761-807. Varshavsky, A. (1992). Cell 69, 725-735. Maurizi, M. R. (1992). Experientia 48, 178-201. Goldberg, A. L. (1992). Eur. J. Biochem. 203, 9-23. Gancedo, J. M. (1992). Eur. J. Biochem. 206, 297-313. Heby, O., and Persson, L. (1990). TIBS 15, 153-158. Luca, F. C, and Ruderman, J. V. (1989). J. Cell Biol. 109, 1895-1909. Murray, A. W., Solomon, M. J., and Kirschner, M. W. (1989). Nature {London) 339, 280-286. Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991). Nature {London) 349, 132-138. Bohley, P., and Seglen, P. O. (1992). Expenentia 48, 151-157. Shapiro, S. D., Kobayashi, D. K., Pentland, A. P., and Welgus, H. G. (1993). J. Biol. Chem. 268, 8170-8175. Mellgren, R. L., and Murachi, T., eds. (1990). "Intracellular Calcium-Dependent Proteolysis," pp. 228. CRC Press, Boca Raton, Florida. Rawlings, N. D., and Barrett, A. J. (1991). Biochem. J. 275, 389-391. Rennex, D., Hemmings, B. A., Hofsteenge, J., and Stone, S. R. (1991). Biochemistry 30, 2195-2203.

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27

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CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Calponin STEVEN J. WINDER MICHAEL P. WALSH MRC Group in Signal Transduction Faculty of Medicine University of Calgary 3330 Hospital Drive N.W. Calgary, Alberta, Canada T2N 4N1

I. Introduction The contractile state of smooth muscle is regulated primarily by the phosphorylation and dephosphorylation of myosin (1). Ca^^, which enters the sarcoplasm (muscle cytoplasm) from the extracellular milieu or the lumen of the sarcoplasmic reticulum in response to a variety of hormones and neurotransmitters, binds to calmodulin (CaM) to form the Ca^^-CaM complex (2). This complex, due to a Ca^^-induced conformational change (3), interacts with the enzyme myosin light-chain kinase (MLCK) to form the ternary complex Ca42^-CaM-MLCK (4); the kinase is thereby converted from an inactive to an active state. The activated kinase catalyzes the transfer of the terminal phosphoryl group of Mg^^ATP^" to serine-19 in each of the two 20-kDa light-chain subunits of myosin. This simple phosphorylation reaction triggers the cycling of myosin cross-bridges along actin filaments and the development of force or contraction of the muscle which is driven by the hydrolysis of Mg^^ATP^"; the sites of ATP hyrolysis are located within the globular head domains of the myosin molecules (1). Relaxation of smooth muscle generally follows the removal of Ca^^ from the sarcoplasm, whereupon the Ca/^-CaM-MLCK complex dissociates to regenerate the inactive MLCK apoenzyme (5). As a result, myosin phosphorylation stops and phosphorylated myosin is dephosphorylated by one or more myosin light-chain phosphatases (6). Myosin heads dissociate from actin filaments, cross-bridge cycling stops, and the muscle relaxes. The evidence supportive of a central role for myosin phosphorylationdephosphorylation in the regulation of smooth muscle contraction is considerable (reviewed in Ref 7). However, numerous physiological and biochemical studies have suggested the existence of additional 33

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

34

STEVEN J. WINDER AND MICHAEL P. WALSH

secondary mechanisms, both Ca^^ dependent and Ca^^ independent, which can modulate or fine tune the contractile state of a smooth muscle cell (reviewed in Ref. 7). Mechanisms of regulation of contraction in diverse muscle types can be divided into two principal groups: thick filament-linked regulation, as exemplified by myosin phosphyorylation-dephosphorylation in vertebrate smooth muscles (1) and the direct binding of Ca^^ to myosin in the striated muscle of the scallop (8), and thin filament-linked regulation, as exemplified by the troponintropomyosin complex of mammalian skeletal and cardiac muscles (9). Precedents do indeed exist for the functioning of both thick and thin filament-linked regulatory mechanisms in the same muscle, e.g., the striated muscle of Limulus (the horseshoe crab) is regulated by both troponin-tropomyosin (10) and myosin phosphorylationdephosphory ation (11). The myosin competition test devised by Lehman and Szent-Gyorgyi (12) is designed to test the existence of a thin filament-linked regulatory mechanism in any given muscle. It is based on the fact that the Mg^^ ATPase activity of a mixture of purified actin and myosin from vertebrate skeletal muscle is independent of Ca^^. We applied the myosin competition test to vertebrate smooth muscle (13). The test involves combining a crude actomyosin preparation from the muscle of interest with excess pure rabbit skeletal muscle myosin at high ionic strength (e.g., 0.6 M KCl) to dissociate actin and actin-binding regulatory proteins which may be present in the crude actomyosin preparation. The mixture is then dialyzed against a low ionic strength buffer to allow hybridization of skeletal muscle myosin with, in this case, chicken gizzard smooth muscle actin and associated proteins. The actinactivated myosin Mg^^-ATPase activity is then measured in the presence and absence of Ca^^. If there is a thin filament-linked Ca^^ regulatory mechanism in the muscle of interest, then the ATPase activity will be higher in the presence than in the absence of Ca^^; on the other hand, if there is no such mechanism, the ATPase activity will be high and independent of Ca^^. The results of the myosin competition test as applied to chicken gizzard smooth muscle are shown in Table I. The Mg^^-ATPase activity of rabbit skeletal muscle myosin reconstituted with gizzard thin filaments (in crude actomyosin) exhibited >50% Ca^^ sensitivity. On the other hand, the Mg^^-ATPase activity of rabbit skeletal muscle myosin reconstituted with purified gizzard actin plus tropomyosin was Ca^^ insensitive. These results suggest, therefore, that chicken gizzard smooth muscle contains a thin filament-linked Ca^^ regulatory mechanism.

CALPONIN

35 TABLE I

IDENTIFICATION OF THIN FILAMENT-LINKED REGULATION IN VERTEBRATE SMOOTH MUSCLE USING MYOSIN COMPETITION TEST Mg2^. A T P a s e rate (nmol Pi/mg myosin/min)" Source Gizzard a c t o m y o s i n + s k e l e t a l myosin Gizzard actin + tropomyosin + s k e l e t a l myosin

Ca^^ s e n s i t i v i t y

+Ca2^

-Ca^^

(%)

255.8

120.8

52.8

308.7

313.2

0

° Chicken gizzard actomyosin (1 mg/ml) and rabbit skeletal muscle myosin (1 mg/ml) were incubated separately and together in 0.6 M KCl, 10 mM imidazole hydrochloride (pH 7.0), and 5 mM MgCl2 with gentle stirring for 6 hr at 4°C to dissociate actin and myosin and associated proteins. Samples were then dialyzed overnight against two changes (2 liters each) of 60 mM KCl, 10 mM imidazole hydrochloride (pH 7.0), 5 mM MgCl2,10 mMNaNa, and 0.5 mM dithiothreitol to allow hybridization of skeletal muscle myosin with gizzard actin and associated proteins. Mg^^-ATPase activities were measured as previously described (13) in the presence and absence of Ca^^. Mg^^-ATPase rates have been corrected for the ATPase rates of the individual components [gizzard actomyosin = 6.0 C+Ca^^) and 0.0 (-Ca^^) nmol P/mg actomyosin/min; skeletal muscle myosin = 5.2 C+Ca^^) and 4.9 (-Ca^^) nmol Pi/mg myosin/min]. Values represent the means of two experiments. Reprinted from Ref. (7) with permission of The National Research Council of Canada.

We proceeded to isolate native thin filaments from chicken gizzard using mild extraction conditions so as to retain actin-associated regulatory proteins. These thin filaments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1) and found to contain, in addition to actin and the two tropomyosin subunits, proteins of Mr 140,000 and 32,000. The larger protein was identified as caldesmon by immunoblotting (13), but, at the time, the identity of the 32-kDa protein was unknown. While this work was under way, Takahashi and co-workers reported the isolation from chicken gizzard of a 34-kDa protein which bound to actin and actin-tropomyosin in a Ca^^-independent manner and to calmodulin in a Ca^^-dependent manner (15). This protein was later named calponin (for caZcium and ca/modulin-binding troponin T-like protein) (16). The 32-kDa protein in native thin filaments (Fig. 1) was identified as calponin by immunoblotting, physicochemical properties.

36 .

STEVEN J. WINDER AND MICHAEL P. WALSH

-Caldesmon

-Actin/Tropomyosiriy^ -Tropomyosins ^Calponln

FIG. 1. SDS-PAGE analysis of native thin filaments prepared from chicken gizzard smooth muscle. Thin filaments were isolated from chicken gizzard as previously described (14) and subjected to 0.1% SDS/7.5-20% polyacrylamide gradient gel electrophoresis and Coomassie blue staining.

and partial amino acid sequence analysis (16; S. J. Winder and M. P. Walsh, unpublished observation, 1989).

II. Biochemical Properties of Calponin A. Size and Distribution Calponin has been purified from avian gizzard (15,17,18), bovine aorta (19), sheep aorta (20), porcine stomach (18), and toad stomach (21) and identified by immunoblotting in several other bovine (22) and human (23) smooth muscle tissues. Figure 2 shows the protein composition of a toad stomach homogenate (Figure 2A) and the corresponding immunoblot using polyclonal antibodies specific for toad stomach calponin (Figure 2B). It is apparent from the stained gel in Fig. 2A that the tissue content of calponin is approximately the same as that of tropomyosin, in agreement with the results of Takahashi et al, (15) with chicken gizzard. Figure 3 depicts the analysis of calponin by twodimensional gel electrophoresis of a homogenate of a toad stomach smooth muscle strip contracted in response to carbachol. Calponin was identified by Western blotting (Fig. 3B). Due to the very basic nature

CALPONIN

37

A

B

Filamin — — —

Myosin H C - B H H I

Caldesmon -^"••iii a-Actinin — ssapr

Actin Tropomyosin -c ' Calponin - ^M

!

ngM —Calponin

I

FIG. 2. SDS-PAGE analysis and immunoblotting of toad stomach proteins. Toad stomach smooth muscle was homogenized in SDS-gel sample buffer and boiled and the proteins were separated by SDS-PAGE prior to Coomassie blue staining (A) or Western blotting with polyclonal antibodies raised against purified toad stomach calponin (B, left lane). The purified antigen is shown in B (right lane) for comparison.

of calponin (see below), isolectric focusing is not appropriate for its analysis; instead, nonequilibrium pH-gradient electrophoresis was used in the first dimension and SDS-PAGE in the second dimension. Four calponin spots are evident in Figs. 3A and 3B. The two most basic spots probably represent different isoforms and the two most acidic spots the corresponding phosphorylated species (see below). Calponin varies slightly in molecular mass (usually 33-35 kDa) depending on the tissue and species. Purified calponin behaves as a monomer under nondenaturing conditions (15). A low Mr calponin (29,000) was detected in human myometrium, ureter, bladder, and vas deferens in addition to the 34-kDa form (23). Interestingly, the 29-kDa calponin was not observed in benign smooth muscle-derived tumors (leiomyoma) or smooth muscle tissues other than those of the urogenital tract. A low Mr form of calponin (23,000) was also detected in bovine platelets (24). Calponin expression is restricted almost exclusively to smooth muscle. Gimona et al. (25) observed no calponin immunoreactivity in extracts of chicken skeletal muscle, kidney, liver, and spleen. Likewise,

38

STEVEN J. WINDER AND MICHAEL P. WALSH

©

©-^

NEPHGE

©

B

UJ

o < Q.

CO O

(0

UJ

o <

Q. I CO

o

CO

FIG. 3. Two-dimensional gel analysis of toad stomach calponin. A toad stomach strip was induced to contract with carbachol. Near the peak of iosmetric force development, the muscle strip was immersed in liquid N2 prior to separation of the proteins by twodimensional gel electrophoresis (NEPHGE/SDS-PAGE). (A) Coomassie blue stained gel; (B) Western blot using anti-(toad stomach calponin).

we did not detect calponin in chicken skeletal or cardiac muscles, lung, brain, kidney, or liver (Fig. 4). Takahashi et al. (22) concluded that bovine atria and ventricles and brain cortex do not express calponin. They did, however, observe a 36-kDa immunoreactive protein in bovine adrenal medulla and cortex; however, it is possible that this protein is actually p36 (calpactin I) rather than calponin since these two proteins are homologous (37% identical and 20% conservative replacements over a 35-residue sequence) (18). Calponin immunoreactive proteins were also detected by immunoblotting and immunoc3rtochemistry in bovine platelets (24), human umbilical vein endothelial cells (26), and fibroblasts (24,26); however, it remains to be unequivocally established that these are indeed calponins. Calponin expression has also been examined during differentiation and dedifferentiation. The level of calponin expression was found to increase steadily between Days 12 and 19 of embryonic development of the chicken gizzard. On the other hand, calponin expression was downregulated within 48 hr of cultivation of 16-day embryonic gizzard cells and practically no calponin was detected following the first passage (25). Similarly, the calponin content decreased five- and ninefold, re-

CALPONIN

39

CaP-

3

S ?

N 3

5* go
Q.

FIG. 4. Smooth muscle-specific expression of calponin. Homogenates obtained from the indicated chicken tissues were subjected to SDS-PAGE and immimoblotting using polyclonal antibodies raised against purified chicken gizzard calponin (the standard is shown in the left lane).

spectively, on cultivation of vascular smooth muscle cells of rabbit and human aorta (26). B. Amino Acid Sequence Electrophoretic analysis of purified calponin suggested the existence of isoforms (15,27). This was confirmed by molecular cloning and the complete amino acid sequences of two isoforms of calponin were deduced from the cDNA sequences (28) (Fig. 5). The encoded isoforms, denoted a and )8, have 292 and 252 amino acids and M^ 32,333 and 28,127, respectively. The sequences are identical with the exception that calponin j8 is lacking a 40-residue sequence corresponding to residues 217-256 of calponin a (Fig. 5). Calponin a and ^ are probably derived from the same gene by alternative splicing: the cDNA sequences are identical except for the 120-base pair insert in calponin a. The predicted isolectric points of calponin a and j8 are 9.91 and 9.95, respectively (28). The extremely basic nature of calponin was already known from isoelectric focusing (15). Consistent with the tissue distribution analy-

40

STEVEN J. WINDER AND MICHAEL P. WALSH

I

M S N A N F N R G P A Y G L S A E V K N K L A Q K Y D P Q T E R Q L R V W I E G

41

A T G R R I G D N F M D G L K D G V I L C E L I N T L Q P G S V Q K V N D P V Q

81

N W H K L E N I G N F L R A I K H Y G V K P H D I F E A N D L F E N T N H T Q V

121

Q S T L I A L A S Q A K T K G N N V G L G V K Y A E K Q Q R R F Q P E K L R E G

161

R N I I G L Q M G T N K F A S Q Q G M T A Y G T R R H L Y D P K L G T D Q P L D

201

QATISLQMGTNKGAS

241

NI I G L Q M G S N K G A S Q Q G

281

Q A G M T A P G T K R Q I F E P S L G M E R C D T M T V Y G L P R Q V Y D P K Y C D A P G L L G

EDGLKHSFYNSQ

FIG. 5. The deduced amino acid sequences of the a and ft isoforms of chicken gizzard calponin. Reprinted with permission from Ref. (28). The underHned sequence is absent from calponin ft.

ses by immunoblotting described previously, Northern blotting revealed a 1.3-kb calponin mRNA in various chicken smooth muscles but no hybridization signal was detected in cardiac or skeletal muscles or various nonmuscle tissues (28). Examination of the sequence of calponin revealed the presence of three 29-31 amino acid repeats in the carboxyl-terminal region of calponin a (Fig. 6); the /3 isoform contains only two repeats (28). Secondary structure predictions suggest the following for calponin a: 13% a helix, 22% j8 sheet, and 26% /3 turn. The regions encompassing residues 17-34 and 144-160 are predicted to be exposed to the aqueous environment on the surface of the molecule. Regions of calponin exhibit significant sequence homology with several other proteins (18,28): SM22a, a smooth muscle protein of unknown function (43% identical and 69% conservative residues in a 183-residue overlap); the product of gene mp 20 of Drosophila, a putative Ca2+-binding protein which is present in every muscle tissue except the asynchronous oscillatory flight muscles (36% identical and 64% conservative residues in a 181-residue overlap); troponin T (in several regions); troponin I; caldesmon; p36 (calpactin I); ras p21; a-actinin; and the candidate unc-87 gene of Caenorhabditis elegans. Unfortu164 204 243

I G L Q M G T N KIJJA S Q Q G M I ( S ] L Q M G T N K G A S Q ( A | G M T A p l G T| K I G L Q M G [ S 1 N K G A S Q Q G M TrvfY GIL P

164 f l l c 3 | L Q M G T N KF A S QQ 204 l l l s l L Q MG T N KG | A S | - Q

G M T A YG G M T V Y G\

T R RJH L L P[RJQ V

FIG. 6. Repeat motifs in calponin a and ft. Reprinted with permission from Ref. (28).

CALPONIN

41

nately, these sequence homologies shed httle Hght on the functional domains of calponin. C. Protein-Protein Interactions Involving Calponin As noted previously, calponin was initially described as an actinand calmodulin-binding protein (15). Later it was also shown to bind to isolated tropomyosin (29). The calponin-actin and calponintropomyosin interactions are unaffected by Ca^^, but the interaction between calponin and calmodulin requires Ca^^. This latter property has been exploited, along with the protein's heat stability and basic nature, in methods of purification of calponin. The binding of calponin to F-actin is commonly examined by a sedimentation assay (15,17): F-actin is sedimented by ultracentrifugation (e.g., 100,000 g for 1 hr), whereas calponin, being a monomer of —34 kDa, does not sediment. When mixed with actin, however, calponin is recovered in the pellet following ultracentrifugation, indicating the interaction between calponin and F-actin. Using such a sedimentation assay, we have estimated the affinity of actin for calponin in the presence and absence of tropomyosin (30): the K^ of smooth muscle actin for calponin was calculated by Scatchard analysis of the sedimentation data to be 4.6 x 10"^ M; the affinity was not significantly different in the presence of tropomyosin. The maximum binding stoichiometry in vitro was determined to be 1 mol calponin/ 3 mol actin. The tissue content of calponin has been estimated at 1 mol/7 mol actin, i.e., the same molar concentration as tropomyosin (15). The fact that calponin binds to F-actin with high affinity and is found in native thin filament preparations suggests that it may be a bona fide thin filament protein in situ. Immunocytochemical studies support this conclusion. Immunofluorescence microscopy was used to demonstrate the colocalization of calponin and actin in primary cultured bovine aortic smooth muscle cells (24), primary cultures of 16-day chick embryo gizzard cells (25), and rabbit aortic smooth muscle cells (26). In collaboration with Gary ICargacin (Department of Medical Physiology, University of Calgary) we have used confocal immunofluorescence microscopy to investigate the localization of calponin in freshly isolated single toad stomach smooth muscle cells (21). Using rhodamine-phalloidin to label actin and indirect immunofluorescence with polyclonal antibodies raised against toad stomach calponin and chicken gizzard tropomyosin, we observed very similar staining patterns for all three proteins, i.e., a filamentous network throughout the sarcoplasm with no staining in the nucleus. Again,

42

STEVEN J. WINDER AND MICHAEL P. WALSH

these results support the conclusion that calponin is a bona fide thin filament protein in smooth muscle. Takahashi et al. (29) first demonstrated the binding of calponin to purified tropomyosin by centrifugation of tropomyosin paracrystals formed in the presence of calponin: SDS-PAGE analysis of the pellets obtained by centrifugation at 10,000^ for 3 min revealed the presence of calponin indicating its binding to tropomyosin. Electron microscopy of tropomyosin paracrystals in the presence and absence of calponin revealed periodic binding of calponin to tropomyosin: the binding of calponin resulted in the appearance of white lines with widths of 8 nm and with 40 nm periodicity, i.e., similar to the binding of troponin T. They concluded that the calponin-binding site is located —17 nm from the carboxyl terminus of tropomyosin; the amino acid sequence in this region of tropomyosin is highly conserved among skeletal and smooth muscle isoforms. The binding of calponin to tropomyosin has been confirmed by affinity chromatrography using columns of immobilized tropomyosin or calponin (17,18). The fact that calponin eluted from a tropomyosin affinity column at an ionic strength equivalent to 100 mM KCl and, similarly, tropomyosin eluted from a calponin affinity column at 100 mM KCl (18) suggests that the calponin-tropomyosin interaction may not be of physiological importance. Indeed, as noted previously, we observed no difference in the K^ of actin or actin-tropomyosin for calponin; if calponin was capable of interacting with both actin and tropomyosin on the thin filament, one would expect a lower K^ in the presence of tropomyosin. Furthermore, as discussed below, phosphorylated calponin retains the ability to bind to immobilized tropomyosin but does not bind to actin or actin-tropomyosin. Calponin interaction with calmodulin was first demonstrated by calmodulin affinity chromatrography and gel-filtration chromatography (15). Calponin bound to immobilized calmodulin in the presence of Ca^^ and was not eluted by 0.3 M KCl or 6M urea; elution was achieved, however, by chelation of Ca^^ with EGTA in the presence of 0.2 M KCl. When subjected to molecular sieve chromatography in the presence of EGTA, calponin and calmodulin eluted in the same position corresponding to Mr —34,000. However, in the presence of Ca^^, a new high -Mr peak containing both calponin and calmodulin appeared confirming the formation of a calponin-calmodulin complex in the presence of Ca^^ (15). Studies of the binding of a fluorescently labeled derivative of calmodulin to calponin indicate a relatively low affinity {K^ —10"^ M) compared with calmodulin-binding proteins like MLCK {K^ ~ 1 nM)

CALPONIN

43

(31). The physiological significance of the interaction of Ca^^-calmodulin with calponin is unclear (see below).

III. Functional Properties of Calponin A. Inhibition of the Actin-Activated Myosin Mg^^-ATPase Several of the properties of calponin described previously suggest that it may be involved in regulation of actin-myosin interaction in smooth muscle: (i) it binds to actin with high affinity, (ii) it is localized on the thin filaments in isolated smooth muscle cells, (iii) it exhibits some sequence homology with the T and I subunits of the troponin complex of striated muscles and is immunologically cross-reactive (albeit weakly) with troponin T (16), and (iv) it is present at the same molar concentration as tropomyosin. We have utilized a well-characterized in vitro contractile system to examine the functional effects of purified calponin (17). The following contractile and regulatory proteins were purified from chicken gizzard: myosin, actin, tropomyosin, and myosin light-chain kinase. Calmodulin was purified from bovine brain; the chicken gizzard protein has an identical amino acid sequence except for two substitutions (Asp for Asn in positions 24 and 97) (32,33), but brain was the tissue of choice for the purification since it provides a significantly higher yield. These purified proteins were recombined in approximately physiological molar ratios. Two parameters were measured in this reconstituted contractile system: the phosphorylation of the 20-kDa light chain of myosin and the actin-activated myosin Mg^^ATPase activity [the latter correlates directly with the unloaded shortening velocity of the muscle (34)]. Reactions were started by the addition of ATP and samples withdrawn at selected times for quantification of myosin phosphorylation (35) and ATPase activity (36) on the same samples. The effects of calponin were investigated by inclusion of various concentrations of the purified protein. The results of one experiment are shown in Fig. 7 and Table II summarizes the results of several such experiments. Calponin had no significant effect on myosin phosphorylation but caused a marked inhibition (—75% at 2 jxM) of the actinactivated myosin Mg^^-ATPase. This inhibition of the ATPase was shown to be due to calponin since it was lost following immunoprecipitation of calponin (17). Furthermore, it was confirmed as a specific inhibitory effect rather than a nonspecific effect resulting from the strongly basic character of calponin since two other basic proteins, ribonuclease A (p/ 9.6) and chymotr3rpsinogen (p/ 9.5), had no effect on the actinactivated myosin Mg^^ATPase at concentrations as high as 10 ^iM (17).

44

STEVEN J. WINDER AND MICHAEL P. WALSH

B

STAINED GEL Myosin heavy chain

1.2

I

90

• C

0.9 E

o

60

0.6

'^^

Tropomyosin y ~ « • * ^ • i ^ ^ ^ ^ Tropomyosin/9— ^—• - - ^ Calponin—

—-• **•

o

>

E o E

Myosin LC20- ^••^

^Ml^ ^HIM»

Myosin LC^r-

«II»

«M»

m^

"5 E -Ca2+ +Ca** +Ca^ -K)alponin

0.3

AUTORADIOGRAM 0

1

2

3

4

5

6

CALPONIN (fiM)

7

8

0 Myosin LC20—

-Ca^*

^HP^

^ H P

+Ca2+

+Ca2+ +Calponin

FIG. 7. Calponin inhibits the actin-activated myosin Mg^^-ATPase without affecting myosin phosphorylation. (A) Actomyosin ATPase rates (D, • ) and myosin phosphorylation levels (O, • ) were measured at 30°C in a reconstituted contractile system (composed of 1 fiM myosin, 6 fiM actin, 2 /JLM tropomyosin, 1 fiM CaM, and 74 nM MLCK in 25 mAf Tris-HCl, pH 7.5, 10 mM MgClz, 60 mM KCl, 1 mM (y-32p]ATP) in the presence of the indicated concentrations of calponin and in the presence of 0.1 mM CaCl2 (O, D) or 1 mM EGTA (•, • ) . (B) Selected reaction mixtures from A were analyzed by SDSPAGE and autoradiography; these are the fully reconstituted system in the absence of calponin and Ca^^ (-Ca^^), in the absence of calponin and the presence of Ca^^C+Ca^^), and in the presence of Ca^^ and 3 /xM calponin C+Ca^* + calponin).

ATPase inhibition by calponin was reversed by increasing the concentration of actin-tropomyosin, suggesting that the inhibitory effect is due to the interaction of calponin with the thin filament rather than a direct effect on myosin (17). In support of this conclusion, we have shown that calponin does not interact with smooth muscle myosin (phosphorylated or dephosphorylated) (17) nor does it inhibit the Ca^^ATPase or K^/EDTA-ATPase activities of skeletal muscle myosin in the absence of actin (36a). Calponin-induced inhibition of actomyosin ATPase activity has been confirmed by several other investigators (20,37-39). Horiuchi and Chacko (38), using smooth muscle actophosphorylated heavy meromyo-

CALPONIN

45 TABLE II

CALPONIN INHIBITION OF ACTIN-ACTIVATED MYOSIN Mg^^ATPase WITHOUT AFFECTING MYOSIN PHOSPHORYLATION

Ca^^

Calponin"

Actin-activated myosin Mg^^-ATPase*" (nmol Pi/mg myosin/ min)

Myosin phosphorylation'' (mol Pi/mol myosin)

7.5 ± 1.0 112.0 ± 5.5 27.8 ± 2.5

0.15 ± 0.04 1.77 ± 0.13 1.70 ± 0.08

" At a concentration of 2 JJLM where present. Other reaction conditions are provided in the legend to Fig. 7. * Values represent the means (±SEM) of 39 determinations. " Values represent the means (±SEM) of 5 determinations.

sin, demonstrated that calponin affects a catalytic step in the ATPase cycle but has only a slight effect on the affinity of heavy meromyosin for actin. A similar conclusion was reached by Nishida et al. (40) using smooth muscle actothiophosphorylated myosin. B. Regulation of ATPase Inhibitory Effect of Calponin

It is reasonable to suggest that the inhibitory effect of calponin must be regulated in some way since it is unlikely that calponin functions to inhibit the actomyosin ATPase in a consitutive manner. We have considered four possible mechanisms of regulation of calponin function based on the known structural and functional properties of the isolated protein: (i) the direct binding of Ca^^ to calponin, (ii) dissociation of calponin from actin by Ca^^-calmodulin, (iii) binding of GTP to calponin, and (iv) phosphorylation of calponin. With regards to the binding of Ca^^ to calponin, Takahashi et al. (41) demonstrated binding of Ca^^ to purified calponin by UV difference spectroscopy and estimated the Kd for Ca^^ to be ~7 /AM. We have also observed the direct binding of Ca^^ to calponin using a ^^CaCl2 overlay method (17). Although the affinity of calponin for Ca^^ appears rather weak to be of physiological significance, we did examine the possibility that Ca^^ may directly affect the ATPase inhibition by calponin. For this purpose, myosin was prephosphorylated and then actin was added with or without calponin in the presence or absence of Ca^^ (Table III). Inhibition of the actomyosin ATPase by calponin was observed in both the presence (65% inhibition) and the absence (72% inhibition) of Ca^^. The lower ATPase rates observed in the absence of Ca^^ were due to partial dephosphorylation of myosin (0.3-0.4 mol P/mol myosin) during

46

STEVEN J. WINDER AND MICHAEL P. WALSH TABLE III

CALPONIN-INDUCED INHIBITION OF ACTOMYOSIN ATPase ACTIVITY INDEPENDENT OF Ca^^

Assay system Actophosphorylated myosin"

Ca2^

Calponin

-

-

+

-

+ Actothiophosphorylated myosin*

-

+

-

+

4-

+

-

+ +

ATPase rate (nmol Pi/mg myosin/ min) 91.4 114.7 25.5 40.4 62.3 66.5 23.0 24.2

° Myosin (1 /xM) was phosphorylated by incubation at 30°C for 8 min in the presence of 1 /Jlf CaM, 74 nM MLCK, and 2 fiM tropomyosin in 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 60 mM KCl, 0.1 mM CaCla, 1 mM ['y-32p]ATP ( - 1 0 cpm/pmol). The following additions were then made simultaneously: 6 /JLM actin ± 5 /JLM calponin in the presence of 0.1 mM CaCl2 or 1 mM EGTA (final concentrations) and samples were withdrawn at 1-min intervals (up to 5 min) for determination of the ATPase rates. * Myosin was thiophosphorylated under identical conditions except for the replacement of radiolabeled ATP with unlabeled ATPyS. At the time of addition of actin ± calponin with Ca2+ or EGTA, 1 mM [y-^^pj^^P ( - 1 0 cpm/pmol) was also added and ATPase rates were measured as described above. Reprinted from Ref. (17) with permission.

the ATPase reactions due to a contaminating myosin phosphatase in the myosin preparation; this did not occur in the presence of Ca^^ since MLCK was maintained in an activated state under these conditions. Nevertheless, to ehminate the problem of dephosphorylation, the same experiments were repeated using prethiophosphorylated rather than prephosphorylated myosin since thiophosphorylated myosin is resistant to the action of myosin phosphatases (42). Calponin inhibited the actothiophosphorylated myosin Mg^^-ATPase by 64% in the presence of Ca^^ and by 63% in the absence of Ca^^. We conclude, therefore, that calponin-mediated inhibition of the actomyosin ATPase is not regulated by the direct binding of Ca^^ to calponin. Other investigators have also shown that calponin-induced inhibition of actomyosin ATPase activity is independent of Ca^' (19,20). The fact that calponin interacts with calmodulin in a Ca^^-dependent manner suggested that calponin inhibition of the actin-activated myosin Mg^^-ATPase may be regulated by Ca^^-calmodulin. This possibility has been investigated in several laboratories. Abe et al. (19) showed that calmodulin, in the presence but not in the absence of Ca^^, reversed

CALPONIN

47

the calponin-induced inhibition of the actothiophosphorylated myosin Mg2+-ATPase. However, the level of ATPase inhibition detected in their experiments was surprisingly low (—28% inhibition at 1 calponin: 7 actin monomers) and complete reversal of inhibition required ~2.5 mol calmodulin/mol calponin; the tissue concentration of calponin, assuming the same molar concentration as tropomyosin, is —150 jxM (1) and that of calmodulin is —50 fiM (43). Using smooth muscle actin and skeletal muscle myosin, Marston (20) observed —36% inhibition of the actomyosin ATPase at 1 calponin/5 actin monomers and this was unaffected by calmodulin (3.75 mol/mol calponin) in the presence of Ca^^. The data in Table IV show that a 15-fold molar excess of calmodulin over calponin caused only a very slight reversal of inhibition of the smooth muscle actin-activated myosin Mg^^-ATPase. We have also found that Ca^^-calmodulin can dissociate calponin from F-actin but that very high molar ratios of calmodulin : calponin are required (halfmaximal release of calponin from actin occurred at —10 mol calmodulin : 1 mol calponin). All these results indicate that, at physiologically relevant molar ratios of calmodulin : calponin (—1 : 3), the association of calponin with the thin filament and ATPase inhibition are unaffected by Ca^^-calmodulin. Takahashi and Nadal-Ginard (28) observed some sequence similarity between residues 18-42 of calponin and residues 24-50 of the GTPbinding protein ras p21 (58% conservative replacements); this region includes the effector domain (site of interaction of ras proteins with putative cellular target proteins) (44). The sequences around Asp-119 and Ala-146 of ras p21, which are important for binding of the guanosine TABLE IV EFFECT OF CALMODULIN ON CALPONIN-INDUCED INHIBITION OF ACTOMYOSIN ATPase ACTIVITY^

Calponin

CaMC/xAf)

ATPase rate (nmol Pi/mg myosin/ min)

+ +

0.6 0.6 30.0

126.5 46.8 57.2

Myosin phosphorylation (mol P/mol myosin) 1.9 2.0 2.0

° Reaction conditions were as follows: 25 mM Tris-HCl (pH 7.5), 60 mM KCl, 10 mM MgCl2, 0.1 mM CaCl2, 1 mM [y-^^pj^TP, l fiM myosin, 6 /JLM actin, 2 fiM tropomyosin, 0.6 or 30 fiM CaM, 74 nM MLCK, ± 2 fxM calponin. ATPase rates (36) and myosin phosphorylation levels (35) were measured as previously described. Values represent the means of three determinations.

48

STEVEN J. WINDER AND MICHAEL P. WALSH

moiety of GTP (45), were suggested to be similar to the calponin sequences around Asp-104 and Ala-131 (28), although the degree of similarity, particularly around Asp-119 of ras p21, is weak. Nevertheless, this raised the possibility that calponin function may be regulated by GTP. We have examined this possibility in two ways. First, we observed no effect of GTP (at concentrations up to 5 mM) on the interaction between calponin and actin as examined by the sedimentation assay described earlier (S. J. Winder and M. P. Walsh, unpublished observations, 1990). Second, we failed to detect any binding of [^^S] GTPyS to purified calponin under conditions which showed significant binding to a preparation of bovine brain G proteins (predominantly Gi) (Table V). Tropomyosin, which is clearly not a GTP-binding protein, was included as a negative control. These results therefore suggest that GTP may not be involved in regulating calponin function. TABLE V

NoNBiNDiNG OF GTPyS TO CALPONIN

Protein

Concentration (/xg/ml)

None Calponin

Tropomyosin

Bovine brain G proteins

—

34 68 68 102 136 34 68 68 102 136 34 68 68

GTPyS (/xM) 1 10 1 1 10 1 1 1 1 10 1 1 1 1 10

pmol ^^S bound" (mean ± SD) 1.55 1.87 1.70 1.92 0.96 1.53 1.73 1.56 1.66 1.08 1.47 1.40 4.98 8.40 10.41

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.55 0.49 0.56 1.06 0.13 0.27 0.81 0.42 0.68 0.26 0.30 0.45 2.03 3.59 2.60

n 16 4 16 10 4 7 3 16 10 4 7 3 16 10 4

" The binding of [^^SJGTPyS (-4000 cpm/pmol) to calponin was examined using a nitrocellulose filtration assay (46) following incubation for 45 min at 30°C at the indicated protein and GTP concentrations in 50 mM Hepes (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, and 10 mMMgCl2 (reaction volume = 0.1 ml). Following the addition of a stop solution [20 mM Tris-HCl (pH 8), 20 mM MgCls, 0.1 M NaCl], incubation mixtures were filtered onto nitrocellulose discs using a Millipore filtration manifold and washed five times (2 ml each) with stop solution. The discs were air dried and ^^S was counted in scintillant. Tropomyosin was included as a negative control and a preparation of bovine brain G proteins (predominantly Gj) as a positive control.

CALPONIN

49

Protein phosphorylation-dephosphorylation is a very common mechanism of regulation involved in the control of diverse cellular functions (47). We considered the possibility that calponin may be subject to phosphorylation and that consequently its ability to inhibit actomyosin ATPase activity may be affected. We addressed this question first by testing purified calponin as a substrate for a variety of purified protein kinases: protein kinase C (PKC), Ca^^-calmodulin-dependent protein kinase II (CaM kinase II), cAMP-dependent protein kinase, cGMPdependent protein kinase, and myosin light-chain kinase. Only two of these kinases (PKC and CaM kinase II) phosphorylated calponin (Fig. 8). The time courses of phosphorylation by the two kinases (Fig. 8A) show that stoichiometric phosphorylation was achieved in each case. Phosphate incorporation into calponin was confirmed by SDS-PAGE and autoradiography at the end of the phosphorylation reactions (Figs. SB and 8C). The expected dependence of CaM kinase Il-catalyzed phosphorylation on Ca^^ and calmodulin and of PKC-catalyzed phosphorylated of calponin on Ca^^, phospholipid, and diacylglycerol is shown in Figs. 8D and 8E. Phosphoamino acid analysis indicated phosphorylation of both serine and threonine residues (Fig. 9). Two-dimensional phosphopeptide mapping suggested that the major sites of phosphorylation by PKC and CaM kinase II are the same (17). Identification of the sites of phosphorylation has been complicated by the difficulty of obtaining a limit tryptic digest of calponin, possibly due to poor accessibility of several tryptic cleavage sites. To date, we have identified two major sites of phosphorylation by PKC: Ser-175 and Ser-254 (27). The latter site is missing from calponin ^ (see Fig. 5). PKC-catalyzed phosphorylation of calponin was also observed by Naka et al. (49) who also showed that phosphorylation was prevented by the binding of Ca^^calmodulin to calponin. What is the effect of phosphorylation of calponin on its ability to inhibit the actin-activated myosin Mg^^-ATPase? Figure 10 compares the effects on the smooth muscle actomyosin ATPase of isolated calponin [which is unphosphorylated (38)] and calponin, which was phosphorylated to various extents by either PKC or CaM kinase II. Phosphorylation by either kinase resulted in the loss of inhibition of the ATPase. Furthermore, phosphorylation to the extent of ~ 1 mol Pi/mol calponin is effective in alleviating ATPase inhibition. As indicated previously, calponin can interact with actin, tropomyosin, and Ca^^-calmodulin. The loss of inhibition of actomyosin ATPase activity on phosphorylation of calponin provided a means of identifying which of these protein-protein interactions is responsible for inhibition of the ATPase. We examined the effect of phosphorylation of

50

STEVEN J. WINDER AND MICHAEL P. WALSH

M c 'c

2.0

-

o CO

O

"o E

~^

r^

^

/ / / 10 /

o Q/*^

5'

^^'^ °

Q

o

QL

_/ ^ .» 1 0.5 // 0

'

^ 20

--

29 20.1- —

^_CaP—g^

€1

-CaM

14.4- — M

CaP + PKC

CaP + CaMKI

Cap + PKC

AUTORADIOGRAMS

D

CaP + CaMKI

g

Calponin- ^m

*

w

+

^AUTORADIOGRAM

T

97.4 66.2 - - — 45- —

Ca^-^ + CaM +

L_ 60

Time (min)

VINED GEL

kPa

Calponin- ^m

L— 40

+ -

Ca2+ + PL/DG +

+

+ -

-

FIG. 8. Phosphorylation of calponin by PKC and CaM kinase II. Calponin was phosphorylated by PKC (O) or CaM kinase II (•) as previously described (17). (A) Time courses of phosphorylation by the two kinases; (B) SDS-PAGE analysis of the two reaction mixtures at the end of the reactions (M = Mr marker proteins); (C) autoradiogram of the stained gel shown in (B); (D) autoradiogram of the calponin region of a gel of calponin treated with CaM kinase II in the presence and absence of Ca^^ and CaM as indicated; (E) autoradiogram of the calponin region of a gel of calponin treated with PKC in the presence and absence of Ca^^ and phospholipid (PL)/diacylglycerol (DG) as indicated.

calponin on its interaction with each of its three target proteins (17). Phosphorylated calponin retained the ability to bind to immobilized tropomyosin and Ca^^-calmodulin. However, the phosphorylated protein no longer interacted with actin (Fig. 11). Unphosphorylated calponin bound to actin and to actin-tropomyosin as shown by the sedimentation assay (Fig. 11 A, lanes 1 and 2 and 5 and 6). Following

CALPONIN

51

A

e

B e

t :S 1 0

2

3

0

4

FIG. 9. Phosphoamino acid analysis of calponin phosphorylated by PKC and CaM kinase II. Calponin phosphorylated by CaM kinase II (lanes 1 and 2) or PKC (lanes 3 and 4) in the presence of ['y-32p]ATP was hydrolyzed with 6 N HCl at 110°C for 2 hr, mixed with standards of unlabeled phosphoamino acids, and subjected to thin-layer electrophoresis as previously described (48) prior to staining with ninhydrin (lanes 1 and 3) and autoradiography (lanes 2 and 4). The three standards (from top to bottom) are phosphoserine, phosphothreonine, and phosphotyrosine.

phosphorylation by CaM kinase II most of the calponin did not bind to actin or actin-tropomyosin (Fig. 11 A, lanes 3 and 4 and 7 and 8). Similar results were obtained following phosphorylation by PKC. Likewise, in the reconstituted contractile system the interaction of calponin with actin was lost on phosphorylation (Fig. IIB, lanes 4-7). We concluded, therefore, that the calponin-actin interaction is responsible for inhibition of the actomyosin ATPase. This interaction is lost on phosphorylation which would account for the loss of ATPase inhibition. If calponin phosphorylation is to be of physiological significance, there must also be a phosphatase capable of dephosphorylating calponin and restoring its inhibitory capacity. We utilized purified [^^PJcalponin to assay for calponin phosphatase activity in chicken gizzard. A cytosolic fraction (SI) was prepared by high-speed centrifugation of a tissue homogenate and the resultant pellet was washed and centriguged twice to yield supernatant fractions S2 and S3 and a final washed pellet (P). Calponin phosphatase activity was assayed in each of these fractions by SDS-PAGE and autoradiography (Fig. 12). Calponin phosphatase activity was highest in SI, lower in S2, and lowest in S3; relatively little activity was detected in the resuspended pellet (Fig. 12C). Quantification of these data (Fig. 12A) revealed that <10% of total calponin phosphatase activity was recovered in the pellet fraction. Therefore, a combination of fractions S1-S3 was used to purify the phosphatase; this was achieved by ammonium sulfate fractionation and sequential chromatography on columns of Sephacryl S-300, DEAE-Sephacel, co-

52

STEVEN J. WINDER AND MICHAEL P. WALSH

3

4

5

Calponin ,(jxM) FIG. 10. Phosphorylation of calponin prevents inhibition of actomyosin ATPase activity. Calponin was phosphorylated by PKC (to 1.47 mol P,/mol, • ; 0.69 mol Pi/mol, A; or 1.66 mol Pi/mol, T) or CaM kinase II (to 1.11 mol Pi/mol, • ) . Open symbols are the corresponding unphosphorylated calponin preparations. Actomyosin ATPase rates were measured at the indicated calponin concentrations under conditions described in the legend to Fig. 7. Results are expressed as percentage inhibition of the actomyosin ATPase rate in the absence of calponin : 130.1 ± 13.5 nmol Pi/mg myosin/min (mean ± SD, n = 5).

aminooctyl agarose, and thiophosphorylated myosin LC2o-Sepharose (50). The purified phosphatase consisted of three polypeptide chains of 60,55, and 38 kDa and was shown by immunoblotting methods to be identical to SMP-I, a type 2A smooth muscle phosphatase isolated by Pato and co-workers from turkey gizzard as a myosin LC20 phosphatase (51); however, SMP-I does not dephosphorylate intact myosin and therefore is not a physiological myosin light-chain phosphatase. Comparison of the dephosphorylation of several phosphoproteins by calponin phosphatase indicated that calponin was the best substrate.

CALPONIN

53 S

P

1

2

S

P

3 S

S

4

P

5

P S

f

S

6 P

P

7 S

8

P

» - — "

c- ' _

1

2

3

4

5

6

7

FIG. 11. Binding of phosphorylated and unphosphorylated calponin to actin, actintropomyosin, and a reconstituted contractile system. (A) Actin (11 fiM) and calponin [4 fiM of unphosphorylated (lanes 1, 2, 5, and 6) or phosphorylated to 1.0 mol Pj/mol by CaM kinase II (lanes 3, 4, 7, and 8)] in 20 mM Tris-HCl (pH 7.5), 0.1 M KCl, 2 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol, and 0.1 mM CaCl2 were sedimented at 100,000 g for 1 hr in the absence (lanes 1-4) and presence (lanes 5-8) of tropomyosin (2.6 IJLM). The resultant pellets (P) and supematants (S) were analyzed by SDS-PAGE. (B) Calponin phosphorylated to 2.3 P/mol by CaM kinase II (lanes 6 and 7) and unphosphorylated calponin (lanes 4 and 5) (2 /xM) were sedimented as described in the presence of actin (6 fjuM), myosin (1 fjuM), tropomyosin (2 /iM), CaM (1 fjbM), and MLCK (74 nM) in 25 mM Tris-HCl (pH 7.5), 10 mM MgClg, 60 mM KCl, 0.1 mM CaCl2, and 1 mM ATP. The reconstituted system without calponin is shown in lanes 2 and 3. Mr markers are shown in lane 1 : a, phosphorylase 6, 97.4 kDa; b, bovine serum albumin, 66.2 kDa; c, ovalbumin, 45 kDa; d, carbonated hydratase, 29 kDa, e, soybean trypsin inhibitor, 20.1 kDa; f, lysozyme, 14.4 kDa). A, Actin; Tm, tropomyosin; CaP, calponin; M, myosin heavy chain; LC20 and LCn, the 20- and 17-kDa light chains of myosin. Reprinted from Ref. (17) with permission.

54

STEVEN J. WINDER AND MICHAEL P. WALSH 1

00 <

I

1

1

1

l_

-

80

\\\ 60

40

20

V-.

" V<, - V--

—

20

- o — — 1

1

30 40 Time (min)

^=3-

AUTORADIOGRAM

STAINED GEL

I I ,0 15 30 60„0 15 30 60„0 15 30 60„0 15 30 60, P SI S2 S3

Mr I P-CaP

,0 15 30 60,,0 153060,,0 15 3060^,0 153060,

P-CaP

SI

S2

S3

P

FIG. 12. Demonstration of calponin phosphatase activity in chicken gizzard smooth muscle. Chicken gizzard smooth muscle was homogenized in 4 vol of 20 mAf Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM dithiothreitol, 0.25 mM PMSF, 10 /ig/ml leupeptin, and 10 iJLg/ml pepstatin (H buffer) and centrifuged at 30,000^ for 30 min (supernatant, SI). The pellet was resuspended in 4 vol of H buffer and homogenized and centrifuged as described previously (supernatant, S2). This step was repeated to yield supernatant S3. The final pellet (P) was resuspended and homogenized in 4 vol of H buffer. Calponin phosphatase activity was assayed by incubation of [^^PJcalponin (1.8 mol P/mol phosphorylated by PKC) at a concentration of 80 /ig/ml with 40% (v/v) of each fraction (SI, S2, S3, and P) in 20 mM Tris-HCl (pH 7.5) and 10 mM EDTA. Samples of reaction mixtures were withdrawn at 0, 15, 30 and 60 min for analysis by SDS-PAGE and Coomassie blue staining (B) and autoradiography (C). The quantitative data shown in A were obtained by laser scanning densitometry of an autoradiogram exposed for a shorter period of time than that shown in (C).

CALPONIN

55

suggesting that phosphorylated calponin may be a physiological substrate of SMP-I. Using the purified calponin phosphatase we were able to demonstrate that dephosphorylation of calponin restored its ability to inhibit the actin-activated myosin Mg^^-ATPase (Table VI). Therefore, chicken gizzard smooth muscle contains all the enzymatic machinery necessary to catalyze the reversible phosphorylation of calponin, and phosphorylation and dephosphorylation correlate with defined activity changes. However, it remains to be established whether calponin phosphorylation occurs in vivo, Barany and co-workers (52) failed to detect phosphorylation of calponin in resting intact porcine carotid arterial strips labeled with [^^PJPi or following contraction in response to K^ depolarization, norepinephrine, histamine, or phorbol ester. However, we have observed calponin phosphorylation in canine tracheal smooth muscle strips treated with carbachol (53) and in toad stomach strips treated with KCl, carbachol, or okadaic acid (a protein phosphatase inhibitor) (53a). Quantification of calponin phosphorylation in canine trachealis during a sustained contractile response to carbachol revealed a significant increase in calponin phosphorylation during the phase of force development and a return toward the resting level of phosphorylation during the sustained phase offeree maintenance (53). TABLE VI EFFECTS OF PHOSPHORYLATION AND DEPHOSPHORYLATION OF CALPONIN ON INHIBITION OF ACTIN-ACTIVATED THIOPHOSPHORYLATED MYOSIN Mg^^-ATPase

Calponin None Untreated Phosphorylated (1.66 mol/mol) Phosphorylated and dephosphorylated (0.05 mol/mol)

Concentration (/xM)

Mg2^-ATPase rate" (nmol Pi/mg myosin/ min)

2 3 2 3 2 3

124.1 25.1 17.1 140.9 134.5 38.0 12.0

° The actin-activated Mg^^-ATPase activity of thiophosphorylated myosin was assayed as described in the footnotes to Table III in the presence of Ca^^ and the indicated concentrations of calponin (untreated, phosphorylated to 1.66 mol/mol by PKC and dephosphorylated to 0.05 mol/mol). Reprinted from Ref. (50) with permission.

56

STEVEN J. WINDER AND MICHAEL P. WALSH

Figure 13 depicts a model that explains the possible physiological role of calponin. This mechanism is based on the in vitro binding properties of calponin, its inhibition of actomyosin ATPase activity, and the effects of phosphorylation. In the resting smooth muscle cell, the sarcoplasmic free [Ca^^] ([Ca^+Ji) is --120-270 nM (54-56). At this concentration, MLCK, PKC, and CaM kinase II are expected to be inactive so that myosin and calponin should be dephosphorylated. Therefore, myosin heads are dissociated from actin, calponin is bound to the actin filament, and the muscle is relaxed. Stimulation of the smooth muscle cell typically results in an increase in [Ca^^Ji to -500-700 nM (55). This would result in activation of MLCK, CaM kinase II, and, if the stimulus triggers the generation of 1,2-diacylglycerol, PKC. Consequently, calponin will be phosphorylated, causing its dissociation from the thin filament, and myosin will also be phosphorylated, activating cross-bridge cycling and the development of force. In these extreme situations, calponin serves no particular function except perhaps to inhibit a low cross-bridge cycling rate RESTING

CONTRACTING

INTERMEDIATE [Cag^1

FIG. 13. A model of the postulated physiological role of calponin in regulation of smooth muscle actin-myosin interaction. Calponin is shown spanning three actin monomers only because this is the maximum binding stoichiometry determined in vitro (30); the calponin content in situ is 1 mol/7 actin monomers. Only the SI regions of myosin are included for simplicity. P denotes phosphorylation. Reprinted from Ref. (7) with permission of The National Research Council for Canada.

CALPONIN

57

which may otherwise exist at rest. However, it has been estabhshed using Ca^^ indicator dyes and the photoprotein aequorin that, in smooth muscle strips or isolated smooth muscle cells, [Ca^^Ji peaks shortly after stimulation and then declines to an intermediate steadystate level during continued stimulation (see, e.g., 55,57,58). It is in this situation that we suggest calponin comes into play. At an intermediate [Ca^^Ji, CaM kinase II and PKC, which are less sensitive to Ca^^ than is MLCK (59-61), will be predominantly inactive whereas MLCK will remain predominantly in the activated state. Calponin dephosphorylation by calponin phosphatase (SMP-I) induces its reassociation with the thin filaments. Even though myosin remains phosphorylated, the cross-bridge cycling rate (equivalent to the actinactivated myosin Mg^^-ATPase) will be inhibited due to the presence of dephosphorylated calponin bound to actin. ATP utilization in stimulated smooth muscle has indeed been shown to be biphasic: upon stimulation the rate of ATP utilization rises rapidly to approximately three times the unstimulated rate and subsequently decreases to a steady-state rate of approximately twice the unstimulated rate during the sustained phase of contraction (62). The cross-bridge cycling rate can therefore be adjusted to any level between resting and maximally activated by precise adjustment of [Ca^^li which will determine the ratio of phosphorylated to dephosphorylated calponin and phosphorylated to dephosphorylated myosin. The presence of two regulatory components (calponin and MLCK) which have different sensitivities to Ca^^ gives the system the flexibility required to explain the physiological properties of the smooth muscle contractile system.

IV. Structure-Function Relations To date, little is known about the structure-function relations of calponin. Site-directed mutagenesis will clearly be the method of choice in this regard now that calponin cDNA has been cloned and sequenced (28). Partial proteolysis of calponin with characterization of the fragments generated has provided a limited amount of information. Cleavage of a mixture of the a and j8 isoforms of calponin at cysteine residues with 2-nitro-5-thiocyanobenzoic acid (NTCB) in the presence of 6 M guanidine hydrochloride generated major fragments of 30 and 21 kDa (27). The a isoform contains three cysteine residues (at positions 61, 238, and 273); the ^ isoform contains only two cysteine residues (at positions 61 and 233) due to the deletion of residues 217-256 of the a isoform (see Fig. 5). The 21-kDa NTCB fragment must therefore correspond to calponin a (61-237) and calponin j8 (61-232). These

58

STEVEN J. WINDER AND MICHAEL P. WALSH

fragments were separated from other NTCB peptides by re versed-phase high-performance hquid chromotography and shown to retain the ability of intact calponin to bind to F-actin and to inhibit the actin-activated Mg^^-ATPase activity of phosphorylated smooth muscle myosin. Further degradation of the 21-kDa fragments with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease resulted in loss of actin binding and actomyosin ATPase inhibition suggesting that regions of the molecule which are far apart in the primary structure are required for interaction with actin (27). Both the 30- and the 21-kDa NTCB fragments bound to immobilized tropomyosin and Ca^^-calmodulin. The tropomyosin-binding site was more precisely located by VancompernoUe et al. (18) who isolated a 13-kDa chymotryptic fragment of turkey gizzard calponin (corresponding to Asn^-Tyr^^ of chicken gizzard calponin) which retained the ability of the intact protein to bind to immobilized tropomyosin. A more precise definition of the functional domains of calponin will require expression and characterization of mutated forms of calponin.

V. Calponin and Caldesmon As noted previously, native thin filaments prepared from smooth muscle contain caldesmon in addition to actin, tropomyosin, and calponin (Fig. 1). The reader is referred to reviews (63-65) for detailed discussions of caldesmon; only those properties relevant to calponin function will be discussed here. Caldesmon shares several properties with calponin: (i) it binds to actin and tropomyosin in a Ca^^-independent manner and to calmodulin in a Ca^^-dependent manner, (ii) it is located on thin filaments in situ, (iii) it inhibits the actin-activated myosin Mg^^-ATPase activity in a reconstituted contractile system, and (iv) its inhibitory function is regulated by phosphorylation. Caldesmon also interacts with myosin, with the actin- and myosin-binding sites on caldesmon being located near opposite ends of this elongated molecule (66-68). In fact, caldesmon can cross-link actin and myosin filaments (67) and this may account for the formation of latch bridges (69), i.e., slowly or noncycling cross-bridges responsible for force maintenance at low levels of myosin phosphorylation which is often observed during prolonged stimulation of smooth muscle. The regulatory properties exhibited by calponin and caldesmon in vitro suggested that they may interact physically and/or functionally. VancompemoUe et al. (18) reported that caldesmon binds to immobilized calponin, but this interaction was disrupted at low ionic strength

CALPONIN

59

(70 mM KCl) and therefore is unlikely to be of physiological significance. However, it is still possible that the two proteins may interact at a functional level. Therefore, several investigators have examined the combined effects of calponin and caldesmon on the actin-activated myosin Mg^^-ATPase in vitro. Using thiophosphorylated smooth muscle myosin, Abe et al. (19) concluded that the inhibitory effects of calponin and caldesmon were unaffected by each other's presence. Similar conclusions were made by Makuch et al. (39) using skeletal muscle actomyosin which does not require phosphorylation for actin activation of its ATPase. In both of these studies, however, the potency of calponininduced inhibition of ATPase activity was low. Using smooth muscle actothiophosphorylated myosin, we observed that calponin was —twofold more potent than caldesmon in inhibition of the ATPase (36a). Given the higher tissue concentration of calponin [1 mol/7 mol actin (15)] compared with caldesmon [1 mol/22-28 mol actin (70)], our results suggest that calponin is a more effective regulator of the actomyosin ATPase than is caldesmon. This raises the question of whether caldesmon actually does play a regulatory role in smooth muscle. Perhaps, through its ability to cross-link actin and myosin filaments, caldesmon serves a structural or organizational role, i.e., it may function in the organization of the contractile filaments into a three-dimensional network in which the actin and myosin filaments have the proper orientation and spatial distribution for effective force development in response to appropriate stimuli (63). With regards to calponin, further study with skinned and intact smooth muscle cells and fibers is required to evaluate its physiological function as a regulator of smooth muscle contraction. ACKNOWLEDGEMENTS Work carried out in the authors' laboratory was supported by a grant from the Medical Research Council of Canada (MRCC). S.J.W. was the recipient of a Fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR). M.P.W. is an AHFMR Medical Scientist. The authors are very grateful to Cindy Sutherland and Elaine D. Eraser for expert technical assistance, to Dr. Andy Braun for assistance and advice in connection with the GTPyS-binding experiments, and to Gerry Gamett for secretarial support. REFERENCES 1. Hartshome, D. J. (1987). In "Physiology of the Gastrointestinal Tract" (L. R. Johnson, ed. ), pp. 423-482. Raven, New York. 2. Walsh, M. P., and Hartshome, D. J. (1983). In *The Biochemistry of Smooth Muscle." (N. L. Stephens, ed.), pp. 1-84, CRC Press, Boca Raton, Florida. 3. Klee, C. B. (1980). In "Calcium and Cell Function" (W. Y. Cheung, ed.). Vol. 1, pp. 59-78. Academic Press, New York. 4. Adelstein, R. S., and Klee, C. B. (1981). J. Biol. Chem. 256, 7501-7509.

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STEVEN J. WINDER AND MICHAEL P. WALSH

Kamm, K. E., and Stull, J. T. (1989). Annu. Rev. Physiol. 51, 299-313. Pato, M. D. (1985). Adv. Protein Phosphatases 1, 367-382. Walsh, M. P. (1991). Biochem. Cell Biol. 69, 771-800. Szent-Gyorgyi, A. G., Szent-Kiralyi, E. M., and Kendrick-Jones, J. (1973). J. Mol. Biol. 74, 179-203. 9. Weber, A., and Murray, J. M. (1973). Physiol. Rev. 53, 612-673. 10. Lehman, W. (1975). Nature (London) 255, 424-426. 11. Sellers, J. R. (1981). J. Biol. Chem. 256, 9274-9278. 12. Lehman, W., and Szent-Gyorgyi, A. G. (1975). J. Gen. Physiol. 66, 1-30. 13. Ngai, P. K., Scott-Woo, G. C , Lim, M. S., Sutherland, C., and Walsh, M. P. (1987). J. Biol. Chem. 262, 5352-5359. 14. Deleted in proof. 15. Takahashi, K., Hiwada, K., and Kokubu, T. (1986). Biochem. Biophys. Res. Commun. 141, 20-26. 16. Takahashi, K., Hiwada, K., and Kokubu, T. (1988). Hypertension 11, 620-626. 17. Winder, S. J., and Walsh, M. P. (1990). J. Biol. Chem. 265, 10148-10155. 18. Vancompemolle, K., Gimona, M., Herzog, M., Van Damme, J., Vandekerckhove, J., and Small, V. (1990). FEBS Lett. 274, 146-150. 19. Abe, M., Takahashi, K , and Hiwada, K. (1990). J. Biochem. (Toyko) 107, 507-509. 20. Marston, S. B. (1991). FEBS Lett. 292, 179-182. 21. Winder, S. J., Kargacin, G. J., Bonet-Kerrache, A. A., Pato, M. D., and Walsh, M. P. (1992). Jpn. J. Pharmacol. 58(SuppL II), 298-348. 22. Takahashi, K., Hiwada, K., and Kokubu, T. (1987). Life Sci. 41, 291-296. 23. Draeger, A., Gimona, M., Stuckert, A., Cells, J. E., and Small, J. V. (1991). FEBS Lett. 291, 2 4 - 2 8 . 24. Takeuchi, K., Takahashi, K , Abe, M., Nishida, W., Hiwada, K., Nabeya, T., and Maruyama, K. (1991). J. Biochem. (Tokyo) 109, 311-316. 25. Gimona, M., Herzog, M., Vandekerckhove, J., and Small, J. V. (1990). FEBS Lett. 274, 159-162. 26. Birukov, K. G., Stepanova, O. V., Nanaev, A. K , and Shirinsky, V. P. (1991). Cell Tissue Res. 266, 579-584. 27. Winder, S. J., and Walsh, M. P. (1990). Biochem. Int. 22, 335-341. 28. Takahashi, K , and Nadal-Ginard, B. (1991). J. Biol. Chem. 266, 13284-13288. 29. Takahashi, K., Abe, M., Hiwada, K., and Kokubu, T. (1988). J. Hypertens. 6, S40-S43. 30. Winder, S. J., Sutherland, C , and Walsh, M. P. (1991). In "Regulation of Smooth Muscle Contraction" (R. S. Moreland, ed.), pp. 37-52. Plenum, New York. 31. Winder, S. J., Walsh, M. P., Vasulka, C , and Johnson, J. D. (1993). Biochemistry 32, 13327-13333. 32. Grand, R. J. A., and Perry, S. V. (1978). FEBS Lett. 92, 137-142. 33. Watterson, D. M., and Sharief, F., and Vanaman, T. C. (1980). J. Biol. Chem. 255, 962-975. 34. Barany, M. (1967). J. Gen. Physiol. 50, 197-218. 35. Walsh, M. P., Hinkins, S., Dabrowska, R., and Hartshome, D. J. (1983). Methods Enzymol. 99, 279-288. 36. Ikebe, M., and Hartshome, D. J. (1985). Biochemistry 24, 2380-2387. 36a. Winder, S. J., Sutherland, C , and Walsh, M. P. (1992). Biochem. J. 288, 733-739. 37. Abe, M., Takahashi, K., and Hiwada, K. (1990). J. Biochem. (Tokyo) 108, 835-838. 38. Horiuchi, K. Y., and Chacko, S. (1991). Biochem. Biophys. Res. Commun. 1 7 6 , 1 4 8 7 1493. 5. 6. 7. 8.

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39. Makuch, R., Birukov, K , Shirinksy, V., and Dabrowska, R. (1991). Biochem. J. 280, 33-38. 40. Nishida, W., Abe, M., Takahashi, K., and Hiwada, K. (1990). FEBS Lett. 268, 165-168. 41. Takahashi, K , Hiwada, K., and Kokubu, T. (1987). Hypertension 10, 360a. 42. Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., and Hartshome, D. J. (1978). Biochemistry 17, 4411-4418. 43. Grand, R. J. A., Perry, S. V., and Weeks, R. A. (1979). Biochem. J. 177, 521-529. 44. Barbacid, M. (1987). Annu. Rev. Biochem. 56, 779-827. 45. Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C , John, J., and Wittinghofer, A. (1989). Nature (London) 341, 209-214. 46. Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988). J. Biol. Chem. 263, 2897-2904. 47. Krebs, E. G., (1985). Biochem. Soc. Trans. 13, 813-820. 48. Hunter, T., and Sefton, B. M. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 1311-1315. 49. Naka, M., Kureishi, Y., Muroga, Y., Takahashi, K., Ito, M., and Tanaka, T. (1990). Biochem. Biophys. Res. Commun. 171, 933-937. 50. Winder, S. J., Pato, M. D., and Walsh, M. P. (1992). Biochem. J. 286, 197-203. 51. Pato, M. D., and Adelstein, R. S. (1980). J. Biol. Chem. 255, 6535-6538. 52. Barany, M., Rokolya, A., and Barany, K. (1991). FEBS Lett. 279, 65-68. 53. Pohl, J., Walsh, M. P., and Gerthoffer, W. T. (1991). Biophys. J. 59, 58a. 53a. Winder, S. J., Allen, B. E., Fraser, E. D., Rang, H.-M., Kargacin, G. J., and Walsh, M. P. (1993). Biochem. J. 296, 827-836. 54. DeFeo, T. T., and Morgan, K. G. (1985). J. Physiol. {London) 369, 269-282. 55. Williams, D. A., and Fay, F. S. (1986). Am. J. Physiol. 250, C779-C791. 56. Williams, D. A., Becker, P. L., and Fay, F. S. (1987). Science 235, 1644-1648. 57. Morgan, J. P., and Morgan, K. G. (1982). Pfluegers Arch. 395, 75-77. 58. Takuwa, Y., Takuwa, N., and Rasmussen, H. (1987). Am. J. Physiol. 253, C817-C827. 59. Stull, J. T., Nunnally, M. H., and Michnoff, C. H. (1986). Enzymes 17, 113-166. 60. Huang, K.-P., Huang, F. L., Nakabayashi, H., and Yoshida, Y. (1988). J. Biol. Chem. 263, 14839-14845. 6L Marais, R. M., and Parker, P. J. (1989). Eur. J. Biochem. 182, 129-137. 62. Paul, R. J. (1989). Annu. Rev. Physiol. 51, 331-349. 63. Walsh, M. P. (1990). In "Progress in Clinical and Biological Research, Vol. 327, Frontiers in Smooth Muscle Research" (N. Sperelakis and J. D. Wood, eds.), pp. 127-140. Wiley-Liss, New York. 64. Sobue, K., and Sellers, J. R. (1991). J. Biol. Chem. 266, 12115-12118. 65. Marston, S. B., and Redwood, C. S. (1991). Biochem. J. 279, 1-16. 66. Hemric, M. E., and Chalovich, J. M. (1988). J. Biol. Chem. 263, 1878-1885. 67. Ikebe, M., and Reardon, S. (1988). J. Biol. Chem. 263, 3055-3058. 68. Sutherland, C , and Walsh, M. P. (1989). J. Biol. Chem. 264, 578-583. 69. Hai, C.-M., and Murphy, R. A. (1989). Annu. Rev. Physiol. 51, 285-298. 70. Haeberle, J. R., Hathaway, D. R., and Smith, C. L. (1992). J. Muscle Res. Cell Motil. 13, 81-89.

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CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Type III Cyclic Nucleotide Phosphodiesterases and Insulin Action VINCENT C. MANGANIELLO* EVA DEGERMAN^ M A S A T O TAIRA*,t

TETSURO KONO^ PER BELFRAGE^ * Laboratory of Cellular Metabolism, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 "^ Second Department of Medicine, Chiba University Medical School, Chiba, Japan ^Department of Medical and Physiological Chemistry, University of Lund Medical School, Lund, Sweden '^ Vanderbilt University School of Medicine, Nashville, Tennessee 37240

I. Introduction cAMP and cGMP are important intracellular second messengers which mediate cellular responses to various extracellular stimuli or signals, including light and a number of hormones, peptides, neurotransmitters, autocoids, therapeutic agents, etc. The adenylyl and guanylyl cyclases (adenylate and guanylate cylases) serve as biological transducers which generate cAMP and cGMP in response to these extracellular stimuli. Initiation of cyclic nucleotide-regulated intracellular signals usually involves interaction with and activation of protein kinases by cAMP and cGMP. Activated kinases catalyze phosphorylation of specific serine/threonine residues in specific protein effectors and initiate the phosphorylation/dephosphorylation cascades that produce cyclic nucleotide-mediated biological responses (1,2). This concept, that virtually all effects of cyclic nucleotides are mediated by protein kinases, may be overly simplistic, however, given the discovery of cyclic nucleotide-gated ion channels in rod photoreceptors and olfactory epithelia and noncatalytic cGMP-binding sites of some cyclic nucleotide phosphodiesterases (3-7). Once formed, cAMP and cGMP either bind to protein kinases (or other receptor/effector proteins) or are hydrolyzed to their respective 5'63

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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VINCENT C. MANGANIELLO et al.

mononucleotides by cyclic nucleotide phosphodiesterases (PDEs). Since PDEs represent the only known mechanism for destruction of cyclic nucleotides, they are very important determinants of cyclic nucleotide concentrations and, consequently, of the biological responses mediated by these important second messengers (8-17).

II. Cyclic Nucleotide PDE Gene Families It is now clear that hydrolysis of intracellular cAMP and cGMP is a highly regulated process that is not dependent on a single PDE, but rather on the coordination and integration of the activities of a diverse and complex group of structurally related PDE isoenzymes. Seven major mammalian PDE gene families with distinctive properties have been identified (8). This chapter concentrates on one, the Type III cGMPinhibited (cGI) PDE gene family, with emphasis on its hormonal regulation and importance in certain actions of insulin. For a more comprehensive review of the different PDE families, see Ref. (8a). The different PDE gene families were named and classified by virtue of their most salient characteristics, i.e., their different substrate affinities, responses to specific effectors, sensitivity to selective inhibitors, structural and biochemical properties, immunological epitopes, and regulatory mechanisms (Table I) (8-17). Multiple types of PDEs are usually found in individual cells where they are differentially regulated and expressed in amounts, proportions, and subcellular locations. In some cells and tissues, however, specific PDEs are found in high concentrations. Because the different PDE isoenzyme families are subject to different modes of regulation in intact cells and because of quantitative uncertainties in determining the total complement of PDEs and distribution of every PDE in individual cells or tissues, it is not possible to relate activity measurements of various PDE types in broken cells to intracellular activities and cyclic nucleotide concentrations. Most, but not all. Type I calcium-calmodulin (Ca2^-CaM)-sensitive PDEs exhibit a higher affinity for cGMP than cAMP; all are activated by Ca^^-CaM complexes (Table I) (18). Although Type I PDEs are widely distributed, immunological studies indicate that in mouse brain CaM PDEs are present in relatively high concentrations in the dendritic fields of cerebellar Purkinje cells and neocortical and hippocampal pyramidal cells (19). Type II PDEs hydrolyze both cAMP and cGMP with positively cooperative kinetics; at subsaturating cyclic nucleotide concentrations hydrolysis of one cyclic nucleotide can be stimulated by the other (Table I) (20-22). cGMP is, however, preferred as both substrate and allosteric effector. Binding of cGMP to allosteric sites induces con-

~

CelVtissue

Intracellu

TABLE I PROPERTIES OF PDE GENEFAMILIES"

CNS (Purkinje cells, pyramidal cells)

Family

CaM sensitive 8-Methoxymethyl-IBMX

Selective inhibitors

I

Ca2+,calmodulin phosphorylation kinase, CAM kin cGMP

Type

I1 I11

CAMP(regulates g expression, i.e., protein amount cGMP transducin phos by G kinase

cGMP phosphorylation kinase, insulinkinase

Adrenal cortex (glomerulosa), CNS Platelets, liver, adipose tissue, myocardium, smooth muscle

IV V and VI

Muscle, lymphocytes

CNS, smooth muscle, inflammatory cells, kidney, testis Retina, smooth muscle, platelets

cGMP stimulated No selective inhibitors cGMP inhibited cGMP Inotropidvasodilator drugs, e.g., milrinone, enoximone, indolidan, cilostamide CAMPspecific RO 20-1724, rolipram denbufylline gCMP specific V. cGMP binding VI. Photoreceptor Zaprinast, dipyridamole CAMPspecific

VII

Information was compiled from Refs. (8-17). CNS, central nervous system; FSH, follicle-stim

66

VINCENT C. MANGANIELLO et al,

formational changes resulting in activation at the catalytic site. Based on studies with purified enzymes, at prevailing intracellular concentrations of cAMP and cGMP, increases in cGMP could activate Type II PDE and increase hydrolysis of cAMP in cells containing Type II PDEs. For this reason, Type II PDEs have been designated as cGMPstimulated PDEs (cGS PDEs) (20-22). Zona glomerulosa cells of the adrenal cortex are very highly enriched in one Type II cGS PDE (23). Type III PDEs have high affinities for both cAMP and cGMP « 1 /^M), with y^ax for cAMP --4-10 times that for cGMP (Table I) (24-26). Type III PDEs are selectively and specifically inhibited by a number of recently developed drugs with positive inotropic and antiplatelet aggregating activities (10-13,24-29). As might be expected, cGMP is a competitive inhibitor of cAMP hydrolysis by Type III PDEs, and for this reason these PDEs are designated "cGMP-inhibited PDEs" (cGI PDEs) or "cGMP-inhibited low K^ cAMP PDEs." Inhibition of cAMP hydrolysis by cGMP also distinguishes Type IIIfi:-omType IV or cAMP-specific PDEs, which exhibit a low K^ ( - 1 - 3 /xM) for cAMP but are not inhibited by cGMP (Table I) (9-11,30,31). Type IV PDEs hydrolyze cGMP poorly and, hence, are called cAMP-specific PDEs. These PDEs are specifically and potently inhibited by RO 20-1724 and roHpram (10-14,30-35). Type V PDEs are classified as cGMP-specific PDEs because they readily hydrolyze cGMP, but exhibit a low affinity for cAMP and hydrolyze cAMP poorly (Table I) (36,37). Photoreceptor Type VI PDEs are present at high concentrations (estimated as much as 30 ixM) in retinal rod and cone outer segments. These enzymes, which hydrolyze cGMP in response to light, play a central role in visual transduction. Light-activated rhodopsin activates the retinal guanine nucleotide-binding protein transducin, which interacts with and activates Type VI photoreceptor PDEs leading to hydrolysis of cGMP, closure of cGMP-gated cation channels, and hyperpolarization of the outer segment cell membrane (37). Type V cGMP-specific PDEs have been isolated from lung and platelets (7,38,39). Type V and VI PDEs contain cGMP-binding sites distinct from substrate sites (7,36-41). These nonsubstrate binding sites may promote interactions of Type V and VI PDEs with other regulatory proteins, e.g., of lung Type V PDE with cGMP-dependent protein kinase (39) and photoreceptor Type VI PDE with its inhibitory subunit (42). Type V and VI cGMP-specific PDEs are selectively inhibited by dipyridamole and zaprinast (36,39,43,44). PDE VII, which is structurally, biochemically, and pharmacologically distinct from previously known PDEs, was identified and isolated by genetic complementation of cAMP PDE-deficient yeast. PDE VII, whose mRNA is expressed in human

67

TYPE III PDE AND INSULIN ACTION

skeletal muscle, has a high affinity for cAMP, does not hydrolyze cGMP, and is not inhibited by cGMP or Type III or IV PDE inhibitors (68). Analysis of amino acid sequences of purified PDEs or deduced sequences of cloned PDE cDNAs indicates that the seven PDE gene families are products of distinct but related genes (45,46). PDEs exhibit a common structural pattern, all containing a conserved catalytic domain (—25-40% amino acid identity) of —270 amino acids (usually found in their C-terminal regions) and divergent N-terminal regulatory domains (Fig. 1) (46-65). In these regulatory domains putative Ca^'^-CaM-binding sites, membrane-association domains, and phosphorylation sites as well as allosteric, nonsubstrate cGMP-binding sites are found. Cterminal prenylation (at a CAXX consensus sequence) of photoreceptor type V cGMP PDEs is presumably important in association of these cGMP PDEs with rod and cone outer segment membranes (66,67). Each PDE family apparently comprises at least two subfamilies which may be products of distinct but closely related genes (with up to 70-80% amino acid identity) or arise by alternative splicing of mRNA, perhaps related to tissue-specific distribution of isoenzyme subfamilies. More than 20 different PDE isoenzymes belonging to seven major gene fami-

^ Regulatory Domain I. CAM Binding Sites A-Kinase, CAM Kinase Phosphorylation Sites II. Non-Catalytic Cyclic Nucleotide (cGMP) Binding Sites III. Hydrophobic Membrane-Association (Dimerization) Domains A-Kinase, Insulin-Sensitive Kinase Phosphorylation Sites V. Non-Catalytic cGMP Binding Sites G-Kinase Phosphorylation Sites Prenylation (C-Terminal) Sites

^ Conserved Catalytic Domain • Different Substrate Affinities • Different Inhibitor Sensitivities, i.e. I. 8-Methoxymethyl-IBMX III. Inotropes/Vasodilators (Milrinone, Enoximone) IV. RO 20-1724, Rolipram V. Zaprinast

FIG. 1. Common structural pattern of PDE isoenzymes. The seven PDE gene families exhibit a common domain organization—all share a conserved catalytic domain and divergent regulatory domains. Reproduced with permission from Degerman, E., Leroy, M.-J., Taira, M., Belfrage, P., and Manganiello, V. C. (1995). In "^Diabetes Mellitus: A Fundamental and Clinical Text" (D. LeRoith, J. M. Olefsky, and S. Taylor, eds.). Lippincott, Philadelphia.

68

VINCENT C. MANGANIELLO et al.

lies have been identified by biochemical, pharmacological, and molecular cloning approaches (45-65,68) (Table I). The rich diversity and heterogeneous tissue and cellular distribution of the several PDE families has generated considerable interest in the development of PDE family-specific inhibitors, both as probes to assess function of specific PDE isoenzymes in cells and tissues and as potential therapeutic agents (Table I). In canine tracheal smooth muscle preparations, inhibitors of Type III and Type IV PDEs, SKF94120 and RO 201724, respectively, potentiated isoproterenol-induced cAMP accumulation and relaxation (69-71) but did not alter the effects of nitroprusside. On the other hand, zaprinast, a Type V and VI PDE inhibitor, potentiated nitroprusside-induced accumulation of cGMP and relaxation. In perfused heart preparations and intact mammals. Type III PDE inhibitors are positive inotropic agents (12,27-29,72-75). In brain cerebral cortex slices. Type IV PDE inhibitors, RO 20-1724 and rolipram, but not Type III or Type V PDE inhibitors, potentiated isoproterenol-induced cAMP accumulation (12,76). In eosinophils. Type IV inhibitors rolipram and denbufylline, but not Type III or Type V inhibitors, inhibited superoxide anion formation and potentiated isoproterenol-induced cAMP accumulation (14-17,77,78). Simultaneous inhibition of both Type III and Type IV PDEs can enhance some biological responses (12,14,7982). TVpe III PDE inhibitors OPC 3911, CI-930, and milrinone were potent relaxants of contracted rat aorta, whereas the Type IV inhibitor rolipram was not. At low concentrations of OPC 3911, however, rolipram produced synergistic effects on relaxation (79). Although both Type III and Type IV inhibitors alone induced bronchodilation in intact guinea pigs and relaxation of isolated tracheal smooth muscle preparations, combinations of Type III and Type IV inhibitors produced synergistic effects (80). In human basophils in which Type IV inhibitors alone, but not Type III inhibitors, reduced antigen-stimulated mediator release. Type III inhibitors (but not Type V inhibitors) potentiated the effects of Type IV inhibitors (81). In isolated human T lymphoc3^es (82) and cultured pig aortic smooth muscle cells (83), combinations of Type III and Type IV inhibitors [but not Type V inhibitors (83)] produced more than additive inhibitory effects on DNA synthesis, most likely mediated by increased cAMP. These results suggest that, in cells and tissues, not only the expression and location/distribution of individual PDE isoenzymes but also their contribution to total cyclic nucleotide hydrolysis might be important factors in regulation of cyclic nucleotide responses. In the case of T lymphocytes, it is of interest that the Type IV cAMP-specific enzyme is thought to be predominantly soluble, whereas the Type III cGI PDE is particulate (82).

TYPE III PDE AND INSULIN ACTION

69

Specific and selective inhibitors of tissue-specific PDE isoenzymes could provide potent therapeutic agents without the side effects of nonselective PDE inhibitors (10-17,27-29). In general, although most of the specific inhibitors exhibit at least 20-fold selectivity for individual PDE isoenzyme families, their specificity is not absolute. It is also uncertain whether all of the biological effects of these compounds are related to PDE inhibition. Finally, none of the known inhibitors distinguishes among individual members of the same PDE gene family—an issue of paramount concern if inhibition of a specific PDE in a specific cell or tissue is to be targeted for therapeutic benefit. Nevertheless, Type IV PDE specific inhibitors such as rolipram are being evaluated as potential therapeutic agents for allergic/inflammatory disorders, such as asthma, since Type IV cAMP-specific PDEs are the predominant PDEs and play key roles in regulation of cAMP concentrations in a number of inflammatory cells (12,14-17,81). Type III cGI PDE-specific inhibitors such as milrinone and enoximone, which are very potent positive inotropic agents, are used therapeutically for short-term control of certain types of refractory cardiac failure (27-29,84). As indicated in Table I, PDEs, and therefore cyclic nucleotide concentrations, are directly or indirectly affected by a variety of hormones and regulatory signals, i.e., Ca^^, cyclic nucleotides, several different protein kinases, receptor tyrosine kinases, cAMP-regulated gene expression, and others not listed. In cells and tissues containing multiple PDEs, the different regulatory properties of the PDE gene families as well as their heterogeneous distribution and expression allow for intergration of multiple physiological regulatory signals and "fine tuning" of cyclic nucleotide concentrations and biological responses. Differences in expression and subcellular location could confer physical or functional compartmentalization of cyclic nucleotide-mediated processes. PDEs also serve as a locus for "cross-talk" between different second-messenger systems. In some cells, regulation of cyclic nucleotide concentrations by calcium is mediated in part via Type I CaM-sensitive PDEs. In 1231N1 human astrocytoma cells, muscarinic cholinergic agents activate a Type I PDE and increase cAMP degradation severalfold, presumably by increasing cell Ca^^, thus inhibiting isoproterenolinduced cAMP accumulation (85-87). It has been suggested that inhibition of cAMP-induced steroidogenesis by ANF in bovine adrenal glomerulosa cells is related, in part, to activation of the Type II cGS PDE and reduction in cAMP. In these cells, ANF activates guanylyl cyclase, increasing cGMP which then may activate the Type II cGS PDE and increase cAMP hydrolysis (23). Similarly, in cultured PC 12 cells, ANF- and sodium nitroprusside-induced increases in cGMP, pre-

70

VINCENT C. MANGANIELLO et aL

sumably by activating a Type II PDE, increase cAMP hydrolysis and counteract adenosine-mediated activation of adenylyl cyclase and cAMP accumulation (88). It has been suggested that nitrovasodilators potentiate inhibitory effects of prostaglandins on aggregation of rabbit platelets by activating guanylyl cyclase and increasing cGMP which in turn inhibits the Type III cGI PDE, leading to increases in cAMP and inhibition of aggregation (89). In hepatocytes and adipocytes, insulin, via activation of the insulin receptor tyrosine kinase, initiates a series of reactions resulting in activation of the Type III cGI PDE and a decrease in cAMP (24,90). The evolutionary significance of the diversity in structure, function, expression, and regulation of the PDE gene families is not understood. It is possible that development of alternative or redundant cyclic nucleotide hydrolytic pathways (Table I) as well as divergence of regulatory domains (Fig. 1) conferred selective advantages as well as enhanced homeostatic control mechanisms in complex organisms. By virtue of their distinct biochemical, pharmacological, and regulatory properties and differences in tissue and subcellular distributions, different PDE isoenzyme families can play predominant roles in regulating cyclic nucleotide concentrations in different cells and tissues and, therefore, in pathophysiology. For example, mutations in the Type VI photoreceptor cGMP-specific PDE gene have been implicated in expression of the retinal degeneration phenotype of the rd mouse, an animal model for human retinitis pigmentosa. Expression of functional bovine Type VI cGMP PDE subunit in transgenic rd mice resulted in partial or complete rescue of the rd phenotype (91-93).

III. Type III cGMP-lnhibited Cyclic Nucleotide Phosphodiesterases A. Purification and Characterization of Type III cGI PDEs

Type III cGI PDEs have been purified from a number of tissues, including rat and bovine adipose tissue (25,94), bovine cardiac ventricle (26), human platelets (95,96) and placenta (105), rat liver (97,98), and bovine aortic smooth muscle (99). All exhibit similar characteristic properties (24). K^ values for both cAMP and cGMP are in the 0 . 1 0.8 /xM range; Vj^ax for cAMP (2-9 /xmol min/mg) is, however, higher (4- to 10-fold) than that for cGMP (0.3-2 /xmol min/mg) (Table II). Another defining feature of Type III cGI PDEs is their sensitivity to inhibition by a number of drugs with positive inotropic and antithrombotic actions, including OPC 3689 (cilostamide), OPC 3911, cilostazol, and other OPC derivatives, milrinone, enoximone, CI-930 (imazodan).

K m

Property

(fl)

Catalytic CAMP cGMP V,, (pmoYmin per milligram) CAMP cGMP ) Inhibitors ( f l ICmb OPC-3911 Milrinone (21-930 RO 20-1724 cGMP From Ref. (24).

nmol/min/mg.

0.4 0.3

2.5 1.6

0.3 0.8

0.054 0.40 0.25 3w 0.25

3.1 0.3

0.16 0.09

Bovine aortic smooth muscle (99)

0.005 0.26d 62d 0.06d

6.0 0.6

0.15 0.10

Bovine heart (26)

TABLE I1 PROPERTIES OF TYPE111 cGI PDEs"

8.5 2.0

0.06 2.2 0.6 1100 0.6

Bovine adipose tissue (94)

0.04 0.6 0.4 190 0.2

by 50%.

Rat adipose tissue (25)

enzyme activity at PHICAMP<

f l was less than 20%.

* ICs0,Concentration which inhibited K,.

The inhibition obtained with 30

72

VINCENT C. MANGANIELLO et al.

LY195115 (indolidan), Y-590, anagrelide, and ICI233188 (10-13, 24-29,72-75,94-103) (Tables I and II). Studies with these specific inhibitors suggest that Type III cGI PDEs regulate cAMP pools important in myocardial contractility, platelet aggregation, vascular smooth muscle relaxation, and lipolysis. As indicated in Table II, the IC50 values for inhibition of several Type III cGI PDEs were generally <0.1 /xM for cilostamide or OPC 3911, and about 0.5 /xM for milrinone and CI930; RO 20-1724, a specific inhibitor of Type IV cAMP-specific PDEs, did not effectively inhibit Type III cGI PDEs. As might be predicted from ifm values for cAMP and cGMP, cGMP competitively inhibited cAMP hydrolysis by Type III cGI PDEs with IC50 values of 0.1-0.6 /xM. There is some evidence that inhibition of Type III cGI PDEs by endogenously produced cGMP is of physiological consequence. Inhibition of rabbit platelet aggregation by nitrovasodilators (which increase cGMP via activation of guanylyl cyclase) may be in part related to inhibition of platelet Type III cGI PDE by cGMP, which leads to an increase in cAMP (89). A third important general characteristic of Type III cGI PDEs involves short-term regulation of these PDEs by hormones. In intact adipocytes, hepatocytes, and platelets, incubation with hormones that increase cAMP increases Type III cGI PDE activity (24,90). This increased activity is thought to be important in "feed-back" regulation of cAMP content and biological processes initiated by hormonal activation of adenylyl cyclase. In adipoc5rtes and hepatocytes, insulin reduces hormone-stimulated cAMP accumulation and cAMP-dependent protein kinase activities at least in part by activating Type III cGI PDEs (24,90). On the other hand, in frog ventricular muscle, glucagon (a positive inotropic agent) is reported to inhibit a membrane-associated Type III cGI PDE via a pertussis toxin-sensitive G protein (104). Attempts to purify Type III cGI PDEs have been hampered by their low tissue abundance, lability, and sensitivity to proteolysis. With the development of specific and selective inhibitors of Type III cGI PDEs, the A/'-(2-isothiocyanato)ethyl derivative of cilostamide (CIT) was synthesized and coupled to aminoethylagarose as an affinity matrix (CITagarose) for purification of Type III cGI PDEs from several tissues, including rat and bovine adipose tissue (25,94), bovine aortic smooth muscle (99), and human platelets (96) and placenta (105). Other types of procedures have been used to purify this enzyme from myocardium (26), platelets (95), and liver (97,98). Even in the presence of protease inhibitors during purification, however, virtually all enzjrme preparations contain degraded PDE products.

TYPE III PDE AND INSULIN ACTION

73

The rat adipocyte Type III cGI PDE was solubilized and purified from particulate fractions (100,000 g) from rat epididymal fat pads (25). The PDE could be partially solubilized by freeze-thawing in the presence of high salt concentrations or by sonification in the presence of nonionic detergents. A combination of moderate salt concentrations, nonionic detergent, and 20% glycerol solubilized in maximal yield and stabilized adipoc3d:e particulate Type III cGI PDE activity. Following partial purification by chromatography on DEAE-Sephacel and Sephadex G-200, a homogeneous preparation (a 62/66-kDa doublet on SDSPAGE) was obtained by chromatography on CIT-agarose (25). Although the rat enzyme was obtained in relatively good yield (20%, purified ~65,000-fold from the initial particulate fractions), only a few micrograms of enzyme protein was obtained from 900 fat pads. To prepare enough pure adipose tissue enzyme for antibody production, Type III cGI PDE was purified to homogeneity (77- and 61/63-kDa polypeptides) from bovine omental adipose tissue (94). Using CIT-agarose chromatography, Type III cGI PDEs have also been purified from human platelets (96) and placenta (105) and bovine aortic smooth muscle (99). Based on SDS-PAGE and reactivity with anti-bovine adipose, anti-human platelet, and monoclonal anti-bovine cardiac Type III cGI PDE antibodies on Western blots, these purified enzyme preparations contained polypeptides of -30-138 kDa. Type III cGI PDEs have been purified from several sources without chromatography on CIT-agarose. The human platelet enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Blue dextran-Sepharose, yielding a polypeptide of 61 kDa (95). Type III cGI PDEs have been purified from bovine cardiac tissue (80-, 67-, and 60-kDa polypeptides) (26) and from rat liver "dense vesicles" (57-kDa polypeptide) (97) by different chromatographic procedures, all of which included Blue dextran-Sepharose as an important step. An hepatic Type III cGI PDE (identified as a 73-kDa polypeptide), different from the 57-kDa form, has been purified from rat liver after solubilization from particulate fractions by limited proteolytic digestion with chymotrypsin (98). Given the apparent sensitivity of Type III cGI PDEs to proteolysis, it is not surprising that the molecular weights of the native enzyme(s) are uncertain. Specific anti-Type III cGI PDE antibodies have been utilized to rapidly isolate Type III cGI PDEs by immunoprecipitation. Based on SDS-PAGE of immunoprecipitated Type III cGI PDEs, the molecular mass of native monomeric forms from intact rat adipocytes (106,107) and cardiac tissue from several species (110) have been estimated at -130-135 kDa; from placenta (105), -138 kDa; from platelets

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VINCENT C. MANGANIELLO et al,

(108,109) and aorta (99), -105-110 kDa. It appears that purified Type III cGI PDEs are predominantly proteolytic fragments of larger native forms. The rat liver "dense vesicle" cGI PDE has a smaller monomeric Mr (—57,000) (97) than those estimated for native platelet, cardiac, aorta, or adipose tissue Type III cGI PDEs (11,24,120). This could reflect proteolysis or the existence of a liver-specific cGI PDE. Although the state of association of intracellular Type III cGI PDEs has not been completely defined, dimeric forms of platelet (95), cardiac (9,26), aortic (99), and rat liver (97,98) Type III cGI PDEs have been reported. B. Molecular Cloning of Two Type III cGI PDE Subfamilies

cDNAs encoding two distinct but related Type III cGI PDEs have been cloned from rat and human adipose and human cardiac cDNA libraries— R(rat)cGIPl, RcGIP2, H(human)cGIPl, HcGIP2 (Fig. 2) (55,56; Taira, Kedev et al., 1994). These cDNAs encode proteins with Mr values of —123,000-125,000, consistent with estimated monomer Mr values for phosphorylated Type III cGI PDEs in intact adipocytes and adipocyte and cardiac muscle microsomal preparations. The deduced primary amino acid sequences contain the structural domain pattern common to all PDEs, with the catalytic domain conserved among all mammalian PDEs in the C-terminal region. The N-terminal regulatory domain contains several consensus cAMP-dependent protein kinase phosphorylation sites (consistent with phosphorylation of Type III cGI PDEs by cAMP-dependent protein kinase) and a hydrophobic putative membrane association domain. The conserved domain is followed by a hydrophilic C-terminal domain. The deduced sequence of RcGIPl is more closely related to that of HcGIPl than RcGIP2, which is more closely related to HcGIP2 (Fig. 2). Within the conserved domain of the four cGI PDEs there is an insertion of 44 amino acids (the "additional region") which does not align with sequences within the conserved domains of other PDE families. The sequence of the insertion is very similar in RcGIPl and HcGIPl and differs from those in RcGIP2 and HcGIP2, which are very similar to each other (55,56; unpublished observations). It is possible that this insertion not only differentiates Type III cGI PDEs from other PDE gene families, but is also important in the identification of individual members or subfamilies within the Type III cGI PDE gene family. RcGIPl and HcGIPl have very httle similarity to RcGIP2 and HcGIP2 in the regulatory domains. All four Type III cGI PDEs have very similar hydropathy plots, consistent with similar structural and functional domains (55,56; Taira, Kedev, et al., 1994). RcGIPl mRNA is highly expressed in rat adipocytes and increases dramatically during differentiation of 3T3-L1 adipocytes (56). Multiple

75

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200 aa FIG. 2. Domain organization and percentage amino acid identities of four Type III cGI PDEs. Four Tjrpe III cGI PDEs representing two different subfamilies have been cloned. There is greater conservatior with the same subfamily in rat and human species than between different subfamilie in the same species. All exhibit the domain organization common to all PDE families. The conserved catalytic region is followed by a hydrophilic C-terminal domain. The N-terminal region contains a hydrophobic putative membrane association domain and several consensus sequences (RRX S/T) for cAMP-dependent protein kinase phosphorylation sites. Numbers refer to the percentage amino acid identities in the different domains. The bars are drawn to scale in which 200 aa represents 200 amino acids. Reproduced with permission from Degerman, E., Leroy, M.-J., Taira, M., Belfrage, P., and Manganiello, V. C. (1995). In ''Diabetes Afe//i^i/s; A Fundamental and Clinical Text" (D. LeRoith, J. M. Olefsky, and S. Taylor, eds.). Lippincott, Philadelphia.

RcGIP2 mRNA species have been detected in Northern blots of total RNA from rat heart, aorta, adipose tissue, and lung (56; Taira, Kedev, et al.y 1994). These results are consistent with the conclusion that RcGIPl represents the hormone-sensitive Type III cGI PDE found in adipocytes and RcGIP2, a cardiovascular Type III cGI PDE. There is apparently more similarity between the same subfamilies in rat and human species than there is between different subfamilies in the same species.

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VINCENT C. MANGANIELLO et al.

C. Hormonal Regulation of Type III cGI PDEs

Effects of hormones on cyclic nucleotide PDE activities have been intensely studied in adipose tissue and liver. In 1970, Loten and Sneyd first reported that incubation of intact rat adipocytes with insulin resulted in increased hydrolysis of cAMP as measured in adipocyte homogenates (111). The insulin-sensitive PDE was found to be primarily associated with particulate fractions of rat or 3T3-L1 adipocytes; these fractions exhibited some but not all characteristics of the endoplasmic reticulum (112-116). Kono and co-workers have consistently reported that incubation of intact adipocytes with insulin results in a 2.5- to 3-fold activation of microsomal Type III cGI PDE activity; others, including us, have generally reported somewhat less (—1.4 to 1.6-fold) activation. Incubation of adipocytes with agents that increase cAMP or with cAMP analogs (115-118) also rapidly increased activity (by as much as 100% within 2-5 min) of a particulate PDE (25,118) that exhibited substrate affinities, kinetic properties, and inhibitor sensitivities (25,118,119) of the Type III cGI PDEs (24,55,56,120). This activation by cAMP is most likely mediated via cAMP-dependent protein kinase. In adipoc3d:es, the concentration dependency for isoproterenol-induced activation of particulate Type III cGI PDE is very similar to that for activation of hormone-sensitive lipase and stimulation of lipolysis (121). In adipocytes exposed to 8-p-chlorothiophenyl-cAMP (which activates cAMP-dependent protein kinase), cAMP content decreased in parallel with increased PDE activity and lipolysis (122). In broken cell systems, cAMP-dependent protein kinase can phosphorylate and activate rat adipocyte particulate cGI PDE (123-125). Thus, in adipocytes there is apparently a very close relationships between isoproterenol-induced activation of adenylyl cyclase, the particulate Type III cGI PDE, and lipolysis, presumably a tight functional coupling via activation of cAMPdependent protein kinase. Combinations of insulin and agents that increase cAMP have been reported to produce synergistic (121), additive (115), or less than additive (126) effects on adipocyte particulate PDE activity. In the last study, the effect of insulin alone (~2.7-fold increase in microsomal PDE activity) exceeded that of isoproterenol alone (~2-fold). Like adipocytes, isolated hepatocytes responded to insulin or glucagon with increased cAMP phosphodiesterase activity (127-129); the time courses and hormone concentration dependencies for PDE activation were similar in both (147). In liver, however, insulin alone was reported to stimulate a peripheral plasma membrane enzyme with characteristics of a Type IV, rolipram-inhibited cAMP-specific PDE

TYPE III PDE AND INSULIN ACTION

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(128,130,131), whereas both insuUn and glucagon activated a Type III cGI PDE associated with the Golgi network (132) or with an incompletely characterized liver microsomal fraction designated as dense vesicles (90,97,98,128,130,131). Activation of hepatocyte Type III cGI PDE by cAMP, like that in adipocytes, is most likely mediated by cAMPdependent protein kinase. In hepatocytes exposed to glucagon or 8-chlorothiophenyl-cAMP, cAMP content decreased in parallel with increases in cAMP-dependent protein kinase, cAMP PDE, and phosphorylase activities (122,133). In broken cell systems, cAMP-dependent protein kinase has been reported to phosphorylate and activate the dense vesicle Type III cGI PDE (134). In intact platelets in which specific Type III cGI PDE inhibitors such as cilostamide inhibit platelet aggregation, presumably by increasing cAMP (100,135), insulin (136) and agents that increase cAMP (108,109,137) activate platelet Type III cGI PDE. In contrast to liver and adipocyte enzymes, however, the platelet Type III cGI PDE is predominantly cytosolic (95,100,108,109). Although it seems paradoxical that both hormones which activate adenylyl cyclase and insulin (which can reduce cAMP) activate the same Type III cGI PDE and increase cAMP hydrolysis, activation of Type III cGI PDE may reflect at least part of the mechanism whereby these hormones regulate cAMP-dependent protein kinase. Although activation of the adipocyte Type III cGI PDE by isoproterenol, for example, clearly represents a type of "feed back" regulation of cAMP generated via activation of adenylyl cyclase, it is not merely a response to excess cAMP, since activation occurs over virtually the entire range of isoproterenol-induced activation of adenylyl cyclase and lipolysis, including conditions in which cAMP-dependent kinase is not saturated with cAMP and maximally activated (121). Coordinate regulation of adenylyl cyclase, cAMP-dependent protein kinase, and cAMP PDE activities may be important in physiological regulation of cAMP turnover and steady-state concentrations of cAMP, and, therefore, the activation state of cAMP-dependent protein kinase and hormone-sensitive triglyceride lipase. In fact, Goldberg, Walseth, and associates have demonstrated that, in intact platelets in which adenine nucleotides were labeled with ^^O, agents that activate adenylyl cyclase increase both synthesis and degradation of cAMP, suggesting that rapid activation of platelet PDE is important in cAMP turnover (138). Increased turnover, perhaps due to the free energy released during hydrolysis of cyclic nucleotides, may somehow increase sensitivity of cAMP-dependent protein kinase to cAMP. This was suggested in a study which demonstrated that in broken cell preparations cAMP-dependent protein kinase activa-

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VINCENT C. MANGANIELLO et al.

tion occurred at lower cAMP concentrations in the presence of active adenylyl cyclase and a Type I CaM PDE than in the presence of active adenylyl cyclase and an inhibited PDE (139). With insulin plus agents that increase cAMP, insulin activation of cAMP hydrolysis could sufficiently decrease cAMP to reduce cAMPdependent protein kinase activity to a new steady state, leading to a reduction, for example, in hormone-sensitive triglyceride lipase and hepatic glycogen phosphorylase activities and inhibition of lipolysis and glycogenolysis. Thus, with insulin, as well as with hormones that increase cAMP, PDE activation may contribute to regulation of cAMPdependent protein kinase and kinase-regulated effector systems. D. Importance of Type III cGI PDE Activation in Insulin Action Lipolysis in adipocytes and glycogenolysis in hepatocytes are stimulated by hormones that activate adenylyl cyclase, elevate cAMP, and activate cAMP-dependent protein kinase, resulting in activation of adipocyte hormone-sensitive lipase and hepatocyte glycogen phosphorylase. Insulin is a physiologically important inhibitor of lipolysis and glycogenolysis. Although the precise mechanisms of these actions of insulin are not completely known, insulin inhibition of hormonesensitive lipase/lipolysis in adipose tissue and phosphorylase/glycogenolysis in liver are related at least in part to insulin-induced decreases in both hormone-stimulated accumulation of cAMP (140-144) and hormone-activated cAMP-dependent protein kinase (145,146) (Fig. 3). Several types of experiments support the notion that activation of adipocyte and hepatoc3rte Type III cGI PDEs by insulin is important in the insulin-induced reduction in cAMP and cAMP-dependent protein kinase activity and inhibition of lipolysis and glycogenolysis (a schematic presentation of these experiments is shown in Fig. 3). The concentration dependence for insulin inhibition of lipolysis is similar to that for activation of Type III cGI PDE (113,116,121,147) and the time course for PDE activation parallels that for reduction of hormone-stimulated cAMP-dependent protein kinase (121). More importantly, perhaps, inhibitors of the insulin-sensitive Type III cGI PDE, such as cilostamide (148), griseolic acid (149), or SKF95654 (150), can block the antilipolytic action of insulin in 3T3-L1 and rat adipocytes, respectively (Fig. 3). In frog ooc3i:es, stimulation of meiosis by high concentrations of insulin or Insulin-like Growth Factor (IGF-1) is associated with inhibition of adenylyl cyclase, activation of a cilostamideinhibitable cAMP PDE, and activation of an intracellular signal chain resulting in activation of protein serine/threonine kinases such as ribosomal S6 kinase (151-153). Injection of activated Ha-ras protein also

79

TYPE III PDE AND INSULIN ACTION

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• Similar concentration dependence for insulin activation of adipocyte cGI PDE and inhibition of lipolysis • Time course for insulin activation of adipocyte cGI PDE correlates with insulininduced reduction in isoproterenol-stimulation of A-Kinase • Antilipolytic effects of insulin and insulin mediator (Pl-Glycan) are blocked by specific inhibitors of the particulate adipocyte cGI PDE, i.e., cilostamide • Insulin effectively inhibits lipolysis stimulated by cAMP analogs that are substrates of adipocyte cGI PDE.

FIG. 3. Activation of adipocyte Type III cGI PDE in the antilipolytic action of insulin. Hormones and lipolytic agents activate adenylyl cyclase, leading to increased cAMP and activation of cAMP-dependent protein kinase. This results in phosphorylation and activation of the hormone-sensitive triglyceride lipase, leading to hydrolysis of stored triglyceride and release of glycerol and free fatty acids. Insulin, an important inhibitor of lipolysis, acts at least in part by reducing cAMP and, consequently, cAMP-dependent protein kinase activity. Several observations that support the notion that insulin-induced activation of the adipocyte Type III cGI PDE is important in the antilipolytic action of insulin are listed.

increases cAMP hydrolysis and promotes meiosis (152). In these cells inhibitors of Type III cGI PDE (but not of Type IV or V PDEs) inhibit the effects of insulin, IGF, and H-ras on meiosis and of IGF-1 on ribosomal S6 kinase activity (153,154). These latter studies suggest that perhaps some of the effects of insulin on regulation of gene expression and growth in certain cells involve activation of a Type III cGI PDE and reduction in cAMP. One current hypothesis for the mechanism of insulin action involves insulin receptor-mediated activation of phospholipase C and production of an inositol phosphate glycan which presumably functions in an incompletely defined manner as a second messenger of insulin action (155). Exogenous addition of inositol glycan from liver, muscle, or adipose tissue reproduces many of the effects of insulin in intact cells and broken cell systems, including activation of the adipocyte particulate

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VINCENT C. MANGANIELLO et al.

cAMP PDE (156,157) and inhibition of lipolysis (158). An analogous inositol phosphate glycan purified from Trypanosoma brucei reproduced several metabolic effects of insulin in rat adipoc3rtes and hepatocytes, including inhibition of isoproterenol-stimulated lipolysis and inhibition of pyruvate-induced glucose production in hepatocytes (159). Imazodan (CI914), a specific inhibitor of the adipocyte particulate Type III cGI PDE, blocked the antilipolytic actions of both insulin and the inositol phosphate glycan (Fig. 3) (159). Beebe et al. (160) reported that, of a series of cAMP analogs which activated cAMP-dependent protein kinase and stimulated lipolysis and glycogenolysis in intact adipocytes and hepatocytes, insulin effectively inhibited responses to those analogs that were substrates and competitive inhibitors of adipocyte and hepatocyte particulate cAMP phosphodiesterases (Fig. 3). These elegant experiments, which demonstrated insulin inhibition of cellular responses stimulated by hydrolyzable cAMP analogs, pointed to the importance of the particulate cAMP PDEs in the antilipolytic and antiglycogenolytic actions of insulin. They further suggested that these actions of insulin did not require either insulin-induced inhibition of adenylyl cyclase or production of a factor that reduced responsiveness of cAMP-dependent kinase to cAMP without changes in cAMP concentrations. E. Mechanisms for Hormonal Regulation of cGI PDEs

Several studies strongly indicate that insulin and agents that increase cAMP activate Type III cGI PDEs via serine phosphorylations induced by intracellular insulin-sensitive and cAMP-dependent protein kinases, respectively. Early studies demonstrated that, in adipocytes, ATP was essential for activation of the adipocyte particulate cAMP PDE by insulin (115,161). More recent studies using immunoprecipitating antibodies raised against the bovine adipose Type III cGI PDE have shown that in ^^P-labeled rat adipocytes incubated with insulin or isoproterenol, a ~135-kDa protein, identified as the monomer of the adipocyte particulate Type III cGI PDE, was phosphorylated on serine (106,107). The time course and concentration dependence for insulinand cAMP-induced phosphorylation correlated with activation of the adipocyte Type III cGI PDE (107). The effect of isoproterenol appeared to be somewhat more rapid than that of insulin. In experiments in which rat adipocytes were incubated with both isoproterenol and phenylisopropyladenosine (to activate both Gg and Gi), insulin and isoproterenol acted synergistically on phosphorylation and activation of the adipocyte particulate Type III cGI PDE which correlated temporally with the reduction of isoproterenol activation of cAMP-dependent pro-

TYPE III PDE AND INSULIN ACTION

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tein kinase by insulin (107,121). In adipocytes, okadaic acid both activated the particulate Type III cGI PDE in an ATP-dependent manner and prevented deactivation of insulin-activated PDE (126). In ^^Plabeled adipocytes, phosphorylation and activation of particulate Type III cGI PDE induced by adenosine deaminase (presumably mediated by increased cAMP content and cAMP-dependent protein kinase) were rapidly reversed by phenylisopropyladenosine, an inhibitor of adenylyl cyclase (107). These studies (107,126) support the notion that, in adipoC3rtes, activation of the particulate Type III cGI PDE is related to kinaseinduced serine phosphorylation(s) and that protein phosphatases are involved in deactivation. Incubation of human platelets with insulin (136) or agents that increase cAMP (108,109,136,137) results in phosphorylation and activation of the platelet Type III cGI PDE. After immunoprecipitation with an anti-Type III cGI PDE monoclonal antibody, the immunoisolated enzjmie was phosphorylated and activated by cAMP-dependent protein kinase (137). In broken cell systems, cAMP-dependent protein kinase can phosphorylate and activate platelet (137), adipocyte particulate (123-125,162), and hepatocyte dense vesicle (134) Type III cGI PDEs. Incubation of rat liver dense vesicles with cAMP-dependent protein kinase was associated with a time-dependent phosphorylation and activation of the dense vesicle Type III cGI PDE, provided the membranes were previously incubated in the presence of Mg^^ (134). Phosphatase inhibitors prevented the kinase-catalyzed phosphorylation and activation, suggesting that Mg2+ stimulated endogenous phosphatases. It was also possible to isolate a phosphorylated dense vesicle Type III cGI PDE from intact ^^P-labeled hepatocytes which could be dephosphorylated in the presence of Mg^^ [presumably via activation of phosphatase(s)] with no effect on enzyme activity (134). Taken together, these results suggested the possible presence of at least two phosphorylation sites on the dense vesicle Type III cGI PDE—one "silent" site preventing phosphorylation at a second or "activating" site (134). A serine residue on the adipoc3^e Type III cGI PDE phosphorylated in solubilized membrane preparations by cAMP-dependent protein kinase has been identified (162). The methodology commonly used to identify sites phosphorylated by cAMP-dependent protein kinase, i.e., proteolytic digestion followed by purification and direct sequencing of phosphorylated tryptic peptides, could not be directly applied to adipocyte Type III cGI PDE because of insufficient amounts of pure enzyme and its susceptibility to proteolytic degradation during purification. An alternative strategy was pursued (162). From the deduced sequence of

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the recently cloned rat adipocyte Type III cGI PDE (56), several possible sites for phosphorylation by cAMP-dependent protein kinase, based on the consensus kinase recognition sequence (-RRXS-), were identified in the regulatory domain of the PDE. Based on preliminary studies and chromatographic properties of phosphopeptides released during trypsin digestion of phosphorylated authentic adipocyte Type III cGI PDE, one synthetic peptide LRRSSGASGLLTSEHHSR, corresponding to amino acids 423-440 in the deduced adipocyte Type III cGI PDE primary sequence, was chosen. The sequence of this peptide was such that the same tryptic peptides could be generated following phosphorylation and trypsin digestion of the phosphorylated synthetic peptide or authentic adipocyte Type III cGI PDE (phosphorylated by cAMPdependent protein kinase after solubilization from rat adipocyte microsomes). After phosphorylation and tryptic digestion of authentic T3^e III cGI PDE and the synthetic peptide, the phosphopeptides from both comigrated in several separation systems (FPLC, HPLC, and SDSurea PAGE). The exact location of the phosphorylated serine was then assessed by radiosequencing; from the known sequence of the synthetic phosphopeptide, serine 427 is a likely site in the solubilized adipocyte T3npe III cGI PDE phosphorylated by cAMP-dependent protein kinase (162). An insulin-sensitive intracellular kinase that phosphorylates and activates Type III cGI PDE has been identified in cytosolic fractions of rat adipocytes (125,163) and human platelets (164) that had been incubated with insulin, and in extracts of rat livers removed after administration of insulin to the animals (165). ATP and Mg^^ were required for PDE activation (163,165). C)^osol from insulin-treated adipocytes increased incorporation of ^^P from [y-^^PJATP into control particulate adipocyte Type III cGI PDE severalfold (Fig. 4) in a timeand concentration-dependent fashion (125). Inhibitor studies suggest that the insulin-sensitive cGI PDE kinase(s), which activates and phosphorylates adipocyte and platelet cGI PDEs, is not cAMP-dependent protein kinase, protein kinase C, or several known insulin-sensitive kinases, including casein kinase II, ribosomal S6 kinase, and proteaseactivated and Mn^^-specific protein kinases (125,163-165). The platelet insulin-sensitive cGI PDE kinase, purified (10-fold) by chromatography on DEAE, phosphorylates a serine residue(s) in platelet cGI PDE in a time- and concentration-dependent manner (164). Maximal phosphorylation by activated insulin-sensitive Type III cGI PDE kinase resulted in increased incorporation of 0.2 mol of ^^P per mole Type III cGI PDE and a 15-20% increase in Type III cGI PDE activity (164). Activation of the insulin-sensitive cGI PDE kinase may reflect covalent modifi-

83

TYPE III PDE AND INSULIN ACTION

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FIG. 4. Phosphorylation of adipocyte Type 111 cGl PDE by the insulin-sensitive protein serine kinase. (Top, A and B) Microsomes from control adipocytes were incubated with or without cytosol from insulin-treated (S^) or control (S~) adipocytes, catalytic subunit of cAMP-dependent protein kinase, or protein kinase inhibitor (PKI) for 10 min at 30°C in the presence of [y-^^PJATP as indicated. (Bottom) Control microsomes were incubated (10 min, 30°C) with [-y-^^pj^rpp g^^^ pjQ ^nd the indicated amounts of cytosol from control ( S ) or insulin-treated adipocytes (S^). Microsomes were sedimented by centrifugation and solubilized. Type III cGI PDE was immunoprecipitated with anti-adipose tissue cGI PDE antibodies and subjected to SDS-PAGE and autoradiography. These experiments indicate that the insulin-sensitive kinase (ISK) phosphorylated Type III cGI PDE in a concentration-dependent manner; ISK was not inhibited by PKI, which did inhibit cAMPdependent protein kinase-induced phosphorylation of Type III cGI PDE.

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cation since the kinase remains activated during partial purification. Since phosphatase inhibitors are required during preparation and partial purification to maintain the cGI PDE kinase in its activated state, and since the kinase itself is not apparently phosphorylated on tyrosine (164), it is possible that the insulin-sensitive intracellular protein serine/threonine kinases whose activation is regulated by upstream kinases/phosphatases, which participate in insulin-induced phosphorylation/dephosphorylation cascades (Fig. 5) (166). The sites phosphorylated in response to insulin and cAMP in intact adipoc5^es and the site(s) phosphorylated by the insulin-sensitive protein kinase are under investigation in our laboratories. The availability of recombinant adipocyte Type III cGI PDE expressed in baculovirusinfected insect cells and mammalian cell lines that overexpress the insulin receptor could facilitate identification of sites phosphorylated in intact cells and broken cell preparations. These cell lines should also yield information on postreceptor events involved in phosphorylation/ activation of the Type III cGI PDE. While considerable evidence relates phosphorylation to PDE activation, a precise quantitative relationship between the two effects has not been established. Quantitatively, activation of Type III cGI PDE by cAMP-dependent and insulin-sensitive kinases is less in broken cell systems than in intact adipocytes (123-125,137,163-165). Perhaps the availability of large quantities of recombinant material will allow identification of phosphopeptide sequences, numbers of sites, and stoichiometry of phosphorylation. It is also possible that some phosphorylations are "silent," i.e., do not in themselves alter activity, but might promote interaction of Type III cGI PDE with other regulatory effectors or factors. Some phosphorylations may directly or indirectly reduce or limit the activation of cGI PDE. Homogenization conditions (pH, temperature, EDTA, redox state, salts, phospholipids, etc.) or experimental treatment of particulate fractions (detergents, salts, dithiothreitol, or trypsin) isolated from hormone-treated cells can dramatically alter the relative magnitude of hormonal activation of PDE as measured in broken cell systems (112,113,118,167-177) and thus make it difficult to relate phosphorylation and activation. For example, although the insulin-sensitive particulate PDE is a member of the Type III cGI PDE gene family, under certain conditions incubation with cGMP before assay (in which cGMP inhibits activity) resulted in marked activation of PDE (174). Stimulation by cGMP was similar to that produced by incubation of intact adipocytes with insulin. The oxidation state of critical thiols also effects activity of adipocyte and hepatic particulate PDEs (113,173,175,176).

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These kinds of perturbations could be partially responsible for quantitative differences observed in different laboratories in the relative effects of insulin and agents that increase cAMP, and with additivity of effects of cAMP and insulin. F. Kinase Cascades in Regulation of Cell Metabolism and Proliferation by Insulin

The exact mechanisms whereby insulin regulates cell proliferation and metabolic pathways are unclear. Similar to a number of other growth factors, these pleiotropic responses are initiated by insulin's interaction with its cell surface receptor and activation of the intrinsic receptor protein tyrosine kinase (177,178). The activated receptor phosphorylates specific tyrosines in specific protein targets, triggering a series of events resulting in activation of cytoplasmic protein kinases (predominantly serine/threonine kinases) and phosphatases and, thus, in alteration of the phosphorylation state and activities of key proteins involved in the regulation of cell matabolism and proliferation (166,177-180). Mitogen-activated (MAP) or extracellular signal-regulated (ER) kinases (MAPKs, ERKs) are considered key components in actions, especially those related to mitogenesis and differentiation of insulin and other growth factors (181-186). MAPKs or ERKs (187,188) phosphorylate transcription factors (189-193) and also phosphorylate and activate ribosomal S6 kinases (RSKs) which phosphorylate ribosomal protein S6 (194,195). Although RSKs have been purified and characterized as protein kinases which utilize ribosomal S6 protein or S6 S3nithetic peptides as substrates, it has been suggested that nuclear proteins and transcription factors may be more relevant substrates than S6 for the pp90 RSKs (185,186,189-192). On the other hand, pp70 RSKs may be more specific for ribosomal S6 and thus exert translational control (186). These two RSK^ are also apparently regulated by distinct signal transduction pathways (196-198). MAPKs are apparently rapidly activated by MAPKKs (MAP kinase kinases or MEKs) which phosphorylate threonine and tyrosine residues in a TEY sequence highly conserved in the MAPK gene family (183,199-201). Activation of the MAPK cascade is produced via Ras-dependent (202,203) and -independent pathways (204-206). Some reports have suggested that Raf-1 kinase functions as the MAPKKK in some cells (207-209) and may mediate regulation of MAPKK and MAPK by insulin in H4 hepatoma cells (207). Based on genetic complementation studies of the kinase cascades involved in pherome signaling in yeast, another MAPKKK (or MEKK) distinct from Raf-1 and related in deduced sequence to yeast protein

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VINCENT C. MANGANIELLO et al.

kinases Byr 2 and Ste II, has also been cloned and characterized (210,211). It was hypothesized that Raf-1 kinase might function as MAPKKK in regulation of MAPKK by tyrosine kinase growth factor receptors and Ras activation, whereas yeast-related MEKK(s) would be regulated by G protein-linked receptors (210,211). At this point, however, one cannot dismiss the possibility that under certain conditions or in certain cells, both Raf-1 kinase and MEKK might be necessary for full activation of MAPK and its downstream signaling events. Although the more proximal steps in regulation of the putative mitogenic kinase cascade are even less certain than these distal steps, a conceptual framework has begun to emerge (212). Ras, with significant structural and functional similarity to other GTP-binding proteins, is perhaps the key element in regulation of growth and differentiation signal transduction pathways, especially those activated by receptors with intrinsic or associated tyrosine kinase activities (212-215). It is clear from a number of studies with cultured cells that insulin activates Ras (216-220), perhaps by stimulation of a guanine nucleotide exchange factor, resulting in the release of GDP and an increase in the steady-state level of GTP-bound Ras (217). Activation of Ras seems to be the critical upstream event in activation of Raf and the MAPK cascade (202,203,219,221,222). Introduction of Ras peptides into intact Xenopus laevis oocytes (221,222) or homogenates (221) and overexpression of active Ras in 3T3-L1 adipocytes (219) activates MAPK; dominant-negative Ras mutants block insulin activation of MAPK in 3T3-L1 adipocytes (219) and activation of Raf-1 and/or MAPK by nerve growth factor in PC12 cells (203,223). Antisense c-Raf-1 RNA or dominant-negative c-Raf-1 mutants block Ras-induced transformation in NIH/3T3 fibroblasts (224). Recent experiments demonstrate direct interactions between effector domains of Ras and a cysteine-finger aminoterminal regulatory domain of Raf-1 (225,226). As discussed previously, Raf-1 can activate MKKK (MEKK), and consequently MKK(MEK) and MAPK(ERK) (207-210). One current hypothesis indicates that activation of Ras by insulin is mediated by insulin receptor substrate-1 (IRS-1), a critical intracellular cytosolic protein which is rapidly phosphorylated on multiple tyrosine residues by the activated insuHn receptor (227-232). This protein is important in insulin action since both phosphorylation of IRS-1 and various metabolic actions of insulin are inhibited in cells transfected with tyrosine kinase-deficient insulin receptors (231). IRS-1 serves as a "docking" protein, allowing ternary complexes of the insulin receptor with proteins critical to the initiation of intracellular insulin-induced signaling (227,233). It has been suggested that phosphotyrosine do-

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mains of IRS-1 bind with high affinity to certain signal-transducing or "adaptor" proteins (212-215,227,233) that contain specific binding sites for t3n:*osine phosphopeptides called SH2 (Src homology) domains and specific proline-rich motifs called SH3 domains (212-215,227,233). By virtue of its multiple distinct phosphotyrosine motifs, IRS-1 can interact with SH2 domains of any of several adap tor molecules which in turn interact with, perhaps via SH3 domainsyand activate downstream elements in the insulin-signaling chain (212-215,227,233), including the Ras-Raf-MAPK phosphorylation and the phosphatidylinositol 3-kinase (PI-3 kinase) cascades. Incubation of responsive cells with insulin also results in activation of PI-3 kinase [composed of a p85a (85,000 kDa) regulatory subunit which contains SH2 and SH3 domains, and a pi 10 (110,000 kDa) catalytic subunit]. Activation of PI-3 kinase results in enhanced formation of phosphatidylinositol3- phosphate (234) and other phosphorylated phosphatidylinositol products thought to be important in cell growth and metabolism and, perhaps, activation of ras (234-236). Although some PI-3 kinase associates directly with the insulin receptor, activation of the catalytic subunit of PI-3 kinase is thought to occur primarily by complex formation with IRS-1 via binding of the regulatory subunit p85a SH2 domains to phosphotyrosine motifs in IRS-1 (232). Whether IRS-1-mediated activation of PI-3 kinase indirectly leads to insulininduced activation of the MAPK pathway via Ras/Raf or other pathways is not known. By analogy, interaction of IRS-1 phosphotyrosine motifs with other adaptor molecules containing SH2 domains, such as the recently described GRB-2/sem 5, could lead to activation of Ras or other intracellular signal cascades. GRB-2/sem 5, a small cytoplasmic protein with SH2 and SH3 domains, seems to be an upstream regulator of Ras (237). In L6 myoblast cell lines, overexpression of GRB2 enhanced the ability of insulin to phosphorylate and activate MAPK; intact GRB2 SH2 and SH3 homology domains were required for these effects of insulin (238). It is possible that GRB2 serves as an adaptor, linking insulin receptor/IRS-1 to the Ras pathway via complex formation with and activation of a guanine nucleotide-exchange protein. An insulin receptor-dependent association of IRS-1 and dSos (a putative Drosophila guanine nucleotide-exchange factor) was recently detected immunochemically in COS cells expressing the human insulin receptor, Ras, and dSos (238). In L6 myoblasts overexpressing GRB-2, insulin induced formation of a complex of IRS-1, GRB-2, Sos, and She (an SH2 domain containing protein) (239). It should be emphasized that at present, the detailed mechanisms for insulin activation of the MAPK cascade via Ras and Raf is primarily

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a conceptual framework. The actual network are almost certainly not linear from receptor to MAPK, but is divergent and subject to multiple upstream inputs as well as downstream feedback regulation of upstream events (185,186,240-242). The MAPK cascade also does not directly account for involvement of protein kinase C (243-246), phosphatidylinositol glycan (or other) "mediators" (155-159), or other signaling pathways (260) in insulin action. Even less is known of the detailed mechanisms whereby insulin regulates carbohydrate and lipid metabolism. An insulin-stimulated protein kinase (ISPK) in skeletal muscle exhibits certain similarities to a RSK from Xenopus oocytes; both kinases are phosphorylated and stimulated by MAPK (247,248). ISPK phosphorylates and stimulates a specific protein phosphatase which dephosphorylates and stimulates glycogen synthase (247,248). This suggests that insulin might regulate glycogen synthase at least in part via a cascade involving MAP-2 kinase, ISPK, and a protein phosphatase. It should be noted, however, that phosphatase inhibitor-1 is active when phosphorylated by cAMPdependent protein kinase (249). In cells containing insulin-sensitive PDE, activation of PDE could, by reducing cAMP and cAMP-dependent protein kinase activity, lead to net dephosphorylation and inactivation of phosphatase inhibitor-1 and, consequently, phosphatase activation. Thus, under some conditions insulin activation of PDE could result in phosphatase activation. Wortmanin, a specific inhibitor of PI-3 kinase, has been reported to inhibit the antilipolytic action of insulin and insulin-stimulated hexose uptake in rat adipocytes (261) as well as insulin-induced differentiation of 3T3-L1 adipocytes (262). We have demonstrated that Wortmanin also blocked insulin activation of cGI PDE kinase and activation/ phosphorylation of cGI PDE (263). The components of the presumed signaling chain between PI-3 kinase and cGI PDE kinase and the glucose transporter are unknown. Dissection of these pathways will be complicated considering that Wortmanin can block insulin-induced activation of both PI-3 kinase and MAPK (262). Whereas Wortmanin blocks insulin activation of p70S6 kinase and cGI PDE kinase, rapamycin, which inhibits activation of the p70S6 kinase, does not block insulin activation of cGI PDE kinase (Rahn et al, 1995) or translocation of glucose transporters (264). These results suggest activation of p70S6 kinase and cGI PDE kinase is independent and may require different signals from PI3-kinase. G. Summary for Mechanisms for Regulation of Adipocyte Type III cGI PDE

Our current understanding and hypotheses concerning hormonal regulation of adipocyte Type III cGI PDE and lipolysis are schemati-

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cally presented in Fig. 5. Agents that activate adenylyl cyclase, e.g., catecholamines, increase lipolysis via cAMP-dependent protein kinasecatalyzed phosphorylation (on Ser-563) (250) and activation of hormone-sensitive lipase (251,252). Activation of lipolysis seems to be functionally coupled to feedback regulation of cAMP concentrations/ turnover via cAMP-dependent protein kinase phosphorylation/activation of adipocyte particulate Type III cGI PDE (24,106,107,121-124). The deduced amino acid sequence of adipocyte Type III cGI PDE suggests that Ser-427 is a likely target for cAMP-dependent protein kinase phosphorylation of the solubilized particulate enz5rme (162). This must, of course, be verified in intact adipocytes. The antilipolytic action of insulin is associated with a net dephosphorylation of the hormone-sensitive lipase (251,252), mediated at least in part by a reduction of cAMP (140,141) and in cAMP-dependent protein kinase (145,146). Insulin-induced inhibition of adenylyl cyclase [(253255) liver], reduced sensitivity of cAMP-dependent protein kinase to cAMP (256,257) and phosphatase activation (146), as well as activation of the Type III cGI PDE have been suggested as possible mechanisms for the antilipolytic action of insulin. As discussed previously, several different kinds of experiments strongly support the idea that insulin-

(Glycerol, Free Fatty Acids)

FIG. 5. Role of Type III cGI PDE in the antilipolytic action of insulin. For recent reviews of mechanisms of insulin action, both general and specifically relating to antilipolysis, see Refs. (259) and (265). Reprinted from Cell Signalling, Type III cGMP-Inhibited Cyclic Nucleotide Phosphodiesterases (PDE3 Gene Family), with kind permission from Elsevier Science Ltd., The boulevard, Langford Lane, Kidlington OXS IGB, UK.

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induced activation of the Type III cGI PDE, associated with serine phosphorylation (106,107,125,163-165) of the PDE, is an important component in the antiUpolytic action of insulin (113,116,121,125, 126,147-150,159-165). This phosphorylation results in activation of the Type III cGI PDE and reduction in cAMP-dependent protein kinasecatalyzed phosphorylation/activation of hormone-sensitive lipase (107,121). The insulin-induced phosphorylation (on serine )/activation of the adipocyte Type III cGI PDE is catalyzed by an insulin-sensitive Type III cGI PDE kinase (ISK) which is itself perhaps activated by a serine/ threonine phosphorylation cascade initiated through PI-3 kinase (106,107,125,163-165,263) (Fig. 5). Initial results suggest that the Type III cGI PDE ISK is not one of several kinases known to be stimulated by insulin, i.e., casein kinase-2, Mn^^-stimulated kinase, proteaseactivated kinase, RSK, protein kinase C, purified RSKs (supplied by J. Avruch), or purified skeletal muscle ISPK (supplied by P. Cohen) (125,126,163-165; Rahn et al., 1995). Identification of the components of the putative T5^e III cGI PDE kinase cascade is also under way in our laboratories. The initial postreceptor downstream events in insulin regulation of Type III cGI PDE are not completely known. Results indicate that the Type III cGI PDE ISK is not phosphorylated on tyrosine and therefore is presumably not a substrate of the insulin receptor tyrosine kinase (165). Whether activation of Type III cGI PDE by insulin is coupled only to receptor-IRS-1 complex formation and activation of PI3-kinase or also involves other signaling pathways, e.g., G protein, phospholipase, PI glycan, is now known. In rat adipose tissue, a number of protein kinase activated by insulin have been described, including protein kinase C (243-245) and acetylCoA carboxylase kinase, ATP-citrate lyase kinase, casein-2, p44 MAPK (ERK-1), p42 MAPK (ERK-2), p70 RSK, and p90 RSK (258). Whether activation of the Type III cGI PDE ISK via PI3-kinase utilizes components of the MAPK cascade or a unique cascade remains to be established. It will be important to determine whether components of the MAPK cascade are involved in activation of Type III cGI, PDE, since in X. oocytes, insulin, insulin-like growth factor, and H-ras stimulate phosphodiesterase activity (and MAPK as well) (152-154). In rat adipocytes, however, PMA, which can stimulate MAPK, has little or no apparent effect on phosphodiesterase activity (Rahn et ai, 1995). The availability of recombinant adipocyte Type III cGI PDE should facilitate characterization and cloning of the Type III cGI PDE ISK as a first step in elucidating the signal chain between the insulin receptor and

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regulation of Type III cGI PDE. This formation could be useful not only in defining specific molecular mechanisms for regulation of the Type III cGI PDE and the antilipolytic action of insulin (an important biological effect of the hormone), but also could shed light on the mechanisms of insulin regulation of other metabolic pathways. ACKNOWLEDGMENTS We t h a n k E. Meacci, A. Rascon, H. Shibata, C. Smith, T. Rahn, and H. Tomqvist for allowing us to include unpublished information, Dr. Martha Vaughan for useful discussions and critical reading of the manuscript, and Mrs. C. Kosh for secretarial assistance.

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CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Mammalian Aminoacyl-tRNA Synthetases DAVID C. H . YANG Department of Chemistry Georgetown University Washington DC 20057

I. introduction Translation and transcription constitute the two major steps of gene expression in all organisms. Aminoacyl-tRNA synthetases (RSs) carry out the key role of the interpretation of the genetic code by the covalent attachment of specific amino acids to cognate tRNAs. In the past few years, a full complement of cDNA and protein sequences of the bacterial RSs was completed and led to the structural classification of synthetases (Eriani et al., 1990). The classification of RSs and recognition of tRNA have also been reviewed (Burbaum and Schimmel, 1991; Carter, 1993). During the same period, the three-dimensional structures of six additional RSs were highly resolved that included Escherichia coli MetRS (Brunie et al., 1990), Thermophilus thermophilus SerRS (Cusack et al., 1990), E. coli GlnRS (Rould et al, 1989), Lys RS (Onesti et al, 1995), TrpRS (DouWie et al, 1995), and yeast AspRS (Ruff et al, 1991). The structures of Asp-, Glu-, and SerRSs were determined as cocrystals with their respective cognate tRNA and adenylate analogs (Beirhah et al, 1994; Biou et al, 1994; CavareUi et al, 1994). The roles of the modified bases in the conformation of tRNA, tRNA identity, and codon recognition have been firmly established in a number of cases (Bjork et al, 1987; Schulman, 1991). Studies of RSs have also contributed to our understanding of fundamental principles in the RNA-protein interactions (Schulman, 1991; Shiba and Schimmel, 1992; Abelson, 1994; Rogers et al, 1994), the architecture of proteins (Rould et al, 1989; Ruffed al, 1991; Biou et al, 1994), gene regulation (Springer et al, 1985; Yanofsky, 1987), enzyme catalysis (Eldred and Schimmel, 1972; Avis and Fersht, 1993), and the evolution of proteins (Eriani et al, 1990; Nagel and Doolittle, 1992). The progress in the studies of mammalian RSs has been relatively slow. Nonetheless, a number of structural and functional features that are absent in bacterial and yeast enzymes have been found. Our understanding of this important family of enzymes in mammalian systems 101

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has been growing increasingly rapidly since they were last reviewed in this series (Yang et al, 1985). A number of mammalian synthetases have been cloned, sequenced, and expressed. Studies of the regulation of these enzymes have begun. Reviews on prokaryotic and eukaryotic synthetases (Lapoint and Giege, 1991; Mirande, 1991; Kisselev and Wolfson, 1994) and phosphorylation of synthetases have appeared (Traugh and Pendergast, 1986; Proud, 1992). This review focuses on recent developments of studies on the structure-function relationship of mammalian synthetases, with emphasis on those features that are unique in mammalian synthetases and are for the most part absent in bacterial and yeast enzymes.

II. Classification of Mammalian Aminoacyl-tRNA Synthetases Studies of mammalian synthetases have been hampered by the presence of the high Mr forms of synthetases (Bandyopadhyay and Deutscher, 1971, 1973; Vennegoor and Bloemendal, 1972). These high Mr synthetases were initially thought to be aggregates of synthetases. Studies of mammalian synthetases were further complicated by the facts that these enzymes occur in multiple forms and are highly susceptible to proteolytic degradation and that aminoacylation activities are unstable (Dang and Yang, 1982). In addition, a number of nonsynthetase proteins or nonprotein materials such as cholesteryl esters (Bandyopadhyay and Deutscher, 1973; Sivaram et al., 1988) were found to associate with the synthetases (Dang et aL, 1982a). It is now clear that 9 synthetases for the activation of Arg, Asp, Gin, Glu, lie. Met, Leu, Lys, and Pro are associated as an RS complex. ValRS uniquely occurs as a separate high Mr ValRS complex with EFIH (a, p, y, 8) (Motorin et al, 1988; Bee et al, 1989; Venema et al, 1991a). Ten synthetases that were not specified in the synthetase complex have been purified from various sources as free soluble enzymes which include CysRS (Pan et al, 1976), GlyRS (Ge et al, 1994; Shiba et al, 1994a), HisRS (Fahoum and Yang, 1987), PheRS (Tscheme et al, 1973; Tanaka et al, 1976), SerRS (Mizutani et al, 1984; Fahoum, 1985; Dang and Traugh, 1989; Miseta et al, 1991), ThrRS (Gerken and Arfin, 1984; Dang and Traugh, 1989), TyrRS (Wolfson et al, 1990), and TrpRS (Kisselev, 1993). Although some of the synthetases, e.g., AlaRS and AsnRS, have yet to be highly purified, they have consistently been found occurring as free soluble enzymes.

III. General Structure of the RS Complex The synthetase complex was first highly purified from sheep mammary gland (Kellermann et al, 1979) by a three-step purification proce-

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dure that included polyethylene glycol fractionation, gel filtration on Bio-Gel A-5m, and affinity chromatography on immobilized E. coli tRNA-Sepharose. This procedure has been successfully applied to the purification of a number of synthetase complexes in other mammalian cells and tissues which include rat liver (Johnson and Yang, 1981; Cirakoglu and Waller, 1985c), rabbit liver (Kellermann et a/., 1982; Godar et ai, 1988), rabbit reticulocytes (Kellermann et al., 1982; Godar et al, 1988), sheep liver (Kellermann et al., 1979), Chinese hamster ovary (CHO) cells (Mirande et al., 1985b), hen (Kerjan et al., 1994), etc. A similar synthetase complex has been purified from fruit flies (Kerjan et al., 1994), suggesting that all animals from the metazoan subgroup of coelomated should possess such a synthetase complex. A different purification procedure was developed for rabbit reticulocytes and yielded the same complex (Kellermann et al., 1982). When the procedure was applied to the purification of the synthetase complex from murine erythroleukemia cells, similar results were obtained (Norcum, 1989). The synthetase complexes from various sources exhibited very similar peptide compositions. Variations in the stoichiometry and subunit molecular weights may have occurred in some cases due to the high susceptibility of synthetases toward endogenous proteases (Siddiqui and Yang, 1985; Kerjan et al., 1992) as well as the weak association of sjmthetases in the complex (Dang et al., 1985). Inclusion of irreversible protease inhibitors such as phenylmethylsulfonyl fiuoride and diisopropyl fiuorophosphate (DIFP) during homogenization is essential to preserve the structural integrity of the synthetases. Despite these difficulties, determination of the stoichiometry of the synthetases (Johnson and Yang, 1981), immunoprecipitation of the entire synthetase complex from cellular extracts using monospecific antibodies (Mirande et al., 1985b), and the electron micrographs of the highly purified complex (Norcum, 1989) provided strong evidence for the existence of a multienzyme complex of RSs in mammalian cells. The synthetase complexes from various sources generally contain nine synthetases specific for Arg (2 x 70K), Asp (2 x 55K), Gin (1 X 94K), Glu/Pro (1 X 160K), lie (1 x 125K), Leu (1 X 135K), Lys (2 X 72K) and Met (1 x 104K). The stoichiometry of the 160K component has been found to be two if cells were homogenized imder gentler conditions (Kerjan et al., 1992) as this component is particularly susceptible to the endogenous proteolysis. The polypeptides in the synthetase complex were identified by assaying activities of proteins extracted from SDS-polyacrylamide gels and by activity inhibition and Western analysis using monospecific antibodies (Mirande et al., 1982) as well as by the purification and characterization of free synthetases dissociated

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from the synthetase complex (Johnson et al., 1980; Cirakoglu and Waller, 1985b; Lazard et a/., 1985; Kerjan et al., 1992; Ting et al, 1992). Three low M, proteins—bands IX (43K), X (38K), and XI (18K)— apparently associated with the synthetase complex, but the functions of these proteins are unknown. The 38K protein was suggested to be a casein kinase (Pendergast and Traugh, 1985), but the partial amino acid sequences (Jacobo-Molina et al, 1988) did not show any similarity to known casein kinase sequences. Recent in vivo studies demonstrated that ArgRS specifically cross-linked to the 38K protein (Filonenko and Deutscher, 1994). The sizes and the composition of the synthetase complex also varied according to the sources (e.g., liver vs reticulocytes), physiological conditions (e.g., livers from starved vs fed rats; amino acid starvation of cultured cells), and ages. However, since the S5nithetases are unstable and highly susceptible to endogenous proteolysis, some of the reported variation in the structures of the synthetase complex was certainly due to changes in the lability of synthetases or in the endogenous protease activities (Godar et al., 1988; Kerjan et al, 1992). Derepression of MetRS in CHO cells by methionine starvation has been observed. The derepression resulted in an increase in the stoichiometry of MetRS from one to two in the synthetase complex without a significant increase of free MetRS (Lazard et a/., 1987). Whether or not the limited enhancement of MetRS under derepression reflects autorepression of mammalian MetRS is not known. Starvation of several other amino acids in CHO cells did not result in any significant changes of synthetase activities (Lazard et al, 1987). The occurrence of high M^ RSs in bacteria has been suggested (Harris, 1990). However, evidence such as the purification and characterization of high Mr complexes has not been reported. A search of yeast synthetase complexes similar to the mammalian synthetase complex was unsuccessful (Cirakoglu and Waller, 1985a). However, a high Mr form of ValRS was found (Black, 1985). Activation of ValRS appears to be regulated by redox of cysteine residues through heme proteins and glutathione cascade and resulting oscillating activities (Black, 1986, 1993). The structures of these reported high Mr synthetases in bacteria and yeast remain undetermined. The organization of synthetases as a multienzyme complex provided an interesting biochemical arrangement of a group of essential enzymes in the highly complex translation process. These synthetases catalyze parallel reactions instead of sequential reactions as in all previously reported multienzyme complexes (Srere, 1987). A number of questions have been invoked: Is the complex required for the enzyme activities?

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Do the synthetases function independently? How are the synthetases assembled and organized in the complex? Why is this particular group of synthetases specified in the synthetase complex? Are there structural and functional differences between those synthetases in the complex and free synthetases? Answers to these questions have emerged but much remains to be learned.

IV. Dissociation and Organization of the Synthetase Complex The synthetase complex dissociated to fully active smaller subcomplexes and free synthetases when subjected to a number of physical and chemical treatments, such as ultracentrifugation, high concentration of salt, extreme pH, neutral and ionic detergents, or combinations of these treatments. Hydrophobic interaction chromatography was the most effective method among various treatments (Dang and Yang, 1979). A number of synthetases that dissociated from the synthetase complex by hydrophobic interaction chromatography were subsequently purified and characterized, and include LysRS (Johnson et al, 1980; Brevet et al., 1982), LeuRS (Cirakoglu and Waller, 1985b), and IleRS (Lazard et al., 1985). Dissociation of the synthetase complex by hydrophobic interaction chromatography suggested that hydrophobic interactions play major roles in the association of these synthetases. Dissociated free Leu-, He-, and LysRSs showed high affinity toward phenyl- or aminohexyl-Sepharose. Bound synthetases were effectively eluted by neutral detergents such as Nonidit P-40 or organic solvents such as ethylene glycol (Cirakoglu and Waller, 1985b). Besides the hydrophobic interactions, such binding may also involve electrostatic interactions, since salt effectively eluted bound LysRS off the gel (Johnson et al., 1980). Important roles of hydrophobic interactions in the association of synthetases were also suggested based on studies of the effects of salts and detergents on the sizes of the synthetase complex (Sihag and Deutscher, 1983). Controlled proteolysis also dissociated the synthetase complex into fully active free synthetases. Met- and LysRSs were dissociated from the complex by controlled trypsinization and elastase digestion, respectively. The loss of a 40K fragment in the dissociated MetRS did not affect the MetRS activity (Kellermann et al., 1978). Similarly, the removal of a 16K fragment from LysRS did not affect LysRS activity (Brevet et al, 1982). The 40K fragment in MetRS and the 16K fragment in LysRS likely contain complementary structures for binding to other synthetases in the RS complex. ArgRS (Vellekamp and Deutscher, 1987), and

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ProRs (Kerjan et al, 1992; Ting et al, 1992) were also purified and characterized after controlled proteolysis by subtilisin and elastase, respectively. Since both hydrophobic interaction chromatography and controlled proteolysis dissociated the synthetase complex, it was hypothesized that N- or C-terminal dispensable domains in mammalian synthetases are involved in the assembly of the synthetases in the synthetase complex (Cirakoglu et aL, 1985). This suggestion was substantiated by the presence of the heparin- and hydrophobic interaction chromatography (HlC)-binding affinity of the full-length synthetases and the losses of the heparin and HIC affinity of the protease-digested synthetases (Cirakoglu et al., 1985; Cirakoglu and Waller, 1985b,c). A systematic study of the rat liver synthetase complex showed that synthetases dissociated sequentially and provided a disassembly scheme (Dang and Yang, 1979). Hydrophobic interaction chromatography was used to dissociate the synthetase complex and sucrose gradient ultracentrifugation to identify subcomplexes and free synthetases. Thus, the 24S synthetase complex, which contains all nine synthetase activities, was dissociated to an 18S synthetase complex that contained only five synthetases: Lys-, Arg-, Leu-, He-, and MetRSs. Complexes containing identical synthetases were reported in early studies of the purification of the synthetase complexes (Vennegoor and Bloemendal, 1972; Som and Hardesty, 1975). Lys- and ArgRS could be isolated as a subcomplex (Dang et al, 1982b), and He-, Leu-, and MetRSs as another subcomplex (Dang and Yang, 1979). Both subcomplexes dissociated into free synthetases by hydrophobic interaction chromatography on aminooctyl-Sepharose. All five synthetases—Lys-, Arg-, He-, Leu-, and MetRSs—were purified and characterized as free enzymes. The disassembly scheme that encompasses these observations is shown in Fig. 1. The synthetases in the complex must be organized such that all active sites are accessible to all substrates. The spatial arrangement 24 S GluProRS GlnRS LysRS ArgRS LeuRS p-^IleRS i MetRS ' ProRS

21 S GluRS GlnRS LysRS ArgRS LeuRS IleRS MetRS

12S 18 S

T* GluRS 9 GlnRS

LysRS ArgRS LeuRS IleRS MetRS

LysRS ArgRS 13 S LeuRS IleRS MetRS

LysRS

ArgRS lOS LeuRS IleRS

LeuRS

IleRS MetRS

FIG. 1. Disassembly of the multienzyme complex of aminoacyl-tRNA synthetases. Reprinted from Dang and Yang (1979) with permission from ASBMB.

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of the synthetases is yet to be determined by methods such as chemical cross-hnking, immunoelectron microscopy, and singlet-singlet energy transfer. To obtain meaningful results, the complex preparations used for such studies should be devoid of the microheterogeneity which results from partial dissociation or endogenous proteolysis. The sizes of the synthetase complex in cell extracts are usually significantly larger than those of highly purified complexes, suggesting loss of some of the structural components during purification. For example, the synthetase complex in the Fraction X was 24S, but the purified synthetase complex was 18S (Johnson and Yang, 1981). A number of nonprotein components were reportedly associated with the synthetase complex (compiled in Dang et aL, 1982a). The presence of 5S RNA in the synthetase complex was suggested (Ogata et aL, 1991a,b), as were lipids (Bandyopadhyay and Deutscher, 1973; Sivaram et aL, 1988). The protease susceptibility of synthetases in the complex was reduced after the extraction of the lipids from the complex and was restored on readdition of lipids (Sivaram, et aL, 1988). It should be noted that very little lipid or nucleic acid were found in the purified complex (Dang and Yang, 1979; Johnson and Yang, 1981). These results underscored some of the complications in the structural studies of the synthetase complex.

V. Primary Structures of Mammalian Synthetases HisRS cDNA was the first mammalian synthetase cDNA that was cloned and sequenced (Tsui and Siminovitch, 1987; Raben et aL, 1992). AspRS cDNA was the first cDNA of the synthetases in the synthetase complex that was cloned and sequenced (Jacobo-Molina et aL, 1989; Mirande and Waller, 1989). The cDNA sequences of GluProRS (Fett and Knippers, 1991), LysRS (GenBank, file cllysrs.gb_ro), ArgRS (Lazard and Mirande, 1993), ValRS (Hsieh and Campbell, 1991; Vilalta et aL, 1993), IleRS (Shiba et aL, 1994b; Nichols et aL, 1995), ThrRS (Cruzen and Arfin, 1991), CysRS (Cruzen, 1993), TrpRS (Lee et aL, 1990; Garret et aL, 1991), GlyRS (Ge et aL, 1994; Shiba et aL, 1994a; Williams et aL, 1995), and GlnRS (Lamour et aL, 1994) have since been determined. Surprisingly, ProRS was found to occur as a fusion protein with the GluRS (Cerini et aL, 1991; Fett and Knippers, 1991). Newly evolved sequences were found in all mammalian synthetases. Among the nine synthetases in the synthetase complex, AspRS(rat and human), ArgRS (CHO), LysRS (OHO), IleRS (human), GluProRS (human and Drosophila), and GlnRS (human) have been cloned and sequenced.

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The sequences of MetRS and LeuRS are not known at present, but they will likely become available in the near future. The sequence data indicated that the core catalytic domains of bacterial and yeast synthetases are largely preserved in all mammalian synthetases with the exception of GlyRS, which does not show significant homology with the E. coli counterpart (Ge et al, 1994; Shiba et al.y 1994a). The identity was more than 50% in core catal3^ic domains. The signature sequences and the structural motifs for synthetases are highly conserved. Several mammalian synthetases, such as AspRS, ArgRS, and GlyRS, do not aminoacylate the bacterial or yeast tRNAs. The newly evolved sequences in the core catalytic domains are likely to play roles in differentiating mammalian tRNA from bacterial and yeast tRNAs. Such sequences have not been well defined in mammalian or yeast synthetases. Several structural motifs, such as the proteolytic signal sequences, PEST sequences, protein kinase phosphorylation sites, and glycosylation sites, were readily recognized. However, the physiological significance of such structural motifs has yet to be examined. Analyses of the known primary structures revealed another distinct structural feature of the mammalian synthetases besides the occurrence of the synthetase complex: the presence of the N- or C-terminal extensions beyond the core catalytic domains. Since studies of the synthetase complex using hydrophobic interaction chromatography and controlled proteolysis indicated that extensions in several synthetases are involved in their association with the synthetase complex, analyses of the roles of the extensions are critically important toward our understanding of the structural organization and the function of the synthetase complex. Extensions varjdng from 30 residues to 40 kDa were found at the N and C termini of mammalian synthetases. Some of these extensions have been shown to be dispensable for enz3mie catalysis and will be discussed later. The structures of the extensions in the known sequences appear to be idiosyncratic. In the case of yeast HisRS, it has been clearly demonstrated that the N-terminal extension was used as a signal peptide for the transport of the nuclear-coded mitochondrial HisRS (Natsouhs et aL, 1986; Chiu et aL, 1992). It should be noted that deletion of the N-terminal extensions in yeast GlnRS by mutagenesis did not reveal any significant effect on cell growth (Ludmerer and Schimmel, 1987) or enzyme properties (Ludmerer et al, 1993).

VI. Distinct Characteristics of N-Terminal Extensions in Mammalian Aminoacyl-tRNA Synthetases On the bases of the primary structures of mammalian synthetases, two distinct features of the extensions have emerged. One is the pres-

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ence of highly charged, putative amphiphiHc heHces, and the other is sequences homologous to subunits of the elongation factor 1. The extension in human AspRS was the first extension which was found to be highly charged and to favor the a-helical secondary structure (Jacobo-Molina et al., 1989). The resulting helices showed the clustering of charged residues on one face of the helix and the clustering of uncharged or hydrophobic residues on the other face (Fig. 2). Similar amphiphilic helices were subsequently found in human ValRS (Hsieh and Campbell, 1991; Vilalta et al, 1993), hamster ArgRS (Lazard and Mirande, 1993), human GluProRS (Fett and Knippers, 1991), and LysRS (Fig. 2). In contrast to common amphipathic helices involved in lipid-protein interactions (Eisenberg et al, 1982; Segrest et al, 1990), the amphipathic helices in the extensions in mammalian synthetases, except that in ArgRS, are not highly hydrophobic (Lazard and Mirande, 1993). These helices can provide the necessary structurally complementary elements in the association of the synthetases. In addition to the occurrence of the highly charged amphiphilic helices in the N-terminal sequences of mammalian RSs, several synthetases contain sequences that are homologous to those in the elongation factor 1 (EFla). These included HisRS, GluProRS, ValRS, and TrpRS. The most dramatic case is the spacer sequence between the core cataIj^ic domains in the fusion protein of Glu- and ProRSs (Fett and Knippers, 1991). There are three 57-amino acid residue repeats in human GluProRS that are homologous to the EFla sequence. Six similar repeats were found in GluProRS from Drosophila (Cerini et al., 1991). The functions of such sequences are unknown but they may be involved in the channeling of aminoacyl-tRNA or in the regulation of protein biosynthesis. In vivo studies of translation using radioactively labeled amino acids or aminoacyl-tRNA have demonstrated the channeling of aminoacyltRNA in translation (Negrutskii and Deutscher, 1991). However, the mechanism and the structural entities involved in the channeling remain unknown. Precise information has been very limited with regard to the mammalian synthetase complex formation, its structural organization, and its interaction with the translation machinery: Are all extensions of different synthetases associated as an organization core in the synthetase complex? Are extensions able to bind RNA, EFla, and synthetases? Do direct transfers of aminoacyl-tRNAs from synthetases to EFla occur in the synthetase complex? If they do, how are the different structural modules in the extensions in synthetases interacting with RNA, EFla, and neighboring synthetases? As the sequences in the extensions of seven s)nithetases have become known, investigations addressing these questions have begun. The results of recent studies

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FIG. 2. Amphiphilic helices predicted in the N-terminal extensions in mammaUan aminoacyl-tRNA synthetases in the synthetase complex.

MAMMALIAN AMINOACYL-tRNA SYNTHETASES Arg RS 41-66

111

Hum Glu RS 691-705

FIG. 2. Continued

of the entire complex and specific synthetases are discussed in the following sections.

VII. Functional Significance of Synthetase Complex The question as to why these synthetases are in a complex is not clear. The amino acids specified by the synthetases in the complex do not correlate with their genetic codons, amino acid transport systems, essential amino acids, or the classification of prokaryotic synthetases.

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The functional significance of the synthetase complex has been examined at four different levels: aminoacylation activity, catalytic activities other than aminoacylation of tRNA, the enzyme compartmentalization, and the regulation of protein S3aithesis. Quantitative differences between free and complexed synthetases were found in the catalytic efficiencies of the signal nucleotide AppppA synthesis and of the lysylation of tRNA (Wahab and Yang, 1985a,b, 1986). Appreciable differences between the free form and the complexed form of various synthetases were reported (Dang et al, 1985) in the aminoacylation activity and thermal and oxidative stability. The synthetases in the complexed form also showed increased fidelity in aminoacylation (Kusama-Eguchi et al, 1991) and better ability in differentiating isoacceptors (Lindqvist et a/., 1989). MetRS in the complexed form was shown to associate with the detergent-resistant endoplasmic reticulum, as judged by immunochemical subcellular localization using monospecific antibodies (Dang et al., 1983,1985). Similar studies of PheRS, which is a free synthetase, revealed that the free synthetase was appreciably less likely than the synthetases in the complex to associate with the detergent-resistant ER (Mirande et al, 1985a). To date, no in vivo studies on the functional significance of the synthetase complex have been reported, with the exception of perhaps AspRS (Escalante et a/., 1994). VIII. Aspartyl-tRNA Synthetase Cloning and sequencing of mammalian synthetases opened new ways of stud3dng the assembly and the function of the multienzyme complex of RSs. Comparison of the sequences of yeast and mammalian AspRS revealed a new N-terminal sequence that was absent in yeast AspRS. The N-terminal sequence in human AspRS was found to favor a-helical secondary structure and the resulting helix surprisingly gave a unique amphipathic helix. The amphipathic helix was proposed to play key roles in the assembly of the S3mthetase complex (Jacobo-Molina et al., 1989). The putative amphiphilic helix contains a highly charged face that may interact with proteins, nucleic acids, or phospholipids. The N-terminal extension in human AspRS could conceivably be functional in several aspects, including the catalytic cycle of aminoacylation, the synthetase-tRNA interaction, the synthetase-synthetase association, the synthetase-elongation factor la interaction, and the synthetasec)^oskeleton interaction. Supporting evidence for the involvement of the N-terminal extension in four of the five above-mentioned aspects

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has been obtained. The experimental approaches and the results are summarized here. A. Bacterially Expressed Human AspRS

Human AspRS and an N-terminal 32-residue-deleted form (AspRSA32) were expressed in E, coli as glutathione S-transferase (GST) fusion proteins linked through a thrombin cleavage site (Escalante and Yang, 1993). The glutathione S-transferase-AspRS fusion proteins were purified by affinity chromatography on glutathione-agarose and were fully active in aspartylation of mammalian tRNA and the aspartic acid-dependent ATP-PPj exchange. After cleavage of GST from the fusion proteins by thrombin, free AspRS was purified by affinity chromatography on tRNA-Sepharose. Successful production of AspRS in E. coli provided the free form of a mammalian synthetase in the synthetase complex for the structural and functional analysis omitting the complications of the microheterogeneity of the synthetase complex and the effects of other synthetases or nonsynthetase components in the synthetase complex. Comparison of the full-length and truncated forms of AspRS should reveal the structure and function of the N-terminal extension in AspRS. B. Effects of N-Terminal Extension on Catalysis of Aminoacylation, Binding Properties, and Thermostabiiity

Both bacterially expressed AspRS and AspRSA32 were present as mixtures of monomeric and dimeric forms. Both forms of AspRS bound to hydrophobic interaction gels such as aminohexyl-agarose. The fulllength form bound to aminohexyl-agarose more weakly than the truncated form in the absence of propylene glycol. However, in the presence of propylene glycol, the full-length form bound tighter suggesting dynamic accessibility of the N-terminal extension in AspRS (Escalante and Yang, 1993). Such d5niamic properties of the N-terminal extension can provide the operative segmental flexibility to play versatile roles in the functioning of the synthetase. Compared to AspRSA32, AspRS showed appreciably greater thermal stability and greater ATP-PPj exchange activity but lower aminoacylation activity. The catalytic constant of AspRS for aminoacylation of tRNA was at least twofold higher than that of AspRSA32. The Michaelis-Menten constants for aspartic acid and tRNA^P were 302 )LtM and 13 nM for AspRS, and 29 fiM and 130 nM for AspRSA32, respectively. These results suggest that the N-terminal extension in AspRS may modulate the enzymatic activity, the stability, and the binding capacity

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of AspRS (Escalante and Yang, 1993). The stabilization of the AspRS by the N-terminal extension result from its binding to and the strengthening of the interaction between the N-terminal domain and C-terminal core catalytic domains, assuming that the three-dimensional structure of human AspRS is similar to that of the yeast enzyme (Ruffed a/., 1991). The results also raised the possibility that the N-terminal extension is involved in the release of Asp-tRNA, which would be consistent with the observed decreases in K^for tRNA and the decrease in the catalytic constant of aspartylation by the full-length AspRS. C. Synthetase-Aminoacyl-tRNA Interaction The kinetics of aspartylation of mammalian tRNA^P by AspRS was further examined to address the question as to how the N-terminal extension affected the catalysis of aminoacylation (Reed et a/., 1994). The single-turnover time courses of aspartylation of tRNA were consistent with the following reaction pathway (Scheme I): E

k, [Asp] [ATP] K [tRNA] h > E^Asp-AMP. ^.Asp-tRNA'AMP - ^ E + Asp-tRNA + AlVIP

where E represents AspRS. A set of rate constants that best fit the single-turnover time courses at var3ring concentrations of the enzyme, tRNA, and AMP was obtained using the simulation and modeling SAAM program (Berman et aL, 1983). The dissociation of Asp-tRNA (^3) was found to be rate limiting. The dissociation of Asp-tRNA from AspRSA32 was much faster than that from AspRS. Yet, the rate constants of aspartyladenylate formation and Asp-tRNA synthesis by AspRSA32 were similar to those by AspRS. These results, in combination with those of AspRS, suggested that the N-terminal extension is capable of binding and slowing down the release of Asp-tRNA (Reed et ai, 1994). The slow dissociation of Asp-tRNA from AspRS prompted the examination of the effects of EFla. EFla and GTP stimulated AspRS. The stimulation depended on the presence of both EFla and GTP. Singleturnover time courses of aspartylation of tRNA in the presence of EFla were consistent with the reaction scheme in which EFla formed a transient complex with the AspRS Asp-tRNA complex and stimulated the dissociation of Asp-tRNA. Direct transfer of Asp-tRNA from AspRS to EFla was demonstrated using the membrane-binding assays (Yams and Berg, 1970) for the binding of Asp-tRNA and using the ribonuclease resistance assay for EF la-specific binding of Asp-tRNA (Knowlton and Yams, 1980). Unlike the full-length AspRS, AspRSA32

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was not affected by EFla (Reed et al., 1994). The results provided the first piece of evidence for the channehng of Asp-tRNA from AspRS to EFla without the dissociation of Asp-tRNA from AspRS into the solvent before the reassociation of Asp-tRNA by EFla. Figure 3 describes such a model. This model proposes that the N-terminal extension keeps Asp-tRNA from releasing from AspRS into the solvent and that the

+ ASP

f

RSASP-TRNA

111

EFla-GTP

I II

t

II I

+ ASP RS

ASP-TRNAEFlaGTP

FIG. 3. Model of the transfer of Asp-tRNA from AspRS to E F l a - G T P complex. The structures of tRNA-AspRS complex and E F l a - t R N A are schematically drawn according to the yeast AspRS crystal structure (Ruff et al., 1991) and the E F l a - t R N A complex model (Kinzy et al., 1992). The zigzag line represents the extension of AspRS, which spans the N domain and C domain of AspRS, interacts Asp-tRNA and EFla-GTP, and mediates the transfer of Asp-tRNA from AspRS to EFla. Reprinted from Reed and Yang (1994) with permission from ASBMB.

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N-terminal extension interacts with EFla such that Asp-tRNA can be released from AspRS. D. Synthetic Peptides of N-Terminal Extension in AspRS

Two N-terminal peptides of human AspRS, AspRS(T5-E26) and AspRS(D12-R27), were synthesized to determine whether the N-terminal extension in AspRS can form an amphipathic helix and whether it can bind to tRNA or EFla (Reed and Yang, 1994). Although the peptide AspRS(T5-E26) did not form a helix as analyzed by CD spectroscopy, AspRS(D12-R27) in which additional features were added to minimize the electric dipole of the helix; showed appreciable a-helical contents in nonpolar solvents. The a-helix formation of the N-terminal peptide is consistent with the hypothesis that the N-terminal extension in AspRS folds as an amphiphilic helix. The synthetic peptides provided new tools for studying the roles of the N-terminal extension in the functioning of AspRS. Both peptides bound to tRNA-Sepharose. Highly purified EFla also bound to immobihzed peptides AspRS (T5-E26). The peptide AspRS(D12-R27) inhibited AspRS but not AspRSA32, suggesting that AspRS(D12-R27) bound to the N-terminal sequence in AspRS. Both peptides, AspRS(T5-E26) and AspRS(D12-R27), were monomeric and oligomerized at a high peptide concentration or in 50% propylene glycol (Reed and Yang, 1994). These results explained the observed dynamic accessibility of the N-terminal extension of AspRS and also suggested that the N-terminal extension preferentially self-associated in nonpolar environments, likely through stronger electrostatic interactions of the charged residues. E. Implications on Functional Significance of Synthetase Complex

The capability of the N-terminal extension to bind to EFla and to mediate the transfer of Asp-tRNA from AspRS to EFla led to the hypothesis that the function of the synthetase complex involves the channeling of aminoacyl-tRNAs from synthetases to EFla. The requirement of 0.5 M NH4CI for a direct in vitro transfer of aminoacyl-tRNA from AspRS to EFla could be facilitated by the clustering of highly charged extensions in vivo from various synthetases in the synthetase complex. Sequences of EFla from various species are highly conserved. The three-dimensional structure of the bacterial elongation factor Tu has been determined (Kjeldgaard and Nyborg, 1992). A structural model for the EFla-tRNA complex was proposed based on studies by chemical cross-linking and controlled proteolysis (Kinzy et a/., 1992). The contact surface in tRNA for EFla binding is apparently different from that

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shown in the 3D structures of the cocrystal of yeast AspRS and tRNA'^p (Russ et a/., 1991) and that of E. coli GlnRS and tRNA^*" (Rould et al, 1989). The mechanism of the transfer of aminoacyl-tRNA from RSs in the synthetase complex to EFla remains to be investigated. The structure and the mechanism for the transfer of charged tRNA in the synthetase complex are probably complex, since the N-terminal extensions are also likely involved in the association of synthetases with other synthetases and components in the synthetase complex (Mirande et ai, 1992). F. Association, Regulation, and Subcellular Localization of AspRS

Most definitive evidence for the involvement of the N-terminal extension in the association of AspRS to the sjnithetase complex was obtained by expressing both full-length and N-terminal truncated forms of AspRS in CHO cells. Only the full-length form was found to associate with the synthetase complex (Mirande et al., 1992) as analyzed by gel filtration and Western blot analysis using monospecific antibodies against AspRS. However, insertion of the N-terminal sequence of rat AspRS to yeast LysRS failed to associate yeast LysRS with the synthetase complex in CHO cells. These results demonstrated that the Nterminal sequence in rat AspRS is necessary but not sufficient for the specific association with the synthetase complex. AspRS of human was also transiently expressed in COS cells (Escalante et al., 1994). Expression of full-length AspRS cDNA in COS cells resulted in a moderate enhancement of AspRS as a low-molecularweight form, as analyzed by gel filtration. This result refuted the skepticism that the existence of the synthetase complex resulted from the nonspecific association of synthetases during homogenization or purification. The nonaggregation of the overproduced AspRS demonstrated that the synthetase complex could accommodate a saturating amount of AspRS, which in turn suggested that the complex had defined stoichiometry and was not a result of nonspecific association. Northern analysis of the AspRS transcripts in the transfected COS cells showed that the level of the transcripts greatly enhanced while the level of the protein only moderately increased. The absence of proportional increases of AspRS suggested the regulation of the synthesis of AspRS at the posttranscriptional level. Autorepression of the synthesis of ThrRS at the translational level was observed in bacteria (Springer et ai, 1985). In mammalian cells, the rate of protein turnover may be an important factor. As analyzed by myc epitope tagging and immunofluorescence microscopy, both AspRSmyc and AspRSA32myc were found to localize in the

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cytoplasm and show diffused patterns. The N-terminal extension in AspRS was Ukely not involved in the expression or subcellular localization of AspRS, but it may play a role in the maintenance of the enzymatic activity of AspRS in COS cells (Escalante et al, 1994).

IX. Arginyl-tRNA Synthetase ArgRS occurs in two structurally distinct forms (Deutscher and Ni, 1982; Cirakoglu and Waller, 1985c; Sivaram and Deutscher, 1990). The larger dimeric form (subunit Mr, 74,000) is an integral component of the sjmthetase complex (Mirande et ai, 1982), and the smaller free form (subunit M^. 60,000) is monomeric (Deutscher and Ni, 1982). The occurrence of two forms of ArgRS and the presence of Met at the N terminus of the 60K form led to the proposal of distinctive roles for the two forms in mammalian cells (Sivaram and Deutscher, 1990). Ciechanover and co-workers (1985) found that tRNA stimulated the ATP- and ubiquitin-dependent protein degradation. The action of tRNA relied on the ability of few aminoacyl-tRNA protein transferases to ligate basic amino acid residues to the N termini of proteins (Ferber and Ciechanover, 1987; Ciechanover et al., 1988). According to the N end rule (Bartel et al., 1990), the proteins modified by certain residues at the N termini became substrates for the ubiquitination enzymes and consequently became substrates for the 26S proteasome (Hochstrasser, 1992). It was proposed that the free form of ArgRS aminoacylates tRNAs that are used in the ubiquitination pathway, while the complexed form of ArgRS is designated for aminoacylation in protein biosynthesis (Sivaram and Deutscher, 1990). Cloning and sequencing of mammalian ArgRS revealed that the free form and the complexed form of ArgRS shared the same transcript (Lazard and Mirande, 1993). The partial N-terminal amino acid sequence of the 60K form of ArgRS was identical to the predicted amino acid sequence of ArgRS initiated at the second methionine residue. Whether the free form was compartmentalized in vivo for arginylation of the N termini of proteins targeted for degradation is yet to be determined. In the connection of ubiquitination, it should be noted that the association of the synthetases with the arginyl-tRNA protein transferase has been reported (Ciechanover et ai, 1988). Two regions (S9-L29 and L41-K66) in the N-terminal sequence of ArgRS were predicted to be a helices, and the resulting helices are amphiphilic and contain highly hydrophobic faces that resemble the leucine repeats in transcription factors (Fig. 2). Because the 60K ArgRS does not associate with the synthetase complex, the N-terminal exten-

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sion in the full-length ArgRS is implicated in mediating the association of ArgRS with the synthetase complex. ArgRS was selectively dissociated from the complex after a brief incubation of the complex in 0.5 M NaSCN (Norcum, 1991). The leucine repeat-like structures may play roles in the oligomerization of ArgRS as demonstrated in DNA-binding proteins (Kerpolla and Curran, 1991) or in the RNA-protein interactions as demonstrated in bacterial SerRS (Biou et ai, 1994). The revelation of the existence of such amphiphilic helices provided additional support for roles of the amphiphilic helices in synthetase-synthetase interactions. Purification of intact free ArgRS dissociated from the synthetase complex is yet to be accomplished. Attempts to purify dissociated ArgRS were complicated by the fact that ArgRS dissociated from the complex was highly hydrophobic and unstable, likely due to the presence of highly hydrophobic N-terminal extension. A comparison of the thermostability and hydrophobic properties of the high- and low molecular weight forms of rabbit liver ArgRS (Berbec and Paszkowska, 1989) confirmed earlier results (Dang et al., 1985) that the complexed form is more stable and more hydrophobic than the free form.

X. Lysyl-tRNA Synthetase LysRS in mammalian cells occurs exclusively in the synthetase complex (Cirakoglu and Waller, 1985c). However, purification of mammalian C5rtoplasmic LysRS yielded at least five different forms with different degrees of association with other synthetases which include a free intact form (Johnson et al, 1980; Cirakoglu and Waller, 1985c), a free truncated form (Brevet et al,, 1982), the subcomplex of LysRS and ArgRS (Hilderman et a/., 1983), the complex of Lys-, Arg-, He-, Leu-, and MetRS (Vennegoor and Bloemendal, 1972; Som and Hardesty, 1975), and the complex with nine s)mthetases. The complexed form of LysRS bound to the hydrophobic interaction gels and evidently contained a hydrophobic extension (Cirakoglu and Waller, 1985b). The complexed form was also more stable but less active in aminoacylation and in AppppA synthesis (Wahab and Yang, 1985a,b) than the free form. The hydrophobic extension appeared to only be present in the mammalian LysRS and not in the yeast LysRS (Cirakoglu and Waller, 1985b). The LysRS cDNA from CHO cells has been cloned and sequenced (cllysrs.gb_ro). Surprisingly, no N-terminal extension beyond the yeast sequence was found. Instead, the N-terminal sequence of CHO LysRS contains a putative amphiphilic helix that does not appear

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to contribute more hydrophobicity than that which is already present in the yeast LysRS.

XI. GluPro-tRNA Synthetase ProRS was one of the few synthetases that were not consistently found in the synthetase complex in early studies (Mirande et al., 1985b). ProRS dissociated early in a disassembly scheme of the synthetase complex (Dang and Yang, 1979) and the amount of the free form increased with the severity of homogenization conditions (Garcia, 1984). Similarly, GluRS was absent in a number of purified complexes (Vennegoor and Bloemendal, 1972; Som and Hardesty, 1975) and dissociated early in a disassembly scheme (Dang and Yang, 1979). Keijan et al. (1992) found that ProRS coeluted with the synthetase complex when livers were homogenized using an electric meat grinder, but it eluted as a free form of ProRS when a Waring blender was used in the absence or presence of a battery of protease inhibitors. The conversion of ProRS to the low-molecular-weight form was associated with the loss of the 160K component in the synthetase complex. The free forms of ProRS purified from sheep and rabbit livers were homodimers with a subunit molecular weight of 85,000. The free form (Mr 85,000) crossreacted to the 160K component in the complex based on the Western blot analysis using antibodies against the free form. The same conclusion was arrived at in a study of ProRS from rat liver except that the free form in rat was found to have a lower subunit molecular weight (Mr 60,000) (Ting et al., 1992). The difference could be because of the omission of the protease inhibitor, DIFP, in the homogenization buffer or because of the use of a different species. Molecular weights close to 60,000 were reported in another study of rat liver ProRS (Bianchi et a/., 1992). Interestingly, ProRS was found to be a heterodimer with subunit molecular weights of 58,000 and 61,000, which were the same as those previously found (Garcia, 1984; Godar et al, 1988). In both studies, DIFP was not included in the homogenization buffer. ProRS cDNA was closed using antibodies against RNA polymerase (Fett and Knippers, 1991) and subsequently using antibodies against ProRS (Ting and Dignam, 1994). The cloned cDNA (4.3 kbp) was initially identified as GlnRS on the basis of the high sequence similarity to bacterial and yeast GlnRS. Activity assays of purified proteins and bacterially expressed proteins identified the protein as GluRS and the cDNA encoding GluRS and not as GlnRS. The long C-terminal extension was later identified as ProRS (Cerini et al., 1991) after the cloning and sequencing of bacterial ProRS when the sequence homology could

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be compared (Eriani et al., 1990). The discovery of the fusion of Gluand ProRS as a single peptide underscored the evolutionary pressure on the physical association of RSs. Fusion of Glu- and ProRSs in Drosophila was also reported by the cloning, sequencing, and expression of the corresponding cDNA (Cerini et al, 1991), by protein purification, and by Western Blot analysis from rat liver (Ting et al, 1992) and sheep liver (Kerjan et ai, 1992). It is now clear that the 160K component in the synthetase complex represents the fusion protein of Glu- and ProRSs. The 160K component in the synthetase complex did not always exhibit ProRS activity (Ting et al., 1992). The fragments of the 160K component from different sources were definitely active in the prolylation of tRNA (Cerini et a/., 1991; Kerjan et al., 1992; Ting et al., 1992). Thus, it is appropriate to use the term GluProRS for the 160K fusion protein. Among many findings regarding the structure of GluProRS, the most intriguing feature is the spacer sequence between the catalytic domains of the two synthetases—a tripartite repeat of 57-amino acid blocks followed by a segment of 33 hydrophilic residues including 12 Lys, 4 Glu, and 4 Asp. High sequence similarity of the repetitive sequence with the translational elongation factor la was recognized. Similar sequences were also observed in Drosophila GluProRS cDNA. The large size of the extension in GluProRS prompted the suggestion that it provides a template for the N-terminal extensions of other synthetases in the synthetase complex; thus the assembly of the synthetase complex (Cerini et al., 1991). No supporting evidence has been obtained thus far. It should be noted that MetRS (Kellermann et al., 1978) and GlnRS (Mirande et al., 1982; Ludmerer et al., 1993) are expected to have large extensions as well. Functions such as channeling of charged tRNA and interactions with ribosomes are also possible. The highly hydrophilic and Lys-rich sequence is expected to be highly susceptible to endogenous proteolytic cleavage due to the predicted high surface accessibility and the abundant Lys residues. The association of GluProRS with the synthetase complex is consequently unstable. This may account for the observations that dissociation of these synthetases occurred early in the disassembly scheme (Figure 1) and that extremely gentle homogenization conditions were required to preserve the structural integrity of the 160K protein. Searches of hydrophobic sequences in GluProRS did not reveal any regions that were particularly hydrophobic. No amphiphilic helices with highly hydrophobic faces could be found either. Thus, it is likely that hydrophobic interaction does not contribute for the most part to the association of these synthetases with the synthetase complex.

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Cloning of the genomic DNA encoding GluProRS has been achieved (Kaiser et al., 1994). The genomic sequence showed that the fusion protein was encoded by one gene spread over at least 90 kbp. The 29 exons encoding the Glu and ProRS were clustered in 8- and 10-kbp sections located at the opposite ends of the gene. The exons are clearly separated into functional domains in accord with the known crystal structures ofE. coli GlnRS (Rould et al., 1989). The transcription initiation site of the gene was preceded by a TATA box, a CCAAT box, and other regulatory signals, but the significance of these sites in the regulation of GluProRS is not clear. Northern analysis indicated that the mRNA levels are very low in resting human embryonic lung fibroblast cells but increase 10- to 20-fold after serum stimulation. The regulation of GluProRS was also examined in the rat salivary gland after isoproterenol treatment. Appreciable stimulation of GluProRS was not accompanied by concomitant increases of GluProRS mRNA (Ting and Dignam, 1994), suggesting the posttranscriptional regulation of GluProRS, such as autorepression (Springer e^ a/., 1985), and reduction of the turnover rate. The former mechanism was supported by the observation that human GluProRS bound to a specific site of its mRNA (Schray and Knippers, 1991). These results suggest that synthetases may be regulated at different levels in response to different physiological stimuli. XII. Valyl-tRNA Synthetase Complex ValRS occurs in a high Mr form that does not copurify with any other synthetases. First purification of ValRS from rat liver yielded a monomeric form with a molecular weight of 140,000 (Godar and Yang, 1988). Subsequent purification of mammalian ValRS showed that the high Mr form of the elongation factor 1 (EFIH) was associated with ValRS (Motorin et al., 1988,1991; Bee et al., 1989). The ValRS complex from rabbit liver comprises five polypeptides: ValRS (140K), a (50K), y (52K), 13 (35K), and 8 (27K) in the molar ratios of 1:1:1:1:1, respectively. The native molecular weight of the ValRS complex was estimated to be 800,000 and thus the ValRS complex contained two copies of each polypeptide. The four low Mr pol3^eptides were identical to those in EFIH as judged by two-dimensional gel electrophoresis. The ValRS complex also supported the ribosome-dependent polyphenylalanine synthesis in the presence of EF2. The subunit molecular mass of the free form of ValRS from rat liver (Godar and Yang, 1988) is approximately 5000 Da smaller than that in the complex (Motorin et al., 1988, 1991; Bee et al., 1989). The major

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difference in the purification procedures that could have made such a difference is the inclusion of the protease inhibitor DIFP in the purification of the complexed form. Apparently, the polypeptide fragment that was cleaved from ValRS is required for its association with the ValRS complex. The ValRS complex is more stable than the RS complex in its enzyme activity and the structural rigidity (Bee et ai, 1989). An electron microscopic study of the ValRS complex showed well-defined spherical and rectangular particles. The ValRS complex from liver dissociated into its component proteins by incubation of the complex in 0.5 M NaSCN at O^'C for 3 hr (Bee and Waller, 1989). The dissociated ValRS, the a and 8 subunits, and the Py subcomplex can be separately purified. Dissociated ValRS was fully active and stable in buffer containing 0.1% Triton X-100 and showed high affinity toward phenyl-Sepharose. These properties are very similar to those previously found for lie- and LeuRSs (Cirakoglu and Waller, 1985b; Lazard et a/., 1985). The dissociation of the ValRS complex and the purification of fully active subunits (Bee and Waller, 1989) paved the way for the reconstitution of the complex. A subcomplex was reconstituted from ValRS, jSy subunits, and the 8 subunit. The reconstitution of the ValRS complex required the 8 subunit and the N-terminal extension in ValRS (Bee et al., 1994). The results strongly suggested that the association of the ValRS complex is mediated by the N-terminal extension in ValRS and the 8 subunit in the complex. Unfortunately, the study did not include the a subunit, which binds the product of ValRS. Cloning and sequencing of mammalian ValRS cDNA has been achieved (Hsieh and Campbell, 1991; Vilalta et al, 1993). The human gene encoding ValRS for no obvious reasons is located in the major histocompatibility complex class III region (Hsieh and Campbell, 1991). The predicted amino acid sequence of ValRS contained 295 additional residues in the N-terminal sequence extended beyond the corresponding £. coli ValRS. Of the 295 residues, the sequence 156-295 is homologous to the sequence of yeast ValRS and the sequence 1-155 in human ValRS is newly evolved. The sequence between the first and the second methionine residues contains two regions that have high hydrophobicity and are homologous to EFly. Since yeast ValRS was isolated and purified as a free soluble enzyme (Borgford et ai, 1987; Jordana et aL, 1987), the N-terminal extension in mammalian ValRS appears to be required for the physical association of ValRS with the elongation factor 1. The necessity of the inclusion of DIFP in the extraction buffer to maintain the integrity of ValRS and its association with the elongation

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factor 1 is consistent with roles of the N-terminal sequence in its association with the elongation factor 1. Both yeast and mammalian ValRSs bound to heparin-Sepharose (Bee and Waller, 1989). A stretch of hydrophihc residues (228-298 was found between the second (M155) and third methionine (M295) residues in human enzyme (Hsieh and Campbell, 1991; Vilalta et al, 1993). This stretch of basic residues likely contributed to the heparin-binding capability (Cirakoglu and Waller, 1985a). Synthetic peptides within ValRS also exhibited high affinity toward the microtubule-associated protein T and enhanced the stability and the bundling of microtubules (Melki et aZ., 1991). The functional significance of the association of ValRS with the elongation factor 1 has been examined by comparing the Michaelis-Menten constants and the catalytic constants of the dissociated and the complexed forms of ValRS. No significant difference was found (Bee et a/., 1989). When the activity of EFl in the ValRS complex was compared with that of the EFla subunit, the ValRS complex was about 10 times more active. This was attributed to the presence of EFljS and EFly in the complexed form (Bee et ai, 1989). Interactions between the elongation factor 1 and ValRS were revealed by phosphorylation of the ValRS complex by protein kinase C which enhanced the EFla activity threefold (Venema et al, 1991a,b; Venema and Traugh, 1991). Although the EFla subunit in the complex was phosphorylated only up to 20%, the (3 and y subunits were phosphorylated to 0.5-0.9 mol of phosphate per mole of subunit; thus, it has yet to be determined if the increase of the activity was due to the phosphorylation of EFl or indirectly through the phosphorylation of ValRS in the complex. The amino acid sequence of ValRS suggested a number of potential sites of phosphorylation (Vilalta et ai, 1993). However, the sites of phosphorylation in ValRS have not been determined. XIII. Tryptophanyl-tRNA Synthetase TrpRS is a dimeric, metalloglycoprotein and the smallest S3nithetase in mammalian cells (Kisselev, 1993). TrpRS was surprisingly found to be present in over 2% of cellular proteins in bovine pancreas (Favorova et al, 1989). The abnormally high concentration of TrpRS in the pancreas of ruminants, but not other mammals, suggested an unknown function of this protein in the digestion systems of these mammals. TrpRS cDNA has been cloned and sequenced from rabbit (Lee et ai, 1990), beef pancreas (Garret et al,, 1991), and human fibroblast (Fro-

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lova et ai, 1991; Rubin et al., 1991). The predicted amino acid sequences of mammalian TrpRS also contain the N-terminal extensions that exhibited amphiphilicity in the resulting helices (Kisselev, 1993). However, the charges in the resulting helices are distributed differently— qualitatively and quantitatively—from those in the extensions of the synthetases in the synthetase complex. Further studies are needed to clarify the roles of these helices in the functioning of mammalian synthetases. The N-terminal extension in TrpRS was attributed to unexpected ATPase activity in TrpRS (Kovaleva et al., 1993). Deletion of the N-terminal extension abolished the ATPase activity, but the isolated N-terminal domain alone, not unexpectedly, did not show ATPase activity. The cDNA sequence and the predicted amino acid sequence of TrpRS were initially found to be identical to those of the translation release factor (Lee et al., 1990). However, the amino acid sequence of TrpRS showed little homology to either the yeast or the E. coli release factors and was lacking the characteristic motifs of the release factors. Recent studies showed conclusively that TrpRS is not the translation release factor (Frolova et al., 1993a; Timchenko and Caskey, 1994). The human TrpRS gene has recently been isolated and partially characterized (Frolova et al., 1993b). The TrpRS gene was activated by both type I and type II interferons (Buwitt et al., 1992). After the activation of the TrpRS gene by interferon, the TrpRS protein was enhanced moderately, while the transcripts showed dramatic increases. The relatively low level of the increase of TrpRS compared to the large increase of the transcripts resembles the observed result of the transient expression of AspRS cDNA in COS cells (Escalante et al, 1994). No other synthetase is known to be stimulated by interferon. The yinterferon response element was located in the sequence flanking the TrpRS transcription initiation site and the involved transcription factors have been identified (Seegert et al., 1994). The effect of interferon on the tryptophan metabolism has been documented (Taylor and Feng, 1991). One consequence of the interferon induction of TrpRS might be a reduced availability of tryptophan, but several other possibilities have been discussed (Kisselev et al., 1993; Kisselev, 1993). Additional gene controls are now found at the level of alternative splicing which resulted in a different form of TrpRS with a C-terminal six-residue extension (Pajot et al., 1994; Tolstrup et al, 1995). XIV. Seryl-tRNA Synthetase SerRS has been purified from a number of sources including bovine mammary gland (Mizutani et al., 1984), rabbit reticulocytes (Dang and

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Traugh, 1989), rat liver (Fahoum, 1985), and hen liver (Le Meur et aL, 1972). SerRS is a dimeric enzyme with a subunit molecular weight of 60,000 for all sources except the bovine mammary gland. SerRS cDNA from mouse was cloned using a probe designed for a component in an mRNP complex (Miseta et al., 1991) that was thought to be involved in either splicing or regulation of protein biosynthesis (Slobin and Greenberg, 1988), and the component in the mRNP was identified as SerRS by Western blot analysis and by assaying of SerRS activity. These results suggested an additional function for mammalian SerRS unrelated to aminoacylation of tRNA. SerRS apparently serylates not only tRNA^"^ but also tRNA^s, which use nonsense codons to encode seleno-cysteine (Wu and Gross, 1993; Ohama et al., 1994), suggesting that the expansion of the genetic code to include seleno-cysteine is largely conserved in mammalian systems.

XV. Threonyl-tRNA Synthetase ThrRS has been purified from rat liver (Dignam et a/., 1980), rabbit reticulocytes (Dang and Traugh, 1989), and CHO cells (Gerken and Arfin, 1984) and is a dimeric enzyme with a subunit molecular weight of 85,000. ThrRS reacted with the PL-7 autoantibody from the myositis patients (Mathews et al., 1984). ThrRS catalyzes the AppppA S3nithesis at a rate comparable to that of LysRS, and the rate of AppppA S3nithesis activity was enhanced up to sixfold on enzymatic phosphorylation by protein kinase A (Dang and Traugh, 1989). The ThrRS gene in CHO cells was amplified in the presence of the ThrRS inhibitor borrelidin (Gantt et ai, 1981). Chinese hamster ThrRS cDNA has been cloned and sequenced, and showed high identity to the bacterial and yeast ThrRS (Cruzen and Arfin, 1991).

XVI. Correlation of the Classifications of Amino Acids and Mammalian Aminoacyl-tRNA Synthetases The functional significance of the association of RSs has been a longstanding question. The question as to why the nine synthetases are chosen remains unanswered. It is first necessary to uncover the correlation of the nine S3nithetases; only then will we be able to formulate testable hypotheses. As noted previously, the nine S5nithetases do not correlate with their genetic codons, the amino acid transport systems, the amino acid nutritional requirements, or the classification of RSs. However, the nine amino acids specified by the nine S5nithetases appear to correlate with the classification of amino acids according to their

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physicochemical properties and the replacement mutation frequencies (Taylor, 1986). The amino acids specified in the synthetase complex are grouped on the left (He, Leu, and Met) and right (Lys, Arg, Glu, Asp, and Gin) sides of the Venn diagrams of the various groups of amino acids. Amino acids that are considered small (Cys, Thr, Asn, and Val), tiny (Gly and Ala), or aromatic (Phe, Tyr, Trp, and His) are not specified in the synthetase complex with the exception of Pro (small). Furthermore, the closely related amino acids in the van diagram are specified by subcomplexes of the synthetase complex. LysRS and ArgRS form a subcomplex, as do LeuRS and IleRS (Dang and Yang, 1979; Hilderman et al., 1983). It is intriguing that the synthetases in the complex correlate with amino acids with similar physicochemical properties. The correlation with the physicochemical properties of amino acids raises the possibility that the S3nithetase complex may contain structures which facilitate the transfer or the discrimination of similar amino acids. The synthetase complex might contain such a structure to presort amino acids according to their physicochemical properties. This hypothesis provides a new framework toward the elucidation of the functional significance of the synthetase complex. This hypothesis also concurs with the observation of channeling of amino acid in protein biosynthesis and the channeling of aminoacyl-tRNA from synthetases to EFl (Negrutskii and Deutscher, 1991; Reed et al, 1994).

XVII. Autoantibodies to Mammalian Synthetases Autoantibodies to nuclear and c3rtoplasmic antigens are found in a high proportion of patients with polymyositis and dermatomyositis (TargoflF, 1992). Many of the autoantibodies were against RSs. AntiJO-1 antibodies were the first autoantibodies found that specifically inhibited RSs and, in this case, HisRS (Mathews and Bernstein, 1983). Autoantibodies against ThrRS (Mathews et ai, 1984; Okada et al, 1984), AlaRS (Bunn and Mathews, 1987), GlyRS, and IleRS in the RS complex (Targoff, 1990) were subsequently identified. Susceptibility to autoimmune diseases is strongly linked to certain major histocompatibility antigen class II sequences (Todd et al., 1988). These autoantibodies were in most cases characterized by the immunoprecipitation of the synthetase and tRNA and inhibition of the aminoacylation activities, and in the cases of HisRS (Anti-JO-1) and AlaRS (Anti-PL7) by epitope mapping (Bunn and Mathews, 1987; Ramsden et al,, 1989). It should be noted that these autoantibodies showed appreciably higher affinity toward synthetases than those produced in rabbits (Fahoum and Yang,

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1987), and that autoantibodies invariably recognize the synthetasetRNA complexes. The ethiology of these autoantibodies has long been speculated to involve host viral RNA (Targoff, 1992).

XVIII. Aminoacyl-tRNA Synthetases as Multifunctional Proteins A number of cytoplasmic synthetases that included HisRS and ValRS are known to be encoded by the same gene that encodes the mitochondrial synthetases (Natsouhs et aL, 1986; Kubelik et al, 1991). LysRS, PheRS, ThrRS, SerRS, and a few other synthetases synthesize signal nucleotides, such as AppppA, which appear to be involved in cell growth and heat shock (Zamecnik, 1983). TrpRS does not S3nithesize AppppA but does synthesize ApppA. MetRS catalyzes the synthesis of cysteine thiolactone as part of the physiologically important editing mechanism (Jakubowski and Goldman, 1992). TyrRS is required for efficient mitochondrial RNA splicing (Kamper et al, 1992). TrpRS was overproduced in bovine pancreas and its fragments were secreted into the pancreatic ducts for unknown biological activity (Favorova et al., 1989). The TrpRS synthesis was selectively stimulated by a- and y-interferon (Rubin et al, 1991; Seegert et a/., 1994). Arg- and HisRSs supply arginyl and histidyl residues for the N-terminal modification of proteins by aminoacyl-tRNA protein transferases (Ferber and Ciechanover, 1987). The N-terminal modified proteins are targeted for the ubiquitin- and ATP-dependent proteolysis and exhibited drastically reduced half-lives (Hershko and Ciechanover, 1986). Age-dependent accumulation of synthetase complexes has been observed (Takahashi and Groto, 1987). Temperature-sensitive mutants of RS in CHO cells were shown to be defective in amino acid transport (Moore et al,, 1977). The extent of aminoacylation of tRNA"^^ has been suggested as a recognition signal in mammalian cellular protein degradation (Scomik, 1984; Ferber and Ciechanover, 1987). Many tRNAs are used as primers for reverse transcriptase during the life cycles of retroviruses including human immunodeficiency virus (Goff, 1990) and many viral RNAs can be aminoacylated by synthetases (Haenni et al., 1982). SerRS is found to be one of the major components in the mRNP complex (Miseta et al., 1991) In view of the fact that S3mthetases are involved in numerous reactions other than aminoacylation of tRNA, the association of synthetases as an RS complex could limit the access of certain substrates to multifunctional S3nithetases and thus prevent undesirable products or side reactions. In cases in which the secondary functions of some of the

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synthetases are needed, free forms or multiple forms of RSs occur for those synthetases to fulfill the physiological needs in mammalian cells.

XIX. Organization of Synthetases and the Protein Biosynthetic Machinery Because of the high demands of translational efficiency, fidelity, and versatility in mammalian systems, the translational machinery must be highly organized in mammalian cells. Inasmuch as the involvement of synthetases, tRNAs, and aminoacyl-tRNAs in protein biosynthesis as well as the ubiquitin- and ATP-dependent proteolysis, compartmentalization of biosynthetic components away from protein-degradative enzymes is necessary to avoid futile synthesis. The organization and compartmentalization of protein bios5nithetic machinery are important in the functioning and regulation of protein biosynthesis. All factors required for protein biosynthesis in mammalian cells including synthetases were shown to bind heparin-Sepharose (Hradec and Dusek, 1978, 1980). During the elongation cycle of protein biosynthesis, the elongation factor la appears to remain ribosome bound (Drews et al, 1977; Crechet and Parmeggiani, 1986). These observations suggest that the proteins and RNAs involved in protein biosynthesis are associated. In contrast to the conventional scheme of protein bios3nithesis involving numerous steps of dissociation and reassociation, it is not inconceivable that most components involved do not actually depart from the site of protein biosynthesis and are held together through the poly anion binding capacity of nearly all components involved in protein biosynthesis. Although the polyanions that hold all the components have not been identified, rRNA and mRNA are the most likely candidates to serve such purposes during active protein synthesis (Mirande, 1991).

XX. Prospects Mammalian RSs are obviously more complex than their bacterial or yeast counterparts. The structure and function of mammalian RSs are not well understood, despite the tremendous progress that has been made at an increasingly rapid pace. Five new sequences of mammalian synthetases (He, Gly, Cys, Gin, and Lys) were all reported in 1994 alone. The occurrence of synthetase complexes provides excellent models for the elucidation of basic principles in molecular interactions for a highly complex machinery that requires efficiency, fidelity, and versatility. Synthetases are critically important in the interpretation of the genetic

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code and are responsible for the biosynthesis of all proteins in all cells and tissues. The study of the evolution of the primary structure of the synthetases has just begun. Besides the surprising findings of the fusion protein of GluProRS and the synthetase complex, the structural divergence of the primary structures of He- and GlyRSs was beyond expectation (Shiba et aL, 1994a,b). None of the three-dimensional structures of mammalian synthetases have been determined. Studies of the synthetase complex should reveal fundamental principles of the biochemical solutions in dealing with the complexities (Srere, 1994) of protein biosynthetic machinery. The study of editing mechanisms by mammalian synthetases is currently nonexistent (Jakubowski and Goldman, 1992). The structural bases in synthetases for the heterologous aminoacylation or lacking of heterologous aminoacylation have yet to be examined. The multifunctional roles of this group of enzymes in the cellular metabolism (Zamecnik, 1983; Kamper e^ a/., 1992), the cellular immunity (Hsieh and Campbell, 1991; Targoff, 1992), and cell growth (Kisselev et aL, 1993) are not understood. Most of the synthetase genes, except those of HisRS and GluProRS, have not been isolated. Investigations of the regulation of mammalian RSs have begun. The mechanisms that coordinate the syntheses of the synthetases in the complex are yet to be elucidated. It is obvious that several mechanisms are involved in the regulation of various synthetases. We have thus far seen the involvement of phosphorylation (Traugh and Pendergast, 1986), gene amplification (Gantt et aL, 1981; Tsui et aL, 1985), activation at the transcriptional level (Rubin et aL, 1991; Seegert et aL, 1994), posttranscriptional regulation (Ting and Dignam, 1994), and perhaps autorepression (Lazard et aL, 1987; Escalante et aL, 1994). The rates of synthesis and turnover of synthetases have yet to be analyzed. At the molecular level, thus far only the specific binding of synthetase to mRNA has been studied (Schray and Knippers, 1991), although its role in regulation was not known. No generalization can be made among the many observations on the regulation of mammalian synthetases. Many amino acids are known to be multifunctional. The relation between the regulation of amino acids and the regulation of mammalian synthetases has yet to be examined. In view of the physiological roles of amino acids, elucidation of such relation is obviously important. It is evident that the research field of mammalian synthetases is wide open and future studies are expected to bring much more excitement. ACKNOWLEDGMENT I am indebted to many excellent present and former colleagues and students for their efforts towards better understanding of mammalian aminoacyl-tRNA synthetases.

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Tsui, F. W. L., and Siminovitch, L. (1987). Nucleic Acids Res. 15, 3349-3367. Tsui, F. W. L., Andrulis, P. C , Murialdo, H., and Siminovitch, L. (1985). MoL Cell. Biol. 5, 2381-2388. Vellekamp, G. J., and Deutscher, M. P. (1987). J. Biol. Chem. 262, 9927-9930. Venema, R. C., and Traugh, J. A. (1991). J. Biol. Chem. 266, 5298-5302. Venema, R. C., Peters, H. I., and Traugh, J. A. (1991a). J. Biol. Chem. 266,11993-11998. Venema, R. C., Peters, H. J., and Traugh, J. A. (1991b). J. Biol. Chem. 266,12574-12580. Vennegoor, C., and Bloemendal, H. (1972). Eur. J. Biochem. 26, 462-473. Vilalta, A., Donovan, D., Wood, L., VogeU, G., and Yang, D. C. H. (1993). Gene 123, 181-186. Wahab, S., and Yang, D. C. H. (1985a). J. Biol. Chem. 260, 12735-12739. Wahab, S., and Yang, D. C. H. (1985b). J. Biol. Chem. 260, 5286-5289. Wahab, S. Z., and Yang, D. C. H. (1986). Arch. Biochem. Biophys. 249, 407-417. Wilhams, J., Osvath, S., Khong, T. F., Pearse, M., and Power, D. (1995). Nucl. Acid Res. 23, 1307-1310. Wolfson, A. D., Motorin, Y. A., Ribkinska, T. I., and Bersten, S. F. (1990). J. Chromatogr. 503, 277-281. Wu, X. Q., and Gross, H. J. (1993). Nucleic Acids Res. 2 1 , 5589-5594. Yang, D. C. H., Garcia, J. V., Johnson, Y. D., and Wahab, S. (1985). Curr. Top. Cell. Regul. 26, 325-335. Yanofsky, C. (1987). Trends Genet. 3, 356-360. Y a m s , M., and Berg, P. (1970). Anal. Biochem. 35, 450-465. Zamecnik, P. C. (1983). Anal. Biochem. 134, 1-10.

CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Regulation of Interaction between Signaling Protein CheY and Flagellar Motor during Bacterial Chemotaxis RiNA BARAK MICHAEL EISENBACH Department of Membrane Research and Biophysics The Weizmann Institute of Science 76100 Rehovot, Israel

I. Introduction One of the major modes of communication between a cell and its environment is by chemotaxis (taxis meaning movement), namely by attraction to some chemicals and repulsion from others (collectively termed chemotactic stimuli). Due to their relative simplicity, bacteria are the organisms in which chemotaxis is most extensively studied and are considered a model system for stud3dng sensory signal transduction at the molecular level (1,67). This review focuses on the regulation of the interaction between two key players in the signal transduction pathway in bacterial chemotaxis: the protein CheY and the "switch" at the base of the flagellar motor. For general reviews of bacterial chemotaxis, the reader is referred to Refs. (2,20,37,68,78,129,136). A. Bacterial Chemotaxis Chemotaxis in bacteria such as Escherichia coli and Salmonella typhimurium is carried out by modulating two main swimming patterns: a run, which is a smooth swimming in rather straight lines, and a tumble, which is a chaotic angular motion that reorients the cell (10,81). In the absence of a chemotactic stimulus, the tumbles are brief and usually occur once every 1-5 sec (the tumbling frequency varies from strain to strain). In an ascending gradient of an attractant (or a descending gradient of a repellent), the runs are prolonged and the tumbles are depressed (the extent depends on the steepness of the gradient). The opposite occurs in a descending gradient of an attractant or an ascending gradient of a repellent. These modes of swimming result from flagellar rotation in different directions: counterclockwise (CCW) 137

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rotation leads to a run, clockwise (CW) rotation or a pause leads to a tumble (4,42,71,77,82,123). A pause seems to result from CCW-to-CW futile switching attempt, and there appears to be no signal for it (42). Attractants increase the probability of CCW rotation, resulting in migration toward higher attractant concentrations; repellents increase the probability of CW rotation, resulting in avoidance of the repellent source (10,71,81,123). Thus, the question of how the chemotaxis process is regulated in bacteria narrows down to how the direction of flagellar rotation is regulated. B. Signal Transduction in Bacterial Chemotaxis Most, if not all, chemical stimuli are detected by receptors. Some stimuli (e.g., galactose, ribose, maltose) interact with periplasmic receptors, and the resultant complex subsequently interacts with a membrane receptor (also termed MCP for methyl-accepting chemotaxis protein) (3,86,127,156). Other stimuh are detected by the MCPs themselves (e.g., serine, aspartate) (29,51,52,96,127) or by other receptors (e.g., mannose) (3,110,143). Signal transduction is accomplished by two processes: excitation and adaptation. The excitation process is the mechanism by which the initial signal from the receptor is transduced to the flagellar motor and affects the direction of flagellar rotation. The adaptation process restores, by a feedback mechanism, the unstimulated mode of flagellar rotation (even though the stimulus is still present). One of the adaptation mechanisms involves MCP methylation. This chapter deals with the regulation of specific processes that occur during excitation in two of the best-studied bacterial species, E. coli and S. typhimurium. For reviews on the adaptation process, the reader is referred to Refs. (20,37,50,104,128,129). The nature of signal transduction in the excitation process was not known until about 10 years ago, when it was shown that the signal is not electrical in nature, but rather chemical (40,87,121). A key component of this transduction mechanism is the chemotaxis protein CheY, a signaling molecule that interacts with the flagellar motor and results in a CW rotation (30,108,114,151). The main currently known steps of signal transduction from the membrane receptors to the flagellar motor during excitation are shown, in a simplified manner, in Fig. 1. The excitation process involves the receptors, a few chemotaxis proteins that reside in the cytoplasm, and the switch-motor complex. The components involved in CheY-switch interaction are reviewed under Section II. The current knowledge on the other signaling components is briefly summarized below.

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FIG. 1. Simplified scheme of the main steps of signal transduction during excitation. For simplicity, constituents involved solely in adaptation to sensory stimuli are not shown. The narrow and wide striped arrows represent weak and strong binding, respectively.

The best-studied membrane receptors are the MCPs (see Refs. 37 and 48 for reviews), recently found to be clustered at the bacterial poles (83). There are four MCPs [Tsr, Tar, Trg, and Tap (or Tcp in S. typhimurium)], all sharing extensive sequence homology in the cytoplasmic domain. The MCPs function as homodimers, each monomer traversing the membrane twice, forming a ternary complex with two of the chemotaxis proteins that reside in the cytoplasm, CheA and CheW (47,120). CheA is also a homodimer, thus the ternary complex actually comprises six subunits: a dimer of MCP, a dimer of CheA, and two monomers of CheW (47,120). It is not currently known whether the chemotaxis receptors other than the MCPs are also clustered at the bacterial poles, appear as homodimers, and form ternary complexes with CheA and CheW. It is also noteworthy that no receptor for repellents has thus far been directly identified. However, data suggest that the MCPs are also low-affinity receptors for at least some repellents (39). CheA is a kinase which undergoes autophosphorylation in vitro at His48 (53,55,153) and readily phosphorylates CheY (54,153). As a result of translational initiation at two distinct in-frame initiation sites, the cheA gene encodes two polypeptides, long and short, with the long one having 97 additional amino acids in the N-terminal domain, including the phosphorylation site (125). The protein comprises distinct functional domains for its various activities, including a domain for phosphotransfer which contains the phosphorylation site and the interaction site with CheY and CheB, a domain which catalyzes the autophosphorylation of His-48, and a domain for regulation of the autophosphorylation rate by the receptor and CheW (21,103,106,139,140,152). CheW appears to couple CheA to the membrane receptor (46,47, 72,94,120). Very little is known about its structure or function. It is

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known that CheW (like CheA) is obligatory for signal transduction (31), and that it stimulates in vitro the rate of CheA autophosphorylation and phosphotransfer to CheY (18,19,100). Based on a consensus sequence, CheW may have a nucleotide-binding site (132), but none has thus far been demonstrated. At the other end of the signal transduction pathway is the flagellar motor located at the base of each flagellum and embedded in the cytoplasmic membrane (34) (see Refs. 27,58,61,80 for reviews). There are 5-12 flagella per cell in E. coli and S. typhimurium scattered in a seemingly arbitrary manner around the cell. Unlike eukaryotic flagella, bacterial flagella rotate using a motor at the base (1). There is no motor action in the external filament iself. The motor is driven by a proton (or hydroxyl ion) current through the motor (13,14,95,113) and the energy source is the protonmotive force (PMF) (see Refs. 11,27,36,60,79 for reviews). The proteins MotA and MotB are involved in the motor rotation. The "gear shift" of the motor, i.e., the site which receives the signal and modulates the direction of rotation of the motor, is the switch, located at the base of the motor (Fig. 1). The switch proteins (Section II,C) are also involved in the motor rotation. Thus, of the chemotaxis proteins known to reside in the cytoplasm and to be involved in the excitation process, only CheY and CheZ appear free to diffuse and communicate between the ternary complex and the switch. The general notion today is that ligand binding to the periplasmic domain of an MCP induces conformational changes in its cytoplasmic domain which, via CheW, regulates the autophosphorylation rate of the kinase CheA. This was nicely demonstrated by Gegner et al. (47), who found that binding of an attractant or a repellent to an MCP does not significantly alter the stability of the ternary complex but modulates the kinase activity of CheA. As described in subsequent sections, phosphorylated CheA (CheA~P) transfers a phosphate group to CheY, thus enabling the latter to interact with the switch and resulting in CW rotation. This interaction is terminated by CheZ. II. Proteins That Participate in CheY-Switch Interaction A. CheY CheY is a 14-kDa globular protein that has 128 residues (91). It is a monomeric protein [although it forms a very stable dimer intermediate while undergoing a temperature-induced unfolding (43)] which belongs to the large family (over 100 members) of regulator proteins that is part of the "two component regulatory systems" (20,66,99,144,147). [The regulators are bacterial proteins that have a conserved region of

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about 120 residues in the N terminus; all these proteins are involved in control mechanisms, and most of them are involved in regulating the expression of specific genes in response to stimuli (66,117,137).] All the regulators share a common mechanism of transient phosphorylation by other members of the family; kinases denoted as sensors (in the case of CheY, the sensor is CheA). CheY is the only regulator that has been crystallized, and its three-dimensional structure solved in both E. coli and S. typhimurium (9,134,135,144,145). The structure of the protein in solution has also been elucidated by NMR techniques and found to be closely coincident with the crystal structure (26). CheY is a doubly wound alj^ protein composed of a central core of five parallel /3 strands surrounded by five a helices. Four residues, Asp-12, Asp-13, Asp-57, and Lys-109, clustered at the C-terminal edge of the parallel j8 strands, are highly conserved among the regulators which are homologous to CheY, indicating a functional role in the common mechanism of signal transduction (73,138,144). Asp-57 is the phosphorylation site of CheY (23,119), and all three aspartate residues together form an acid pocket (135,145). The acid pocket binds a single divalent metal ion (75), probably Mg^^, in vivo (98). The binding of Mg^^ causes significant conformational changes in CheY resulting in modification of the surface domain which, as has been shown by genetic analysis, interacts with the switch (9). Mg^^ is essential for CheY phosphorylation, dephosphorylation (both spontaneous and CheZ dependent), and CheZ binding to CheY (15,74,75,148,149). Phosphorylation of the Asp-57 carboxyl moiety transmits a long-range conformational change through the molecule, but does not significantly displace the Lys-109 side chain (35). CheY is not only similar to the prokaryotic proteins mentioned previously, but it is also structurally homologous to the eukaryotic GTPbinding protein Ras p21 (5,28). This suggests that the two proteins might share a common signaling mechanism. CheY can be phosphorylated in vitro either by CheA (54,153) or by low-molecular-weight phospho donors (74). The extent of CheAmediated phosphorylation of CheY can be modulated in vitro by chemotaxis stimuli or methylation of the MCPs in a system consisting of MCP-containing vesicles and the chemotaxis proteins CheY, CheA, and CheW (17-19,100). Furthermore, it was recently found in vitro that phosphorylation of CheY (or, more correctly, conditions which favor phosphorylation of CheY) leads to its dissociation from the quaternary complex MCP-CheA-CheW-CheY; this dissociation, demonstrated for the MCP Tar, is abolished in the presence of aspartate (an attractant recognized by this MCP) (120). The low-molecular-weight phospho donors that can phosphorylate CheY are acetyl phosphate (Ac~P), phos-

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phoramidate, and carbamoyl phosphate. Other response regulators of the two-component systems (e.g., CheB, PhoB, OmpR, NtrC, ArcA, and AlgR) can also be phosphorylated in vitro by these low-molecularweight phospho donors (93). [There is, however, a certain degree of specificity; for example, both CheY and CheB can be phosphorylated by phosphoramidate, but only CheY, and not CheB, can be phosphorylated by Ac~P or carbamoyl phosphate (74).] The CheA-independent phosphorylation indicates that CheY, like the other response regulators, can catalyze its own phosphorylation. Unlike CheA~P, which is stable, phosphorylated CheY (CheY~P) is quickly and spontaneously dephosphorylated in vitro (54,153). This dephosphorylation is enhanced by CheZ (54). Even though CheY phosphorylation has not been detected in vivo, the in vitro results summarized previously strongly indicate that phosphorylation is a mechanism which regulates CheY activity in vivo. Whether CheA~P is the only phospho donor for CheY in vivo or whether Ac~P is also a physiological phospho donor has yet to be determined. Some evidence suggests that Ac~P can act as a phospho donor in vivo for other response regulators. For example, Ac~P apparently phosphorylates NRi, the response regulator of the nitrogen assimilation system (111). B. Chez CheZ is a hydrophilic, very acidic homopol3nner, which may comprise over 20 monomers, each having a molecular size of 23.9 kDa (130,133,136). CheZ enhances the dephosphorylation of CheY (54) with a high specificity, evidenced by the fact that it fails to dephosphorylate CheB (54), even though the N-terminal portion of the latter is homologous to CheY as a whole (131). Because the direction of flagellar rotation in mutants deleted for CheZ is CW biased, it was initially presumed that CheZ is a CCW signal in the same way that CheY is a CW signal. However, a number of observations appear to indicate that the role of CheZ is to enhance the termination of the CheY-switch interaction rather than to be a CCW signal (114). These observations are (i) the default direction of rotation (when the chemotaxis proteins, including CheZ, are missing) is CCW (30,38,42,105,112,151); (ii) cells can respond to attractants and become CCW biased even in the absence of CheZ (122,151) [albeit slower (16,121), possibly reflecting the longer time required for spontaneous dephosphorylation of CheY in the absence of CheZ]; and (iii) intracellular production of CheZ increases the CCW bias only when CheY is also present intracellularly (CheZ cannot increase the CCW bias in a strain that lacks CheY but that, nevertheless.

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rotates its flagella in both directions due to a mutated gene of a switch protein) (151). C. Switch Complex The switch is a complex of three proteins: FhG,* FHM, and FUN (154). They were recognized as switch proteins because in each of them a mutation can lead either to a CCW-biased or to a CW-biased rotation (in other chemotaxis proteins most mutations lead to a bias in only one direction) (33,64,104). The switch proteins are essential for the early stages of flagellar assembly and for flagellar rotation because null mutations lead to a nonflagellated ( F l a ) phenotype, and amino acid substitution in a number of positions in each of these proteins leads to a paralyzed (Mot") phenotype (57,79,126,155). The switch proteins were initially found to be associated with the C3^oplasmic membrane (112) and then—when a bell-like extension from the base of the flagellar motor into the cytoplasm was discovered—they were considered to be associated with this extension (59,62). The bell-shape extension (later denoted as the C ring) is not detected in preparations made from nonmotile alleles of the switch genes, but it is detected in preparations made from motile strains containing nonchemotactic alleles of the switch genes or from mot or che mutants (49,59). The presence of the switch proteins on the C ring was verified by immunoblots (44,45). The switch appears to be attached to the lowermost part of the motor (the MS ring) via FliG (44). On the basis of genetic evidence, FUG and FliN are thought to be the switch components most involved in the mechanism of rotation, with FHM most involved in CheY binding and initiation of the subsequent switching event (57,126) (see ref. 78A for a recent comprehensive review on the switch structure and function).

III. Regulation of CheY-Switch Interaction A. CheY-Switch Interaction The first indication that CheY interacts with the switch came from genetic second-site suppression analyses made by Parkinson et al. (108) and followed by other groups (57,84,116,126,154). In these studies pseudorevertants were isolated from nonchemotactic cheY mutants and found to have a second mutation which phenotypically compensated for the original mutation in cheY. The second mutation mapped at one of * Note that the nomenclature of the gene products of the bacterial flagella in E. coli and S. typhmurium was changed in 1988. Thus, earlier references cite different names than those used in this review. The reader is referred to lino et al. (56) for a nomenclature conversion table.

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the switch genes, mostly in fliM but also in fliG; very few compensating mutations were found in/ZiN (57,126). Similarly, mutations in fliM and fliG were found to be compensated by mutations in cheY (116). This suggested that the main interaction is between CheY and FliM, and possibly also with FliG. Macnab and co-workers raised reservations about this interpretation when they found that the compensating mutations are not allele specific; accordingly, they raised the possibility that the restoration of chemotaxis by a second mutation may be achieved by nonspecific adjustment of the switch bias rather than by specific structural compensation, and therefore should not be taken as evidence for direct physical interaction between CheY and the switch (57,126). In both FliM and FliG, the sites of compensating mutations were found to be segregated in clusters (57). This also held for CheY in which physical mapping of the amino acid substitutions that compensate mutations in fliM or fliG revealed a high degree of spatial clustering on a surface of CheY physically distinct from the phosphorylation site (116,126). Another line of evidence for CheY-switch interaction came from physiological studies in which CheY was overproduced in strains lacking the C5rtoplasmic chemotaxis proteins (denoted as "gutted strains"). Because of the at least partial absence of the chemotaxis machinery, the flagella of gutted strains rotate only in the default direction, CCW. Overproduction of CheY in these strains generates CW rotation, indicating an interaction between CheY and the switch (30,69,124,151). Yet another approach was that of Ravid et al. (114) who inserted purified CheY into cytoplasm-free bacterial envelopes with functional fiagella (38,41). The presence of CheY in the envelopes caused some of them to rotate CW (114). The absence of cytoplasm (and hence the absence of the cytoplasmic chemotaxis proteins) was verified in each envelope studied, indicating that the interaction of CheY with the switch is direct, without mediators. The cloning (65,85,lOOA, 115) and expression of the switch proteins to high levels (100A,115) allowed examination of the CheY-switch interaction by direct biochemical means. By immobilizing purified CheY onto a solid support and adding labeled switch proteins, Welch et al. (148) demonstrated that only FliM specifically binds CheY and that the extent of binding is unaffected by the other two switch proteins. B. Regulation of Interaction by Phosphorylation

Posttranslational modification is a common way of regulating the activity of a protein, and phosphorylation is perhaps the most abundant modification. The findings described previously that CheY can be phos-

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phorylated in vitro and that the phosphorylation level can be modulated in vitro by chemotactic stimuli (17,18,100) made this chemical modification the primary candidate for regulating the activity of CheY at the switch. Welch et al. (148) have found that, indeed, the binding of wild-type CheY to FliM is increased under phosphorylating conditions (Fig. 2). Such an increase was not observed in a nonphosphorylatable mutant CheY. This is the first direct proof that phosphorylation activates CheY and significantly increases its ability to bind to the switch (Fig. 3). Interestingly, the CheY~P-FliM binding is Mg^^ independent, unlike the process of CheY phosphorylation and dephosphorylation (149). Phosphorylation is required for CW generation under physiological conditions; however, when the intracellular concentration of CheY is elevated to above-physiological levels, either in vivo (30,31,69,124,151) or in vitro (6,41,114), CW rotation can be observed even in the absence of a phosphate donor. This was initially attributed to crosstalk phosphorylation by other histidine kinases or low-molecular-weight phospho donors (such as Ac~P) that reside in the cell (76,93,147), but then CW rotation was shown to occur even under conditions that do not 0.15 O

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3 O

0.05

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0.2

0.4

0.6

0.8

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Membrane

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FIG. 3. A scheme of CheY binding to the switch. The organization of the proteins in the switch-motor complex is based on Irikura et al. (57).

allow phosphotransfer (6). The low-level binding of nonphorphorylated CheY to FliM (148) may account for this phenomenon. C. Phosphorylation Alone Not Sufficient for CW Generation On the basis of the observations summarized thus far, it may appear that phosphorylation of CheY (and consequently binding of CheY to the switch) is all that is required under physiological conditions for the generation of CW rotation. However, a number of observations and considerations appear to indicate that this may not be the case, and that CheY phosphorylation alone is not sufficient for the generation of CW rotation (see below). 1. ADDITIONAL CYTOPLASMIC CONSTITUENT REQUIRED FOR CW GENERATION

Because phosphorylation increases CheY binding to the switch, it might be expected that phosphorylation will also increase the efficiency of CheY in causing CW rotation. This expectation was directly tested by phosphorylating CheY within cytoplasm-free envelopes either by CheA and ATP (or caged ATP) (6) or by Ac~P (Y. Blat, R. Barak, and

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M. Eisenbach, 1993, unpublished observations). Unlike the prediction, phosphorylation of CheY did not increase the extent of CW rotation. This result seemed intriguing in view of the observations (mentioned previously) which implicated phosphorylation as a mechanism for regulating CheY activity in vivo. The dilemma was solved when it was found that in semienvelopes, i.e., in "envelopes" intentionally containing remnants of cjrtoplasm but no chemotaxis proteins other than CheY, phosphorylation by CheA and ATP (6) or by Ac~P (Blat et al, 1993, unpublished observations) does increase the extent of CW rotation. This indicated that although phosphorylation of CheY increases its binding activity to FliM, it is not sufficient for switching the motor to the CW state. An additional cytoplasmic constituent(s) is required for this purpose. This constituent is not a chemotaxis protein and is probably involved in a subsequent step of CheY or switch activation. 2. CCW-TO-CW SWITCHING INVOLVES AT LEAST TWO STEPS Kuo and Koshland (70) examined the behavioral effect of CheY on single flagellar motors by means of a computerized video processing system. From the dependence of the direction of rotation and the switching frequency on the CheY concentration, they excluded a two-state (one CCW and one CW) model of the motor and demonstrated that switching from CCW to CW involves at least two steps. The data are best described by two CW and two CCW states of the motor. The phenotypic behavior of a number of substitution mutants of CheY is difficult to understand without assuming at least two steps of switching and/or CheY activation. Two examples follow: (i) Four mutants—CheY13DK57DE (the product of site-directed mutagenesis that has replaced the conserved Asp-13 and Asp-57 residues with lysine and glutamate residues, respectively), CheY13DK57DA, CheY13DR57DE, and CheY13DR57DA—are unable to be phosphorylated in vitro, but nevertheless can generate CW rotation in vivo (22). These results, taken together with ^^F NMR chemical shift measurements indicating that the activating mutations generate a relatively localized perturbation in the active site (unlike phosphorylation that triggers a global conformational change), led Bourret et al. to suggest a two-step model of CheY activation and binding to the switch in which CheY phosphorylation is the first step (22). (ii) The CheY109KR protein is highly phosphorylated but, nevertheless, the mutant does not tumble at any level of induction of this protein (73). The protein is not dephosphorylated by CheZ (73) and its binding to FliM is not enhanced under phosphorylating conditions (149). These observations, taken together with the notion that Lys-109 is involved in the function of CheY (be-

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cause of being a highly conserved residue), raised the possibihty that Lys-109 plays a central role in an event or a step following phosphorylation (73,76,149). 3. SIGNAL TRANSDUCTION PATHWAY PROBABLY CONTAINS ADDITIONAL AMPLIFICATION STEP(S)

Theoretical simulations of bacterial behavior carried out by Bray et al. (24) have revealed discrepancies between the simulation and real bacteria in the phenotypic behavior of several mutants and in the gain of the chemotactic response. In other words, the known amplification steps of signal transduction in bacterial chemotaxis cannot account for the actual measured, much higher amplification. Therefore, Bray et al. suggested that additional, as yet unidentified interactions in the in vivo signal processing pathway exist. To conclude, CW generation involves the following steps: CheY phosphorylation, CheY~P binding to FliM, and at least two additional, as yet unknown steps of switching. A defect in any of these steps will lead to a nonchemotactic phenotype. Additional processes and factors that might be involved in regulating CheY-switch interaction and the subsequent steps are described below. D. Regulation by CheZ Since CheZ enhances dephosphorylation of CheY in vitro, it is likely to interact with CheY in vivo and to be involved in at least the regulation of CheY activity, if not in additional steps. Information on the interactions of CheZ with additional components of the chemotaxis machinery is gradually accumulating. However, close to nothing is known about the way (if any) in which the activity of CheZ itself is regulated. A direct interaction of CheZ with CheY was biochemically demonstrated by immobilizing CheY onto a solid support either in a column (90,94) or in a batch (15). The binding of CheZ to CheY~P was found to be two orders of magnitude higher than to CheY (15). The binding of CheZ to both CheY and CheY~P is enhanced by Mg^^ (15), indicating that Mg^ is required not only for phosphorylation and dephosphorylation of CheY (74,75), but also for the binding of CheY to CheZ (15) [unlike the binding of CheY to FliM (149)]. Under phosphorylating conditions, several molecules of CheZ bind to 1 CheY~P molecule (15). This may reflect the polymeric nature of CheZ (133). The mutant CheY proteins 13DK, 57DE, and 109KR bind CheZ as well as nonphosphorylated wild-type CheY, but phosphorylation conditions do not increase the binding (15). These data indicate that neither an active conforma-

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tion in vivo (as in the case of CheY13DK) nor the abiHty to be phosphorylated (as in the case of CheY109KR) is sufficient for maximal binding. As in the case of CheY interaction with the switch, CheZ was also found by second-site suppression analysis to possibly interact with the switch: mutations in cheZ were phenotypically suppressed by mutations in any one of the three switch genes (57,107,108,126). Here, too, the suppression of the mutations in cheZ was not allele specific (i.e., a given mutation in cheZ could be suppressed by a number of different mutations in a switch protein) (57,126). If CheZ interacts with the switch it may do so in three possible ways: (i) directly, independent of CheY (in which case CheZ should have two separate functions: one on free CheY and one on the switch); (ii) directly, but dependent on the presence of CheY bound to the switch; and (iii) indirectly, via CheY. It is not known which, if any, of these possibilities is correct. The regulation mechanism of CheZ activity is also not known. One of the clues for such a regulation is perhaps the interaction of CheZ with the short form of CheA (CheAs), the function of which is still obscure (152). CheAs, but not the long form of CheA (CheAL), can constitute a complex with CheZ in vitro (90). Recent results of Wang et al. (146) indicated that, indeed, the phosphatase activity of CheZ is increased in the presence of CheAs. Interestingly, the stoichiometry of the complex is 10-30 molecules of CheZ per 1 molecule of CheAs (90). This observation, taken together with the observation that several molecules of CheZ bind to 1 CheY~P molecule (15), raises the possibility that CheAs-mediated polymerization of CheZ is involved in regulating the activity of the latter. To conclude, CheZ interacts with at least two components of the signal transduction pathway in chemotaxis: CheY~P and CheAs (the physiological significance of the latter interaction is still obscure). Any one of these interactions may be involved in the regulation mechanism of CheZ activity. E. Regulation by Calcium Ion The involvement of Ca^^ in chemotaxis was a controversial issue for a long time (see, e.g., 40,92,101,102,121; see also 37 and 40 for reviews). Tisa and Adler have found that photorelease of Ca^^ from a caged Ca^^ within E. coli cells causes tumbling and that this phenomenon requires CheA, CheW, and CheY, but not the MCPs (141). Furthermore Ca^^ channel blockers were found to inhibit chemotaxis (92,142). Ca^^ may stimulate CheY (or CheA) phosphorylation or it may stabilize CheY~P (141). Whether Ca^^ is involved in regulation of CheY-switch interaction awaits further study.

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ACS PTA

Ac~P ADP CoA FIG. 4. Parallel pathways of acetate metabolism to acetyl-CoA.

F. Regulation by Acetylation An intriguing phenomenon, discovered several years ago by Wolfe et al. (150), was that acetate causes a strong and prolonged CW bias in a gutted strain containing CheY but devoid of receptors and the other cytoplasmic chemotaxis proteins. This phenomenon (denoted as the "acetate effect") was attributed to the effect of an intermediate of acetate metabolism on CheY. Acetate is activated to acetyl-coenzyme A (Ac-CoA) by two different pathways (Fig. 4). One utilizes the acetateinducible enzjmfie acetyl-CoA synthetase (ACS; acetate-CoA ligase) (25) and proceeds through an acetyladenylate (AcAMP) intermediate (12). The second pathways uses two enz3rmes, acetate kinase (ACK) and phosphotransacetylase (PTA; ATP: acetate phosphotransferase) and proceeds through an Ac~P intermediate (118). The intermediate responsible for the acetate effect was first thought to be AcAMP (150). However, it was later suggested that Ac--P is the intermediate responsible for this phenomenon (32). At about the same time that the ability of Ac~P to phosphorylate CheY was studied (74), Barak et al. (8) used the acetate effect as one end of a thread for investigating whether there are other ways to regulate CheY activity or the switch function. They looked for chemical modifications of CheY or the switch proteins caused by acetate and ACSt (i.e., by AcAMP). Only CheY, and not the switch proteins, was chemically modified, and the modification was acetylation (Eqs. [1] and [2]) Ac + ATP + ACS ;^ AcAMPACS + PPi

[1]

AcAMPACS + CheY ^ AcCheY + AMP + ACS

[2]

The acetylation was found to be specific to CheY, to be on a lysine residue and/or the AT-terminus of CheY, to occur only on the native t Because ACS had not been isolated by then from E. coli, commercially available ACS of yeast was used.

CheY-MOTOR INTERACTION

151

conformation of the protein (8), and to have a stoichiometry of two acetyl groups per one CheY molecule (R. Barak and M. Eisenbach, 1993, unpublished observations). Chemical modification of a protein is meaningless without demonstration of its physiological relevance. As discussed under Section II,A, phosphorylation by Ac~P may be physiologically significant. Does this also hold true for AcAMP-mediated acetylation of CheY? The finding of mutant CheY proteins (with substitutions in conserved residues) that are unable to become acetylated (8) seems to favor this possibility. Physiological relevance can be demonstrated by an effect of the modification on the function of the protein. And, indeed, the CW-causing activity of CheY is increased four or five orders of magnitude by acetylation conditions within envelopes (8). Unlike the effect of CheY phosphorylation, which can be observed only in the presence of cytoplasmic constituents (Section III,C,1), the effect of acetylation is observed in envelopes in the absence of such constituents. Thus, the current understanding appears to be that both AcAMP and Ac~P can increase the activity of CheY at the switch, one by acetylation and the other by phosphorylation, and either one of them can be involved in the acetate effect mentioned previously. This conclusion is still controversial. For example, Dailey and Berg did not observe an acetate effect in an ack mutant and concluded that ACK, not ACS, is responsible for the acetate effect (32). However, using a gutted Mack pta) mutant, R. Barak, A. Wolfe, and M. Eisenbach (1993, unpublished observations) found that when the cells are grown under ACS-inducing conditions (i.e., the presence of acetate) and/or contain a high level of CheY, an acetate effect is clearly observed. This is in line with the notion that both routes of acetate metabolism, ACS (forming acetylated CheY) and ACK + PTA (forming CheY~P), are involved in the acetate effect. Similarly, since Dailey and Berg did not detect an ACS activity in a Mack pta) mutant in a wild-type background and the mutant appeared to be normally chemotactic, they concluded that neither ACS nor ACK + PTA is involved in chemotaxis (32). However, very recent observations indicated that a Mack pta) mutant can grow on acetate and does have ACS activity (68A). G. Regulation by Fumarate Level A relatively new potential player in the regulation of CheY-mediated switching is fumarate. Initially it was identified as an important factor in phototaxis of Halobacterium salinarium (formerly known as Halobacterium halobium), where it serves as a "switching factor", i.e., as a factor that restores switching ability to a mutant defective in motor

152

RINA BARAK AND MICHAEL EISENBACH

switching. Fumarate is membrane bound in H. salinarium and is released on phototactic stimulation by either an attractant or a repellent light (88,89,97)). Fumarate was also found to be effective in S. typhimurium (7) and E. coli (7A) where it restores to cytoplasm-free envelopes the ability to spontaneously switch the direction of flagellar rotation. [Prior to the addition of fumarate, the flagella of envelopes always rotated either CCW or CW (in the presence of intracellular CheY), but they never switched from one direction of rotation to the other (41,112,114).] No cytoplasmic constituents other than CheY are required for the fumarate effect. The effect is specific for fumarate, but malate, maleate, and succinate are also effective to a lesser extent (7A). The mechanism of function of fumarate in E. coli or S. typhimurium is obscure. It may either be a cofactor, the presence of which is required for switching (just as the presence of Mg^^ is required for most functions of CheY), or a factor directly involved in the regulation of signal transduction. Further studies are required in order to distinguish between these possibilities and to determine whether fumarate is involved in chemotaxis in vivo. It is also not clear at this stage why enzymes that affect the fumarate level in the cell (e.g., succinate dehydrogenase or fumarase) have not been identified as chemotaxis proteins. (The enzjrmes which affect the level of AdoMet—the precursor of MCP methylation—have also not been identified as chemotaxis proteins.) In analogy to the involvement of metabolic intermediates in chemotaixis ofRhodobacter sphaeroides (109), fumerate might be the connecting point between the metabolic state ofE. coli or S. typhimurium and the chemotaxis system. H. Regulation by Protonmotive Force Level Khan and Macnab (63) have demonstrated that, in addition to being the driving force for fiagellar rotation, the PMF exerts a regulatory effect on switching the direction of rotation. A reduction in the PMF causes the motor to spend a higher fraction of time in CCW rotation; when the PMF level becomes so low that the motor speed goes down to ~80% of the maximum speed under any given external load condition, the rotation becomes exclusively CCW. The reason for this seems to be that the CCW state is energetically lower than the CW state, resulting in higher activation energy for the CCW-to-CW switching than for the CW-to-CCW switching (42,63). This type of regulation, which circumvents the conventional signal transduction pathway, may be effective under extreme conditions of metabolic deprivation. Under such conditions tumbling is suppressed due to the decrease in CW

CheY-MOTOR INTERACTION

153 TABLE I

CHEMICAL MODIFICATIONS AND FACTORS THAT ACTIVATE CHEY

Chemical modification or factor

CheY-FliM binding

CW generation

CheA-mediated phosphorylation Ac~P-mediated phosphorylation AcAMP-mediated acetylation Fumarate Ca2^

ND^ + ND

+' +' + + +

c

ND

Switching generation

-

+ ND

^ ND, not determined. Plus (+) and minus ( - ) signs denote whether or not the phenomenon has been observed, respectively. ^ Only in the presence of additional (yet-unidentified) cytoplasmic constituents, other than the known chemotaxis proteins. ''Based on unpublished observations of M. Welch (1993).

rotation, and consequently a bacterial population can disperse more rapidly from a region of scarcity of food or oxygen (63). IV. Concluding Remarks Apparently, the interaction of CheY with the switch, a key step in signal transduction during chemotaxis, may be regulated in a number of ways. A summary is shown in Table I. CheY phosphorylation is an established mechanism of regulation, but the experimental results indicate that this alone is not sufficient. AcAMP, Ac~P, Ca^^, fumarate, and perhaps additional compounds may be involved in the regulation. AcAMP causes an additional chemical modification of CheY— acetylation; the mechanism of function of fumarate is not known. How all these potentially regulatory reactions are integrated is still a mystery. It also remains to be seen whether phosphorylation and acetylation are functional in parallel and complementary to each other, or whether each of these processes is functional under different conditions. It is reasonable to assume that while the phosphorylation activates CheY to bind to the switch and CheZ causes it to detach from the switch, the acetylation and fumarate affect a switching step(s) subsequent to the binding. Since CheY is only one member of a large family of homologous signaling proteins, understanding the mechanism regulating the CheY-switch interaction may have implications for understanding a large number of similar regulatory systems. ACKNOWLEDGMENT Dr. R. M. Macnab is acknowledged for reading the manuscript. M. E. is an incumbent of Jack and Simon Djanogly Professorial Chair in Biochemistry. The research done in

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130. Stock, A. (1988). In "Advances in Post-Translational Modifications of Proteins and Aging" (V. Zappia, P. Galletti, R. Porta, & F. Wold, eds.), pp. 387-399. Plenum, New York. 131. Stock, A., Koshland, D. E., Jr., & Stock, J. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 7989-7993. 132. Stock, A., Mottonen, J., Chen, T., & Stock, J. (1987). J. Biol. Chem. 262, 535-537. 133. Stock, A., & Stock, J. B. (1987). J. Bacteriol. 169, 3301-3311. 134. Stock, A. M., Martinez-Hackert, E., Rasmussen, B. F., West, A. H., Stock, J. B., Ringe, D., & Petsko, G. A. (1993). Biochemistry 32, 13376-13380. 135. Stock, A. M., Mottonen, J. M., Stock, J. B., & Schutt, C. E. (1989). Nature (London) 337, 745-749. 136. Stock, J. B., Lukat, G. S., & Stock, A. M. (1991). Anna. Rev. Biophys. Biophys. Chem. 20, 109-136. 137. Stock, J. B., Ninfa, A. J., & Stock, A. M. (1989). Microbiol. Rev. 53, 450-490. 138. Stock, J. B., Stock, A. M., & Mottonen, J. M. (1990). Nature (London) 344,395-400. 139. Swanson, R. V., Bourret, R. B., & Simon, M. I. (1993). Mol. Microbiol. 8, 4 3 5 - 4 4 1 . 140. Swanson, R. V., Schuster, S. C , & Simon, M. I. (1993). Biochemistry 32,7623-7629. 14L Tisa, L. S., & Adler, J. (1992). Proc. Nat. Acad. Sci. U.S.A. 89, 11804-11808. 142. Tisa, L. S., Olivera, B. M., & Adler, J. (1993). J. Bacteriol. 175, 1235-1238. 143. Titgemeyer, F. (1993). J. Cell. Biochem. 51, 69-74. 144. Volz, K. (1993). Biochemistry 32, 11741-11753. 145. Volz, K., & Matsumura, P. (1991). J. Biol. Chem. 266, 15511-15519. 146. Wang, H., McNally, D., & Matsumura, P. (1993). Bact. Locomotion Signal Transduction Meet., Austin, Tex. Abstr. 147. Wanner, B. L. (1992). J. Bacteriol. 174, 2053-2058. 148. Welch, M., Oosawa, K., Aizawa, S.-I., & Eisenbach, M. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 8787-8791. 149. Welch, M., Oosawa, K , Aizawa, S.-I., & Eisenbach, M. (1994). Biochemistry 33, 10470-10476. 150. Wolfe, A. J., Conley, M. P., & Berg, H. C. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 6711-6715. 151. Wolfe, A. J., Conley, M. P., Kramer, T. J., & Berg, H. C. (1987). J. Bacteriol. 169, 1878-1885. 152. Wolfe, A. J., & Stewart, R. C. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 1518-1522. 153. Wylie, D., Stock, A., Wong, C.-Y., & Stock, J. (1988). Biochem. Biophys. Res. Commun. 151, 891-896. 154. Yamaguchi, S., Aizawa, S.-L, Kihara, M., Isomura, M., Jones, C. J., & Macnab, R. M. (1986). J. Bacteriol. 168, 1172-1179. 155. Yamaguchi, S., Fujita, H., Ishihara, H., Aizawa, S., & Macnab, R. M. (1986). J. Bacteriol. 166, 187-193. 156. Zukin, R. S., Strange, P. G., Heavey, L. R., & Koshland, D. E., Jr. (1977). Biochemistry 16, 381-386.

I

CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

I

Chemical Biology of Nitric Oxide: Regulation and Protective and Toxic Mechanisms DAVID A. WINK^ INGEBORG HANBAUER^ MATTHEW B . GRISHAM^ FRANCOISE LAVAL^ RAYMOND W . NIMS^ JACQUES LAVAL^ J O H N COOK^ ROBERTO PACELLI^ JAMES LIEBMANN^ MuRALi K R I S H N A ^ PETER C. FORD^'^ JAMES B . MITCHELL^

I. Introduction The discovery that nitric oxide (NO)* is endogenously formed throughout the animal kingdom has led to intense interest in the vari^ Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702. ^ Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, Bethesda, MD 20892. ^ Department of Physiology, Louisiana State University Medical Center, Shreveport, LA 71130. ^ Groupe 'Radiochimie de I'ADN', U347 INSERM, 80, rue du General Lectere 94276, LE.Kremilin Bicetre Cedex, Villejuif, Cedex, France. ^ Groupe Reparation des lesions 'radio et chimio-induites'URA 147 CRNS Institut Gustave-Roussy PR II, 94805 Villejuif, Cedex, France. ® Radiation Biology Branch, National Cancer Institute, Bethesda, MD 20892. ^ Department of Chemistry, University of CaHfomia, Santa Barbara, CA 93106. * Abbreviations used: BCNU, iV,Ar'-bis(2-chloroethyl)-i^-nitrosourea; BSA, bovine serum albumin; CHO, Chinese hamster ovary; DEA/NO, (C2H5)2N[N(0)N0]-Na*; DHR, dihydrorhodamine; EDRF, endothelium-derived relaxing factor; ESR, electron spin resonance; Fapy, 2,6-diamino-4-hydroxy-5-iV-methylformamidopyrimidine; Fpg, formamidopyrimidine-DNA glycosylase; GADPH, glyceraldehyde-3-phosphate dehydrogenase; GSNO, S-nitrosoglutathione; His, histidine; LDL, low-density lipoproteins; LPS, lipopolysaccharide; cNOS, constitutive nitric oxide synthase; iNOS, inducible nitric-oxide synthase; PAPA/NO, NH3*(C3H6)N[N(0)N0-](CH2)(CH3); RNOS, reactive nitrogen oxide species; ROS, reactive oxygen species; SIN, 1,3-morpholinosyndnonimine; SNAP, S-nitrosoiV-acetylpenicillamine; SNP, sodium nitroprusside; SOD, superoxide dismutase; TPPS, tetra(persulfonato)phenylporphyrin; XO, h)^oxanthine/xanthine oxidase. 159

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ety of roles this unique molecule plays in vivo. NO is involved in a wide variety of regulatory mechanisms ranging from blood pressure control to neurotransmission, yet it is also a cytotoxic agent. This dichotomy has raised the question of how this potentially toxic species can be involved in so many fundamental physiological processes. Endogenously generated NO is an important biomolecule and plays key roles in the regulation of a variety of physiological functions (1,2) including vascular tone, neurotransmission, platelet aggregation, and bronchodilation. NO, formed from the oxidation of L-arginine via nitric-oxide synthase, has been shown to exist throughout the animal kingdom (3,4). The biology of NO has resulted in an explosion in the literature; currently every medical field is examining possible roles for NO in almost every tissue and organ (5,6). Despite its physiological functions, this molecular radical is also a well-known toxic agent, being a constituent of air pollution and cigarette smoke (7). In addition to being an environmental toxin, endogenously formed NO is thought to be a potential causative agent in a number of diseases (8-10). For example, in an aerobic environment, NO can form reactive nitrogen oxide species (NO^) which can cause DNA damage (11-15), inhibit a variety of enzymes (16-22), and initiate lipid peroxidation (23). The question remains: How can this molecule be unstable in an aerobic environment, generating toxic intermediates, yet have so many physiological functions? The answer lies in understanding the chemical biology of NO, which can be defined as the fundamental chemistry that pertains to specific biological conditions. Consideration of these chemical reactions can provide a road map which can distinguish NO's regulatory processes from its potential toxic effects. This chapter will discuss, from a chemical perspective, those processes which are involved in interactions with key cellular components, as well as detoxification and control of NO in vivo. Defining the chemical, biochemical, and cellular pathways of NO quantitatively can provide insights into the role NO plays in the etiology of various diseases which in turn can provide a basis for the development of new therapeutic agents. The chemical biology of NO will provide the understanding as to how NO can be regulatory, toxic, and protective in biological systems. The chemical biology of NO can be categorized into two effects: direct and indirect (Scheme 1). Direct effects are those which NO itself directly reacts with the biological target. Indirect effects are defined as those mediated by the reactive intermediates formed from NO in an aerobic biological environment. The concentrations of NO define the category within which a particular chemical reaction may dominate. For in-

CHEMICAL BIOLOGY OF NITRIC OXIDE

Direct Effect O2-

Indirect Effects

161

^ ^ Biological Target

^Q Q

RNOS

Physiological Effect

^

Biological Target

(Reactive Nitrogen Oxide Species) SCHEME 1. Direct and indirect effects of NO.

stance, direct effects, as discussed below, generally occur at concentrations less than 10 /xM, One example is the binding of NO to a heme iron in the enzyme guanylate cyclase which can result in lowering of blood pressure. Indirect effects occur at concentrations higher than 10-20 jxM, Examples of these are described below. There are two types of nitric-oxide synthase: constitutive (cNOS) and inducible (iNOS) (3,4). Since cNOS generates low levels of NO, direct effects rather than indirect effects of NO would be particularly relevant. In the case of iNOS, considerably higher concentrations of NO are formed for longer periods of time; therefore, both direct and indirect effects could be relevant. This chapter will discuss important aspects of the solution chemistry of NO and reactive nitrogen oxide species (RNOS), biochemical targets of NO and intermediates in the autoxidation (NO;c), and the effect of NO in the presence of other toxic molecules such as reactive oxygen species (ROS). II. Chemical Aspects of Nitric Oxide One of the key aspects to defining the biology of NO is to understand the fundamental aqueous chemistry of this molecule. NO is a diatomic molecular radical that has solubility and diffusion properties which are similar to those of oxygen under biological conditions (24,25). Radical compounds are usually very reactive, yet NO, under anoxic conditions, is relatively unreactive (26,27). It will react with nucleophiles such as amines (28) and sulfhydryls (29), but only under conditions of high NO pressure. However, NO does react rapidly with many transition metals, with other free radicals, and (under certain conditions) with molecular oxygen (26,27). A. Reactivity of NO with Metal Complexes The chemical interaction between NO and metal complexes is important in understanding NO's regulatory roles as well as its target sites

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in vivo. NO reacts with some transition metal complexes to form metalnitrosyl adducts. For example, it reacts with different oxidation states of iron, copper, cobalt, or nickel, but not with diamagnetic ions such as zinc (26). Thus, the reaction between NO and metal complexes depends on both the valence state of the metal and the ligands in the coordination sphere. Iron serves as a good example of the important impact of the valence state and ligand field on NO metal chemistry. Aqueous ferric ion does not react with NO to form a stable nitrosyl (Eq. [1]). Fe^^(aq) + NO -^ no reaction

[1]

However, if it is reduced to ferrous ion, NO will react to form a nitrosyl complex (Eq. [2]) with a second-order rate constant of 1 x 10^ M-^sec-i (30). Fe^^ (aq) + NO -^ Fe2^-N0(aq)

[2]

As a rule ferrous complexes have a higher affinity for NO than ferric ion complexes. Studies on the influence of the ligand field on NO binding to metal complexes show that porphyrin ligands tremendously increase the affinity of NO for the metal. As noted previously, ferric ion does not form a nitrosyl complex (Eq. [1]). Yet when a porphyrin is ligated to the ferric center, NO will form a stable Fe-NO adduct (31), with an equilibrium constant of 1.1 x 10^ M"^ Like the aqueous ion, reduction of these ferric porphyrin complexes to the ferrous state substantially increases the reaction rate between NO and iron (in this case by 10^) as well as the equilibrium constant by >10^^ (31). Fg3.(Tpps) + NO ^ Fe^^-NO(TPPS)

[31

Fe2^(TPPS) + NO -^ Fe2^-N0(TPPS)

[4]

Some ferrous heme proteins bind NO with bimolecular rate constants as high as >10^ M"^sec~^ (31). These high reaction rates suggest that ferrous heme proteins such as guanylate cyclase serve as receptor sites for NO, and they should be considered as one of the major reasons that NO is involved in many biological processes. NO not only reacts with metals to form nitrosyl complexes, it can also interact with dioxygen heme complexes such as oxyhemoglobin and oxymyoglobin. Doyle and Hoekstra showed that NO reacts with oxyhemoglobin (Eq. [5]) at a rapid rate of 3 X 10'^ M'^sec"^ to form methemoglobin and nitrate (32). Hb-02 + NO -^ metHb + NO3-

[5]

CHEMICAL BIOLOGY OF NITRIC OXIDE

163

Because of this rapid reaction, oxyhemoglobin is used to standardize NO solutions and to scavenge NO in biological experiments (33). Since the concentration of oxyhemoglobin is as high as 7 mM in blood, this reaction may play a key role in the control of the concentration and chemistry of NO in vivo. Another important reaction involving heme proteins is the reaction between NO and high-valent heme proteins (34,35). Exposure of metmyoglobin or hemoglobin to hydrogen peroxide forms a n cation radical complex which can decompose to form free iron (36-38). This free iron can then further oxidize other biomolecules via the Fentontype reaction, facilitating DNA damage (39). NO will react at a rate >10®M~^sec"^ to convert the hypervalent hemeprotein to the original oxidation state, preventing protein degradation (Scheme 2) (35). This reaction may be important for NO's antioxidant properties. B. Reactivity of NO with Radicais NO can react with a variety of radical molecules. Huie and Padmaja have studied the rate constants with superoxide and peroxy radicals (40). Both reactions occur at or near diffusion control. In the case of peroxy radicals (Eqs. [6] and [7]). ROO + NO ^ ROONO (2 x 10^ M'^sec"^)

[6]

ROONO -> RONO2 (1.5 sec-^

[7]

ROO + LH -^ L- + ROOH (10^ M'^sec"^)

[8]

Normally, ROO reacts relatively slowly with other lipids (Eq. [8]), facilitating radical chain propagation attributed to lipid peroxidation (41). As shown in Eqs. [6]-[8] ROO reacts 10^ faster with NO than with lipids; this reaction with NO could result in chain termination of lipid peroxidation. Such organic nitrates have been detected in lipids imder lipid peroxidation conditions (42). H2O2

Fe^"'(heme) —^^^

•

Fe=0(heme)

^^^

•pe^'^'Cheme)

' Released iron and bilirubin SCHEME 2. Mechanism for the scavenging of high-valent heme complexes with NO.

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C. Peroxynitrite

Another important radical reaction is the near-diffusion controlled reaction between superoxide and NO forming peroxynitrite (43) (Eq. [9]), which is thought to be a powerful oxidant. NO + O2- -> OONO-

(6.7 X 10^ M-^sec-^)

[9]

Initial studies suggested that protonation of this species (pi^a 6.7) results in the formation of hydroxyl radical and nitrogen dioxide (44). It was later concluded that the powerful oxidizing intermediate was protonated trans isomer of this molecule (45). However, in the absence of appreciable substrate concentrations, peroxynitrous acid rapidly rearranges to nitrate (46). It has been argued that peroxynitrite plays a role in the oxidation of biological macromolecules contributing to potential deleterious processes (47), or in the detoxification of superoxide, thus preventing damage normally associated with ROS (35,42,48). Beckman et al. (49) showed that sulfhydryls can scavenge the relatively inactive unprotonated form, suggesting that thiol-containing peptides, such as glutathione, may be important in the detoxication of peroxynitrite. In our laboratory, we have compared the oxidizing intermediates in the Fenton reaction, via the use of various chemical probes (50,51), with those for peroxynitrite anion and found that those oxidants associated with the Fenton-type chemistry are far more powerful. An interesting paper by Pryor et al, (52) has shown that peroxynitrite can undergo both oneand two-electron reactions, which cannot be explained on the basis of hydroxyl radical or nitrogen dioxide chemistry. Although peroxynitrite can oxidize, or as a possible NO2 donor, nitrate a substrate, it does not nitrosate (NO^^ donor) substrates as do intermediates formed in the NO/O2 (46,53). It appears that this molecule has its own signature and spectrum of oxidation. D. Chemistry of RNOS Formed from NO/O2 Reaction

Part of the answer to the dichotomy of NO's contradicting physiological roles lies in the understanding of the NO/O2 reaction, i.e., the autoxidation of NO (54,55). Even at micromolar concentrations of NO, this reaction was shown to occur via third-order kinetics with the rate equation, d[NO]/d^ = yfe[NO]2[02] ()fe = 6 x 10^ M'^sec-^ (54-58). One of the fascinating aspects of this reaction is that the rate constant is affected little by pH, temperatures between 20-37°C, or solvents, suggesting that the uncatalyzed reaction between NO and O2 would occur at nearly identical rates in any biological medium (55,56,58,59).

CHEMICAL BIOLOGY OF NITRIC OXIDE

165

The third-order nature of this rate equation reveals a second-order dependence on NO, hence making the half-Ufe or longevity of NO in aqueous solution proportional to its concentration (54). For instance, if the NO concentration is 1 fjuM, the first half-life would be 800 sec or 13 min; yet, as the NO concentration increases to 1 mM, the halflife would be less than 1 sec. Under normal physiological conditions, enz5mfiatically generated NO diffuses from the cell and as it jnigrates away from the cell it dilutes, thereby increases its lifetime. This allows NO to find its biological target with minimal interference from the NO/ O2 reaction. However, under conditions of much higher NO concentration, the rate of autoxidation also exponentially increases. This would lead to higher fluxes of reactive intermediates to which some of NO's deleterious effects in vivo have been attributed. The RNOS formed from the NO/O2 reaction in hydrophobic media are NO2, N2O3, and N2O4 (7,27). These are commonly associated with air pollution, and their deleterious effects on biological systems are well characterized (7). These intermediates nitrosate sulflhydryl and tjrrosine residues (29,53,60). The intermediates formed from the autoxidation in the gas phase are often thought to be the same as those formed in aqueous solution. However, several studies suggest that the intermediates formed from the NO/O2 reaction in aqueous solution are clearly different from those in the gas phase (54). In aqueous solution, there appears to be one primary intermediate which has an empirical formula of N2O3 (unpublished observations). However, it appears to differ from the isomer (possibly O = N - O - N = O) that is formed during the gas phase autoxidation of NO or from acidic nitritel (0 = N-N02) (59). However, the true nature of the NO;^ found in aqueous autoxidation of NO remains a topic of vigorous debate. It has been shown that nitrogen dioxide, which is the quintessential intermediate in the autoxidation of NO in the gas phase and in hydrophobic media, is not formed in the autoxidation of NO in aqueous solution (54,61). Although the exact structure of this reactive nitrogen oxide species is unclear, its chemistry with various bioorganic molecules has been characterized. The NOx species is uncharged and rapidly hydrolyzed in aqueous media to nitrite. This intermediate is capable of biomolecular reactions such as oxidation of redox-active complexes (54,62) and nitrosation of amines (54,62) and thiol-containing substrates (53). The scavenging of these intermediates by various immunosuppressive agents has been examined. In a recent report, Grisham and Miles (62) demonstrated that 5-aminosalicylic acid was a good scavenger of NO;^ and concluded that the oxidation potentials of the immunosuppressive agents examined were low enough to afford efficient scavenging of the intermediate.

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Compounds with higher oxidation potential such as 4-aminosaUcyUc acid were not as efficient and underwent nitrosation rather than 1 electron oxidation, thereby indicating that the effective oxidation potential of NO;c was less than 0.7 V (62). Further studies in our laboratory have shown that dopamine and other catecholamines can also be oxidized by this RNOS. Ascorbate was shown to be a very efficient scavenger of this intermediate and plays a role in the protection of bio-molecules. Hydrolyses to nitrite, oxidation, and nitrosation appear to be competitive reactions of NO^^; this allows an opportunity to assess the relative selectivity of various biologically important substances (Scheme 3). Similarly, these competitive studies have provided information as to the potential biological targets of NO^^. It has been shown that sulfhydrylcontaining peptides such as glutathione have a high affinity for NO^c to give S-nitrosothiol adducts (53) (Eq. [10]). RSH + NO, -> RSNO Direct Effects

ROONO RONO

Fe-NO(heme)

Met-heme + NO3

[10]

A Fe(heme)

Fe=0 Fe,2+

NO

Indirect Effects

Oxidation

02

Immunosuppressive Agents

Catecholamine Oxidation ^

NOv RSH

Nitrosamine

RSNO N02' SCHEME 3.

CHEMICAL BIOLOGY OF NITRIC OXIDE

167

These adducts are formed endogenously in the cardiovascular and pulmonary system (63,64). Thiol-nitrosyl adducts release NO over a period of time and activate guanylate cyclase in vivo. S-Nitrosothiols have even been proposed as an alternative chemical species to NO as an endothelium-derived relaxing factor (65). The affinity of sulfliydryls for NOjc is 10^ greater than that of nucleic acids and 10^ greater than those of amino acids, with the exception of tyrosine (53). It is proposed that glutathione can serve as a scavenger of NO^, playing a critical role in detoxification, and that proteins containing thiol residues critical to their function might be adversely affected (53,64).

III. Biochemical Targets for Nitric Oxide The effects of NO on various biomolecules can provide insights into the toxicological mechanisms of this unique molecule. As noted previously, NO can directly interact with metals (predominantly heme complexes) and radicals. Most of the damage to macromolecules is mediated through the reactive nitrogen oxide species, NO^c and OONO". The following sections of the chapter will describe some of the aspects of macromolecule alteration. A. Inhibition of Enzymes by NO and RNOS NO has been shown to interact with various macromolecules, including proteins. As mentioned previously, NO interacts with metalloproteins to form metal-nitrosyl adducts. Guanylate cyclase contains a heme cofactor to which NO binds to activate this enzyme (1). Iron sulfur clusters also react with NO. It has been shown that Cys2Fe(NO)2 is formed in bacterial and tumor cells in the presence of activated immune cells (66,67). However, in two papers it has been shown that inhibition of aconitase, the enz3ane often thought to be one of the primary targets of NO in vivo, is not inhibited by NO itself, but rather by RNOS such as perox3niitrite (20,21). As will be discussed below, the intermediates from the autoxidation of NO modify cysteine residues bound to metals which destroys the structural integrity of the complex by labilizing the metal. It is likely that aconitase inactivation is mediated by both peroxynitrite and NO^cAs previously stated, NO reacts at near-diffusion controlled rates with oxyhemoglobin and oxymyoglobin (32) providing an important mechanism for the in vivo depletion of NO. Ribonucleotide reductase has been suggested to be inhibited by the presence of NO by interaction at tyrosine residues, sulfhydryl residues, or at the metal center (19). Protein kinase C was shown to be inhibited by NO donor compounds.

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This report implied that nitrosating agents are responsible for the inactivation suggesting that this enzyme is inhibited by reactive nitrogen oxide species and not NO. ADP ribosylation of glyceraldehyde 3phosphate dehydrogenase, which inhibits this enzyme, has been proposed to be the result of nitrosation of a cysteine residue (68-70). Since neither NO nor perox5niitrite efficiently nitrosates thiols directly, the intermediates in autoxidation of NO most likely mediate this process. Mammalian P450 enzymes were shown to be inhibited by NO (18,71,72) (Scheme 4) by two different mechanisms. One involves direct interaction with NO which is reversible, whereas the other is irreversible and attributable to the action of reactive intermediates. NO has been shown to bind strongly to P450, forming a metal nitrosyl analogous to metal carbonyls formed from CO (Scheme 4). Unlike CO, NO binds to both the ferric and ferrous states. NO was shown to inhibit dealkylase activity of P450 lAl and 2B1 at concentrations as low as 3 /xM. This was shown to inhibit substrate oxidation completely, indicating that NO is 100 times more potent as an inhibitor than CO. This reversible inhibition was proposed as a regulatory mechanism for hormone production and other substrate oxidation pathways mediated by P450. One proposed mechanism for reversible inhibition is the reduction of NO to N2O, NH2OH, or NH3 analogous to nitrite reductase (18) (Scheme 5). It has been known for years that NO, when bound to a metal center, can be reduced to other nitrogenous products (73-75) (Scheme 5). It NO [3^lM]

Fe^+(NO) Dealkylation product

NO -,. ,2+ ^ = ± ^ Fe2+(NO)

Alkylated substrate

i NO ^

N^O

Fe^+ + N03-

SCHEME 4. Mechanism for reversible inhibition of cytochrome P450 (18).

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CHEMICAL BIOLOGY OF NITRIC OXIDE

N2O + Fe,3+ 3+

Fe2+

A +N0 Fe^^-NO"

Fe3^-N0 ^ ; = ^ Fe^^-NO

2e3H^ 2e' Fe-'-'+NHj

Fe'^^-NHjOH

2H^

Fe-'"^ + NH2OH SCHEME 5. Mechanism for NO reduction mediated by metallo proteins.

was shown that NO can also be reduced by a bacterial P450, lending support to this proposal (76). At higher concentrations of NO, irreversible inhibition of P450 occurs where the velocity of substrate oxidation is less than that before the exposure to NO (18). It was proposed that the cause of this inhibition was the reaction of NO^c with the protein, which compromised the structural integrity. Bovine serum albumin (BSA) was shown to abate this effect, presumably by scavenging the NO^^ species (Scheme 6). Because NO [100 ^iM]

NO

Bovine Serum Albumin Glutathione ^

\

Irreversible Inhibition of Cytochrome P450

" Scavenging

SCHEME 6. Mechanism of irreversible inhibition of P450 activity.

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DAVID A. WINK et al.

BSA in the presence of RNOS such as NO;^ and NO2 forms a S-nitrosothiol adduct (63), it seems Hkely that the thiol groups protect against the degradation of P450 (Scheme 6). Because NO;^ preferentially reacts with sulfhydryl-containing amino acids more readily than with any of the other bioorganic substances, proteins with functionally critical thiol residues may be irreversibly inhibited. Many enzymes which interact with DNA as regulatory or repair proteins contain sulfhydryl residues. Several classes of DNA-interacting proteins contain critical thiol residues, such as transferases (77), and proteins containing leucine zippers (78) and zinc-finger motifs (79,80). DNA alkyltransferase proteins involved in the repair of 0^-methylguanine and O'^-methylthymine residues contain a thiol group in their active site (77,81). It was shown that NO inhibited the DNA methyltransferase activity not only in the mammalian purified protein but also in whole cells (17). As shown in Scheme 7, the methyl group of 0^-methylguanine is repaired by the DNA transferase protein by simply transferring the methyl group from the O^ position of the methylated guanine to a cysteine residue within the protein. Exposure to NO in an aerobic solution results in nitrosation of the thiol, thereby preventing the methyl transfer (Scheme 7). Furthermore, the presence of NO also potentiated the toxicity of bis-iV,Ar'-bis(2-chloroethyl)-Ar-nitrosourea, presumably by inhibition of this enzyme (17). This is a good illustration as to how NO might potentiate the toxicity of various agents. Another important class of DNA-interacting proteins includes those containing zinc-finger motifs (80). Zinc-finger motifs contain either 2 or 4 cysteine residues. One DNA-repair protein, formamidopyrimidineDNA glycosylase (Fpg protein), which repairs oxidative damage to guanine, such as 2,6-diamino-4-hydroxy-5-iV-methylformamidopyrimidine dG

Toxicity - ^ Mutations RSNO "No Repair"

_

Methylating Agents

MeOMG ^

NO,

RSH

RSMe ^ ^ ^

Alkyltransferase (RSH)

SCHEME 7. Mechanism for O^-methyl-guanine-DNA-methyltransferase by NO.

inhibition

171

CHEMICAL BIOLOGY OF NITRIC OXIDE

(Fapy) and 8-oxoguanine, by glycosylase activity or incises DNA at abasic sites by abd elimination reaction (82) contains a zinc-finger motif which is mandatory for the various activities of the Fpg protein (82). It was shown that both activities of the Fpg protein were inhibited in the presence of aerobic NO (16). It was suggested that NO;^ nitrosates the thiol residue, resulting in the ejection of the zinc. This degradation of the structural integrity of the protein does not allow its interaction with DNA and, hence, inhibits repair in vitro and in vivo (Scheme 8). Another study showed that the zinc-finger protein, LAC9, was degraded by the presence of NO (84). Using Raman spectroscopy, it was shown that S-nitrosothiol adducts were formed (84). Another important aspect of the NO^-mediated destruction of zincfinger motifs may be in the immune system's ability to fight viruses. The life cycle of viruses depends on key proteins which contain the zinc-finger motifs, and thus NO^ may play a role in the immune system's antiviral activity. It has been shown that NO will prevent infection by the herpesvirus (85,86). The ligand field of zinc is similar to that of cadmium or copper sequestered in metallotionein (87). Metallothionein protects cells from the toxic effects of various metals (88). A recent report showed that, in the presence of NO, metals such as cadmium can be released from metallothionein (83). These data imply that RNOS may mediate intracellular metal release and thereby enhance the toxicity of some metals. The reaction of NO^^ with sulfhydryls bound to metals may play an important role in the toxicology of various metals. B. DNA

Bacteria (11,13) and some mammalian cells (12) treated with NO have shown increased mutagenicity. It was demonstrated that NOcaused genotoxicity was due to nitrosative deamination (11,12) (Scheme

+

Protein Denaturation

Zn^.2+

Protein Degradation

SCHEME 8. Proposed mechanism for the degradation of zinc-finger motifs in the presence of NO.

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DAVID A. WINK et al. NH2 H N T V ^ ^

I

R Melhylcytosine

OH

NQ/Q2

HrT"^^^

I

R Thymine

SCHEME 9. Deamination of nucleic acids by aerobic NO.

9). This type of lesion has been suggested to be responsible for spontaneous deamination in vivo. In addition to nitrosative deamination, DNA strand breaks were detected in cells treated with either aerobic NO (12) or nitrogen dioxide (15). We found that treatment of supercoiled DNA with either NO or nitrogen dioxide did not result in these strand breaks (89). An insightful proposal suggests that NO-induced strand breaks observed in cells may be due to the change in kinetics of unwinding and winding DNA by ligase. It was suggested that NO did not directly cause strand breaks (90), but the protein responsible for recoiling of the DNA was inhibited, thus increasing the number of breaks. The ligase may contain a DNA-binding domain similar to a zinc-finger motif which is susceptible to inhibition by NO. IV. Extracellular and Intracellular Metabolism of Nitric Oxide The question arises as to whether or not there are processes in biological systems which control NO and its chemistry (Scheme 10). The autoxidation of NO governs the lifetime of this molecule in aqueous solution. As previously noted, kinetics for the disappearance of NO at physiological temperature and pH has been shown to be third order (55). This predicts that, in excess oxygen, a l-/xM NO solution should be long lived. This is contrary to the lifetime reported in vivo which is <5 sec (1,2,24,91). The discrepancy between the in vivo and in vitro observations suggests that there are other pathways which consume NO in biological systems. A. Role of Hemoglobin in Control of NO Concentrations Several papers have suggested that the reaction with oxyhemoprotein to form nitrate may in part explain this phenomenon (1,2,24,91). Lancaster has used a mathematical model to discuss the influence of oxyhemoglobin in erythrocytes (24). Other studies suggest that reaction

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CHEMICAL BIOLOGY OF NITRIC OXIDE Direct Effects

Mitochondrial Consumption

Met-heme + NO3

Activation of Guanylate Cyclase

Reversible Inhibition of P450 Inhibition of Ribonucleotide^ Reductase?

Indirect Effects

Aconitase Inhibition Ascorbate Scavenging ^•Irreversible Inhibition ofP45()

Zn Finger Proteins Metal Release

Glutathione Scavenging Inhibition of Alkyltransferases

Protein Kinase C Inhibition

ADPRibosylation of GAPDH

SCHEME 10. Summary of the biochemical reactions of NO in terms of direct and indirect effects.

with superoxide may account for the shorter hfetime in vivo (1), though there probably is not enough superoxide available to account for all the NO consumption. B. NO and Mitochondrial Respiration A recent study has used an electron spin resonance technique which is capable of monitoring intracellular NO (92). It was found that mitochondrial respiration consumes NO at a rate comparable to that of oxygen in Chinese hamster ovary cells. In this study, a comparison of the predicted half-life of NO between oxidation mediated by oxygen and consumption mediated by mitochondrial respiration was conducted (Fig. 1). In addition, this mathematical model showed that when intracellular NO concentrations were below 0.020 mAf, NO was primarily consumed by mitochondrial respiration and not by autoxidation (Fig. 2). According to this model, no RNOS are derived from NO/O2 reaction at low cellular NO concentrations.

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CM

SB B NO concentration (|LiM) FIG. 1. A comparison of the half-life of NO in aqueous solution with that calculated for the intracellular environment which includes mitochondrial consumption. (A) Calculated half-life of NO in an aqueous solution. (B) Half-life of NO, taking into consideration the rates of autoxidation and mitochondrial consumption. The model is based on a volume of a sphere with a radius of 10 /im and on mitochondrial consumption rate of 3.2 /JLM/ sec with the autoxidation reaction of-d[NO]/dt = AJ[NO]2[02], where AJ = 6 X 10^ Af-^sec^ [O2] = 200 fiM, and the NO concentration given is the initial concentration.

To evaluate the role of RNOS on biological systems it is essential to know the concentration of NO that is required to form these species intracellularly. As previously shown, levels of 5-20 fiM NO will begin to generate significant amounts of intracellular NO^c- When does peroxynitrite form to an appreciable extent intracellularly? This can be evaluated from the comparison of the rate constants of the reactivity between NO/O2" and superoxide dismutase (SODVOg-. Since SOD and NO react with superoxide at similar rate constants, the amount of steady-state NO concentrations must be caparable to those of SOD to form peroxynitrite intracellularly. Intracellular concentrations of SOD are thought to range from 10 to 50 fjuM; therefore, 5-20 /xAf NO would be required to form this RNOS intracellularly. It appears that, in general, the formation of RNOS would require local concentrations above 5 JJLM to have significant effects on intracellular components. A note of caution; the cell is a highly organized structure and local fiuxes and diffusion of various biomolecules may differ from this simplistic model.

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200Mitochondrial Consumption

C

NO

a

I looH

NO^ Formation

o z 10

0.1

25

100

50

200

NO concentration (|iM) Cardiovascular Function

Neurotransmission (Catecholamine Regulation)

Immunological Response

FIG. 2. Calculated intracellular NO, formation. This model applies for conditions which include the volume of a sphere with a radius of 10 ^tm, a mitochondrial consumption rate of 3.2 fiM/sec; the autoxidation reaction of-d[NOVdt = )fe[NO]2[02], where k = 6x W ^sec^; [O2] = 200 /LLM. The NO concentration is the initial concentration.

V. Nitric Oxide and Oxidative Stress NO and ROS are both components of the immune system (Scheme 11). The respiratory burst, induced by various cytokine-stimulated cells of the immune system resulting in the formation of peroxide/superoxide, often precedes the formation of nitrite from L-arginine. For example, lipopolysaccharide stimulation of macrophages shows two distinct time intervals for the formation of peroxide/superoxide and NO (9,93). Induction of NO formation does, in many cases, require a specific combination of immune system-activating factors, but there are also conditions under which this activation is not linked to NO (4). For these Biological Reduction of Oxygen

NO Generation

(Xanthine oxidase, NADH oxidase mixed function oxidase)

L-Arginine

02 NO-synthase

^^2^2

02"

T

itruUine

NO

SCHEME 11. Biological formation of both ROS and NO.

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conditions, such as with phorbol ester stimulation of alveolar macrophages (94), eukaryocytes (95), or neutrophils (96), there is concurrent formation of peroxide/superoxide and NO. As discussed previously, NO and superoxide can react to form peroxynitrite (Eq. [9]), which, when protonated, forms a powerful oxidant that can potentially damage cellular components. Peroxynitrite is often invoked as a major causative agent in NO-mediated toxicity (44,97,98), yet hydrogen peroxide is also produced (96) under these biological conditions. Therefore, the reactions involving Fenton-type chemistry cannot be ignored. A key question is what effect does the formation of NO have on ROS? In a number of disease states, such as ischemic reperfusion injury, NO has been proposed to be a key causative agent in the destruction of tissue (8,98). However, other groups have shown that the presence of NO under these conditions is protective (99-103). Is NO protective or toxic in the presence of ROS? We decided to take a chemical approach to this problem. In the case of the biological reduction of oxygen, both peroxide and superoxide are generated (Scheme 11). In the absence of NO, superoxide dismutates to hydrogen peroxide (Eq. [11]), 2 O2- -> H2O2 + O2 (1 X 10^ M-^sec-^)

[11]

which in vivo is accelerated by SOD where SOD reacts with superoxide with a rate constant of 2 x 10^ M'^sec ^ A. Effect of NO on Cytotoxicity by Chemically Generated ROS

We tested the presence of a sustained source of NO on peroxidemediated toxicity. A series of compounds known as NONOates has provided a unique opportunity to examine the effect of a sustained source of NO (104) on various biological conditions (12). These are salts of amine adducts to nitric oxide (RiR2N[N(0)N0]") which have been developed for a number of applications in this field (Eq. [12]). RiR2N[N(0)N0]- + H^ -^ 2 NO + R1R2NH

[12]

It was shown that exposure of Chinese hamster V79 lung fibroblast cells to increasing hydrogen peroxide concentrations resulted in a marked increase in cytotoxicity (105). In the presence of the NONOate, DEA/ NO ((C2H5)2N[N(0)NO]-Na^), there was dramatic protection (48). It was surmised that the NO released from this compound was the source of protection. In a commentary by Ohio accompanying this report (106), it was suggested that various vasodilators should be compared to the NONOates. Extension of these studies shows that compounds containing thiol-nitrosyl functional groups were also protective against peroxide-mediated toxicity (unpublished observations). Yet, other nitrovaso-

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dilators, such as 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), potentiated hydrogen peroxide toxicity (manuscript in preparation). When NO concentrations were measured over the time course of the experiment, via a NO-sensitive electrode in buffered solution, it was found that compounds which released IJLM levels of NO were protective (NONOates, S-nitrosoglutathione, and Snitroso-AT-acetylpenicillamine), but those that did not release NO, such as SIN-1 and SNP, increased hydrogen peroxide-mediated cytotoxicity. From these measurements, we found that a minimal NO concentration of 1-5 jxM throughout the peroxide exposure was protective. NO also protected against peroxide-mediated cell damage in other cell systems, including neuronal and hepatoma cells (35). We also examined the effect of NO on organic peroxide-mediated toxicity which is thought to be mediated by oxidation of lipophilic membranes (submitted for publication). In the case of DEA/NO, which has a lifetime of about 2 min, no protection was observed. However, when the NONOate, PAPA/NO (NH^3(CH2)3N[N(0)NO]-(CH2)2(CH3)), whose ti is 15 min, was used, marked protection was observed against both tert-hutyl hydroperoxide and cumene hydroperoxide. Organic peroxides require a longer exposure time to exert cytotoxicity. Since NO is required to be present simultaneously with peroxide treatment, the longer NO-releasing agent is protective, while the shorter acting agent rapidly exhausts its NO and affords no protection. From the studies with hydrogen peroxide and organic peroxide, it can be concluded that NO is protective against Fenton-type-mediated toxicity. In support of these cell experiments. Freeman and co-workers (42) investigated the effect of NO on hypoxanthine/xanthine oxidase (XO)-mediated lipid peroxidation and found that NO acted as an antioxidant. Can these results be understood in terms of the direct and indirect effects of NO? In the Fenton-type chemistry, Kanner et aL (34) first suggested that NO could play an antioxidant role. When peroxide enters a cell, it quickly reacts with heme proteins to form hypervalent complexes. These hypervalent heme complexes can oxidize biological substrates to cause lipid peroxidation (36). Furthermore, decomposition of these complexes either increases the amount of released intracellular low-molecular-weight iron complexes which facilitates DNA damage and cytotoxicity (36,107) or serves as peroxide/superoxide generator. The reaction between these hypervalent metalloprotein species and NO occurs at near-diffusion controlled rates and returns the heme to the ferric form, thereby preventing further decomposition (34,35). Thus, it appears that NO can prevent the formation of powerful oxidants

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normally associated with Fenton-type chemistry as well as scavenge these heme protein intermediates. B. Effect of NO on Enzymatically Generated ROS Biological conditions which facilitate reduction of oxygen also promote the formation of superoxide. For instance, oxygen reduction catalyzed by hypoxanthine/XO forms not only peroxide but also superoxide. Because superoxide and NO can react to form peroxynitrite, we examined XO-mediated cytotoxicity in the presence of NO releasing agents. It was found that, in the presence of XO, both mesencephalic neurons and Chinese hamster V79 cells showed increased cytotoxicity with increased exposure time (48). Yet, in the presence of DEA/NO, cells were protected from both peroxide- and XO-mediated toxicity. From these results, we concluded that NO was protective in the presence of biologically mediated reduction of oxygen. It should be noted that, together, catalase, XO, and DEA/NO showed no toxicity, which suggested that any peroxynitrite formed under these conditions was not toxic. Therefore, it was concluded that extracellular formation of peroxynitrite was not destructive, but instead could play a key role in detoxifying superoxide. Because NO or reactive nitrogen oxide species can inactivate some enzymes, we have examined the effect of NO-generating compounds on xanthine oxidase enzyme activity to determine whether inactivation of XO by NO was the reason for protection. It was shown that NO does not inhibit the oxidation of hypoxanthine to urate (35,42,48,108). Clancy et al. (108) demonstrated that NO could reversibly prevent the reduction of ferric5^ochrome c by NO which was verified in a later study (35,48). It was suggested that NO intercepts superoxide to form peroxynitrite, which subsequently rearranges to nitrate. We have examined the converse reaction. NO will nitrosate amine substrates via the NO/O2 reaction (54). When an NO-generating compound is in the presence of XO, nitrosation is inhibited (109), presumably via scavenging of NO. Again, it appears that superoxide intercepts NO thereby preventing nitrosation. It seems safe to conclude that the diffusion controlled reaction between NO and superoxide (Eq. [9]) could account for inhibition of nitrosation. Further studies in our laboratories have demonstrated that XO in the presence of NO-generating compounds forms an intermediate capable of oxidizing dihydrorhodamine. Because peroxynitrite can oxidize dihydrorhodamine (110), it is reasonable to assume that peroxynitrite is formed in the presence of XO and NO and accounts for the oxidizing

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behavior (Scheme 12). Yet, as the amount of NO flux increases, the extent of oxidation decreases. It appears that the oxidizing intermediate generated from perox3niitrite can be scavenged by NO, thus Umiting the extent of peroxynitrite-mediated oxidation. This suggests that peroxynitrite formed by enzymatic reduction of oxygen is hmited by the NO/0 2 ratio, thus reducing peroxynitrite's abihty to oxidize biological substances in vivo. One ofthe major mechanisms for peroxide-mediated cytotoxicity is oxidative damage to DNA such as double-strand breaks. When supercoiled plasmid is exposed to NO or NO-donor compounds, no single- or doublestrand breaks occur. However, in the presence of 1 mM hydrogen peroxide or xanthine oxidase and 1 mM Fe(His), extensive strand breaks are observed. In contrast, under the same conditions and in the presence of anaerobic NO or DEA/NO, dramatic abatement of DNA damage is observed (89). In further studies, we have found that the XO/NO combination is a very poor hydroxylating agent. In fact, hydroxylation of salicyclic acid or benzoic acid mediated by XO/Fe(His) is reduced by 99% in the presence of NO donors (111). These observations support the contention that NO is protective against reactive oxygen species. C. Effect of NO on Lipid Peroxidation NO has been shown to prevent lipid peroxidation of low-density lipoproteins (LDL). It was proposed by Hogg et al. (41) that NO may play a beneficial role in preventing the progression of arteriosclerosis. In this report, it was demonstrated that LDL oxidation mediated by copper was inhibited by the presence of NO and NO-donor compounds. This NO-generator

XO

o , s l ^'^Hypoxanthine/xanthine oxidase

Ferricytochrome c reductionV. Urate P^2+ Hydroxylation J ^ " ^ ^ ^ DNA breaks Inhibition of Fenton-mediated reactions Nitrosation Scavenging

Oxidation of DHR

SCHEME 12. Mechanism of reactive intermediate formation from an NO generator and xanthine oxidase.

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group suggested that NO may have multiple beneficial effects: (i) inhibition of metal-mediated lipid oxidation (41,42), (ii) inhibition of lipoxygenase (112), and (iii) prevention of platelet aggregation (113) and leukocyte adhesion (99,114). However, in a previous report this group also demonstrated that peroxynitrite was capable of oxidizing LDL; therefore, NO could also play a role in the exacerbation of atherosclerosis (115). Although the metal-mediated oxidation reactions are decreased, NO may enhance O2-dependent oxidation in the absence of metals under specific conditions. It is important to know where, when, and how much NO and RNOS are formed to better understand the role that NO plays in various disease states. D. Effect of NO on Biologically Generated ROS

As discussed previously, the effect of NO on various reactive oxygen species clearly shows that mammalian cells are protected from the toxic effects mediated by the Fenton-type intermediates. In addition to the chemically protective mechanisms, in vivo data show that NO prevents the adhesion of neutrophils to the vascular wall, providing a biologically protective effect (103). Several studies have shown that inhibition of NOS increases leukocyte adhesion (95,114). In the same studies it was demonstrated that superoxide stimulated leukocyte adhesion. It was concluded that under ischemia-reperfusion conditions NO scavenged superoxide and that diminished availability of NO was the cause of increased damages during oxidative stress. Lefer et aL (99) have also shown that NO plays a vital role in the regulation of neutrophil adherence in ischemia-reperfusion injury (117). Their results suggested that NO can prevent the destruction mediated by toxic chemical species by inhibiting their formation. E. Effect of NO on Ionizing Radiation

Are there conditions under which NO could enhance the cytotoxicity of ROS? One logical comparison is between ionizing radiation-induced cytotoxicity and peroxide toxicity. Both conditions generate powerful oxidants, such as hydroxyl radicals, which can evoke significant damage to vital cellular components. As clearly shown above, low concentrations of NO can have a beneficial effect in protecting against peroxide-mediated damage from both a biological and a chemical perspective. Under aerobic conditions, cells are approximately threefold more sensitive to radiation than under hypoxic conditions. This is a problem in radiation therapy in that there are often hypoxic regions of tumor which are resistant (119). It is thought that this hypoxic population of cells is responsible for tumor regrowth and the limited efficacy of radiation

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therapy. We examined the effects of NO on the cytotoxicity of ionizing radiation. Treatment of hypoxic cells with NO showed a marked increase in the radiosensitization of hypoxic cells (120,121). Contrary to hydrogen peroxide-mediated toxicity, NO clearly increases cytotoxicity under these conditions. This is presumably due to the rapid reaction between carbon-centered radicals on DNA with NO to "fix the damage" (Scheme 13). The comparison between NO's ability to protect against peroxide/superoxide- and radiation-mediated damage demonstrates the complexity of effects NO can exert in biological systems. F. Effect of NOxOn ROS-lnduced Cytotoxicity

Another consideration is the effect that NO;^ species formed from the NO/O2 reaction will have on the toxicity of reactive oxygen species. At low concentrations, NO clearly protects mammalian cells from peroxide-mediated toxicity. However, at higher concentrations, the NO/O2 reaction is facilitated, resulting in NO^ and nitrite formation. NO^ can mediate the inhibition of various thiol-dependent proteins. In particular, it can destroy the structural integrity of zinc-finger proteins (16,83) or cause dysfunction of the mitochondria (67). NO;^ can also labilize transition metals from thiol-rich environments, such as metallothionein, and thus provide additional low-molecular-weight transition metal catalysts for the Fenton-type reactions. Another possibility is that nitrite, the autoxidation product of NO, can be oxidized by powerful oxidants formed in the Fenton reaction to form the toxic species nitrogen dioxide (NO2). Nitrogen dioxide is known to be the quintessential toxic intermediate in air pollution, causing lipid peroxidation and thiol oxidation. To test this hypothesis, we exposed cells to nitrite and peroxide and found a 10-fold increase in peroxide-mediated toxicity due to the presence of nitrite (35). Klebanoff (121) proposed that high valent heme complexes formed from peroxide within the cell could catalyze the oxidation of nitrite to nitrogen dioxide (Eq. [13]). "fixed damage"

02

-CH

•

-coo

-C-^

•-GH

^*^

NO

repair

-CNO "fixed damage"

SCHEME 13. Mechanism for the radiosensitization of hjrpoxic cells by NO and O2.

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NO2- + strong oxidant (ROS) -^ NO2

[13]

Most likely, nitrite can play a key role in the enhancement of cell toxicity, and experimental results which attribute toxicity to NO should be examined carefully to account for the effects of nitrite.

VI. Conclusions: Direct versus Indirect Effects of Nitric Oxide on Biological Systems As was previously discussed, NO can be regulatory, protective, or toxic. The chemical reactions can be grouped into two different categories. Direct effects consist of reactions in which NO itself interacts with the biological target. This results in activities such as (i) guanylate cyclase regulation, in which NO directly binds to the heme moiety; (ii) the protection afforded by NO against ROS, in which NO directly scavenges and prevents formation of reactive oxygen species; and (iii) the abatement of lipid peroxidation. NO can be directly consumed by the mitochondria and oxyhemoglobin. NO can directly react (reversibly) with enzymes such as P450 resulting in inhibition of drug metabolism. NO can also directly react with radicals formed from ionizing radiation on DNA-formed adducts, which in turn fixes the damage and enhances radiosensitization of hypoxic bacteria and mammalian cells. Indirect effects are those that result from the chemistry of NO^. For instance, NO^, can cause inhibition of DNA-interacting and repair proteins, which could play a role in enhancing the toxicity and genotoxicity of alkylating agents. NO^ can also labilize metals from sulfhydrylbinding sites, increasing the concentration of intracellular low-molecular-weight transition metal complexes and thereby promoting cellular toxicity. As direct reactions of NO with components of the mitochondria and oxyhemoglobin play a role in preventing the formation of these intermediates, so too will endogenous antioxidants such as glutathione and ascorbate play a critical role in scavenging these NO^^ or OONO" intermediates thereby preventing toxicity. Since cNOS generates low levels of NO, direct effects and not indirect effects of NO would be more relevant under normal physiological conditions. In contrast, iNOS, which responds to some forms of physiological stress, forms considerably more NO for longer periods of time; therefore, both direct and indirect effects could be generated. With these classifications, one can categorize the biological effects and results with respect to either toxicology or to the development of therapeutic strategies involving NO. Direct effects generally require fluxes of NO in the range of 1-5 /xM, whereas indirect effects generally require local concentra-

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tions as high as 20 JJLM, CNOS activity, therefore, would be associated only with direct effects, while iNOS would be associated with both indirect and direct effects. Defining the chemical, biochemical, and cellular pathways of NO in a quantitative way provides insights into the role that NO plays in the etiology of various diseases. The underlying chemical biology of nitric oxide provides an understanding of how NO can have seemingly contradictory regulatory, toxic, and protective roles in biological systems, and even therapeutic applications. REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11.

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CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Nutritional and Hormonal Regulation of Glutathione Homeostasis CARLA G . TAYLOR Department of Foods and Nutrition University of Manitoba Winnipeg, Manitoba Canada R3T2N2 LAURA E . NAGY Department of Nutritional Science University of Guelph Guelph, Ontario Canada NIG 2W1 TAMMY M . BRAY Department of Human Nutrition The Ohio State University Columbus, Ohio 43210

I. Introduction Glutathione (GSH, L-y-glutamyl-L-cysteinylglycine), a substrate for GSH peroxidase [EC 1.11.1.9] and GSH S-transferases [EC 2.5.1.18], plays an important role in the antioxidation and detoxification of reactive oxygen species, free radicals, and xenobiotic compounds. GSH has many other physiological functions including the storage and transport of cysteine, leukotriene and prostaglandin metabolism, deoxyribonucleotide S3mithesis, immune cell response, and cell proliferation (for reviews see IMeister, 1991; DeLeve and Kaplowitz, 1990; Shan et aL, 1990; Deneke and Fanburg, 1989). The potential involvement of GSH in cellular regulation and metabolism was recognized in the 1950s and 1960s when it was demonstrated t h a t several enzymes involved in intermediary metabolism could be regulated by thiol: disulfide exchange between protein thiols and low-molecular-weight disulfides (for reviews see Gilbert, 1984; Zeigler, 1985). Thiol: disulfide exchange provides a mechanism for the sulfhydryl oxidation state of proteins to be in equilibrium with the thiol status of the cellular environment. It has been demonstrated t h a t cellular processes such as signal transduction and gene transcription are dependent on the redox status of critical sulfhydryl groups for interactions among proteins and between tran189

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scription factors and DNA. For example, it has been demonstrated that alkylation of sulfhydryl groups with N-ethylmaleimide (NEM) inhibits receptor-mediated signal transduction in T lymphocytes (Kanner et ai, 1992), and NEM or the sulfhydryl oxidizing agent diamide inhibits the DNA binding activities of fos and jun (Abate et ai, 1990). Furthermore, manipulation of thiol redox status by oxidative stress can alter cellular metabolism and gene transcription. For instance, depletion of lymphocyte GSH in vitro by conjunction with l-chloro-2,4-dinitrobenzene inhibits receptor-mediated T-lymphocyte signal transduction (Kavanagh et ai, 1993), and in vitro depletion of intracellular thiols by reactive oxygen intermediates activates the NF-KB transcription factor and transcription of human immunodeficiency virus (HIV) (Staal et al., 1990; Schreck et al., 1991). Thus, there are many in vitro examples which suggest that GSH may be modulating cellular metabolism and gene expression by affecting cellular thiol redox status. One of the current challenges, however, is to evaluate the potential importance of this concept in vivo. Tissue concentrations of GSH, like many other metabolically important compounds, are highly regulated. For example, it is difficult to deplete hepatic GSH to less than 30% of control values even with xenobiotic challenge or prolonged starvation (Maruyama et al., 1968; Jaeger et al, 1973; Tateishi et al., 1974,1977; Cho et al., 1981; WiUiamson et al, 1982; Jaeschke and Wendel, 1985; Hazelton et ai, 1986). Also, it is difficult to exceed the physiological maximum concentration for hepatic GSH with supplementation of GSH precursors unless hepatic GSH stores have been previously depleted with xenobiotics or by fasting (Williamson et al, 1982; Hazelton et al., 1986). Similarly, tissue concentrations of oxidized glutathione (GSSG) are tightly controlled in vivo (Meister, 1991). GSSG is produced during oxidative stress when peroxides are detoxified by GSH peroxidase. GSSG is recycled back to GSH by GSH reductase at the expense of NADPH. In the absence of adequate reducing equivalents, however, cellular thiol balance is preserved by export of GSSG and uptake by the kidney (Mclntyre and Curthoys, 1980). This emphasis placed on maintaining tissue GSH and GSSG concentrations and GSH/GSSG ratios within a tightly regulated physiological range supports the concept of an important regulatory role in vivo for tissue thiols and thiol redox status. Although many factors influence GSH status, the regulation of tissue GSH by nutrition and hormones is important for understanding in vivo GSH homeostasis, particularly when malnutrition and oxidative stress are present. Malnutrition is a complication of many diseases and occurs during prolonged or repeated bouts of infection. The disease process

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or infection, and their treatments with drug or oxygen therapies, can contribute to increased oxidative stress and decreased tissue GSH concentrations. These same factors, i.e., malnutrition, disease, infection, and drug treatments, can alter hormonal status, and thus modulate tissue GSH metabolism. One of the current clinical interests in the GSH field is the development of therapies to replete or enhance tissue GSH concentrations for optimal antioxidant and immune functions. This strategy may have positive effects on many other cellular processes which are believed to be regulated by tissue thiols and thiol redox status. The focus of this review is to discuss the regulation of tissue GSH homeostasis by nutrition and hormones and to assess the physiological relevance of altered thiol status using the vicious cycle of disease, infection, and malnutrition as an example. We believe that an understanding of the nutritional and hormonal regulation of GSH homeostasis is important for developing strategies to enhance tissue GSH and to intervene in this cycle of disease, infection, and malnutrition.

II. Glutathione Synthesis and Interorgan Homeostasis GSH S3mthesis is a tightly regulated process. In the initial ratelimiting step, glutamate and cysteine are substrates for y-glutamylcysteine synthetase (glutamate-cysteine ligase) (DeLeve and Kaplowitz, 1990). Plasma cysteine concentrations are relatively low compared to other plasma amino acid concentrations and additional sources of cysteine are from cleavage of the disulfide cystine and by synthesis from methionine via the cystathionine pathway. In extrahepatic tissues, uptake of plasma GSH via y-glutamyltranspeptidase (glutamyltransferase) provides an additional source of cysteine for GSH synthesis (Hahn et a/., 1978; Griffith and Meister, 1979). The y-glutamylcysteine synthetase step is regulated in vitro by feedback inhibition of GSH (Richman and Meister, 1975). Feedback inhibition is considered to be an important regulatory mechanism which limits the maximum tissue concentration of GSH in vivo. In the second step of GSH synthesis, GSH S3nithetase catalyzes the reaction between glycine and 7glutamylcysteine to form GSH. The interorgan homeostasis of GSH is summarized in Fig. 1. Hepatic GSH is exported into the plasma for uptake by extrahepatic tissues, and it is also released into the bile (Lauterburg et al., 1984). Other sources of GSH in the intestinal lumen probably include the diet, desquamated epithelial cells, and GSH export from epithelial stomach and intestinal cells (Meister, 1991). Although direct absorption of intact GSH in vascularly perfused small intestine of the rat has been reported

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GSH GSSG GSX

dietary GSH

FIG. 1. Interorgan homeostasis of GSH. After Deneke and Fanburg (1989); in Bray and Taylor (1993).

(Hagen and Jones, 1987), in vivo studies with oral glutathione have not demonstrated a sustained effect of increasing tissue GSH concentrations except in the small intestine (Vina et al., 1989; Martensson et al, 1990; Hagen et ai, 1990; Aw et al,, 1991). In the intestinal tract, GSH can be cleaved by y-glutamyltranspeptidase and dipeptidases to yield dipeptides and free amino acids which are absorbed and enter the circulation (Fig. 1). Alternative forms of GSH such as GSH ester and cysteine prodrugs which bypass intestinal digestion are often used as dietary supplements. The locational specificity of the enzymes of the GSH cycle provides the framework for intracellular maintenance of tissue GSH concentrations and interorgan transport of GSH. However, tissue GSH concentrations are regulated by other factors including the diet, nutritional status, and hormonal balance.

III. Regulation of Tissue Glutathione Concentration by Diet and Nutritional Status The availability of substrate, specifically the sulfur amino acid content of the diet, is a major determinant of hepatic GSH concentration within the physiological range. Hepatic GSH concentration is signifi-

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cantly decreased in rats fed diets deficient in sulfur amino acids or low protein diets or during fasting; supplementation of low protein diets with sulfur amino acid or refeeding fasted rats increases the hepatic GSH concentration (Edwards and Westerfield, 1952; Tateishi et al, 1974, 1977; Cho et a/., 1981; Boebel and Baker, 1983; Jaeschke and Wendel, 1985; Bauman et al, 1988a). This rise and fall in hepatic GSH concentration is strictly a response to the availability of substrate, especially cysteine, in the diet for GSH synthesis (Beck et al., 1958; Maruyama et al, 1968; Jaeger et a/., 1973; Tateishi et aL, 1974, 1977; Cho et al, 1981; Jaeschke and Wendel, 1985; Bauman et ai, 1988b). Generally, this rise and fall in hepatic GSH concentration is within a tightly regulated physiological range. For example, hepatic GSH concentration did not fall below 3 /xmol/g of tissue when rats were fasted for 24 hr or fed a diet containing almost no protein (0.5%) for 2 weeks (Taylor et ai, 1992). Hepatic GSH concentration did not reach beyond the normal physiological maximum of 8-10 fjumoVg when rats were fed high protein (30 or 45%) diets with a sulfur amino acid content which is two- or threefold above the normal protein (15%) diet (Bauman et ai, 1988a). These data also support the literature hypothesis that maximum GSH concentration is regulated by feedback inhibition of yglutamylcysteine synthetase by GSH (Richman and Meister, 1975). Although cysteine is normally the limiting amino acid for GSH synthesis, glutamine supplementation of total parenteral nutrition (TPN) solutions during severe trauma may be beneficial for increasing availability of substrate and energy for GSH synthesis. TPN formulations often contain methionine as the source of sulfur amino acid, glycine, and glutamate, but they do not include glutamine. Glutamine-supplemented TPN solutions have been shown to maintain tissue GSH and improve survival after acetaminophen toxicity, chemotherapy (Hong et ai, 1990, 1992), inflammatory stress (Welboume et ai, 1993), and bone marrow transplantation (Zeigler et ai, 1992). Availability of the amino acid substrates for GSH synthesis may also be influenced by the amino acid transport mechanisms for cysteine, cystine, methionine, and glutamate. A diet that can cause an imbalance of the plasma amino acid proflle may influence the uptake of amino acids which compete for the same transport systems (Christensen, 1990). In agreement with this hypothesis, it has been demonstrated that the GSH concentration of endothelial cells decreases when they are cultured in a glutamate-enriched medium because cystine uptake is inhibited competitively by glutamate (Miura et al, 1992). This raises the possibility that elevated plasma glutamate concentrations (up to sixfold the normal concentration) in HIV-infected individuals (Eck et

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al., 1989) and in patients with advanced tumors (Droge et a/., 1988) may negatively influence cyst(e)ine uptake and synthesis of GSH. The potential role of nutritional status, particularly wasting malnutrition concomitant with diseases such as acquired immunodeficiency syndrome (AIDS) and cancer, on plasma amino acid profile and transport requires further investigation. The amino acid substrates for GSH synthesis in extrahepatic tissues are also provided by efflux of hepatic GSH into plasma and the uptake of plasma GSH via the y-glutamyltranspeptidase reaction into extrahepatic tissues. Unfortunately, very little is known about the specific effects of nutritional status, especially protein-energy malnutrition, on the hepatic efflux mechanism of extrahepatic y-glutamyltranspeptidase activity. Adachi et al, (1992) have reported that the calculated efflux rate of hepatic GSH in mice fed a low protein diet was significantly lower than the control group. It is possible that decreased hepatic efflux of GSH may account for the high concentrations of hepatic GSH which we have observed in rats fed 0.5 or 7.5% protein diets and supplemented with sulfur amino acid (Bauman et a/., 1988a; Taylor et al., 1992). In addition to substrate availability, effects of diet and nutritional status on activities of the GSH synthetic enzymes need to be considered. Unfortunately, there is very little data available on the activities of GSH synthetic enzymes in in vivo models of altered nutritional status or malnutrition. Indirect evidence provides support for the literature hypothesis that an important regulatory mechanism for limiting the maximum GSH concentration in tissues is feedback inhibition of yglutamylcysteine synthetase by GSH (Richman and Meister, 1975). The Ki for this enzyme is in the range of physiological concentrations of GSH found in kidney and liver, and excess dietary protein or sulfur amino acid does not increase the maximum GSH concentration beyond the level found when a diet adequate in protein is fed (Bauman et al., 1988a,b). The GSH synthesizing enzymes are reported to be maintained during starvation (Tateishi et al., 1974), and it appears that the GSH synthetic enzymes are maintained in severe wasting malnutrition for readiness of GSH sjnithesis on substrate availability. In a rat model of severe wasting malnutrition (0.5% protein diet for 2 weeks), oral supplementation of the cysteine prodrug, L-2-oxothiazolidine 4-carboxylate, produced a rapid increase in hepatic GSH to a concentration even higher than normally found at the peak concentration of the diurnal rhythm of rats fed a normal protein diet (Taylor et al, 1992). In AIDS patients with the wasting syndrome, an oral dose of AT-acetylcysteine increased GSH in mononuclear cells (de Quay et al., 1992)

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and this suggests that the GSH synthetic machinery is maintained in these patients. Although the amount of substrate in the diet is an important determinant of tissue GSH concentrations, the previous dietary protein status also affects the response of hepatic GSH concentration to sulfur amino acid supplementation. For example, rats previously fed a normal protein (15%) diet for 2 weeks and then supplemented with OTC had the same rate of increase and peak concentration of hepatic GSH during the diurnal cycle compared to the unsupplemented group (Bauman et al., 1988b). However, rats previously fed a low protein (7.5%) diet for 2 weeks and then supplemented with OTC increased their hepatic GSH concentration more rapidly and sustained it at a higher concentration compared to rats which were previously fed a normal protein (15%) diet for 2 weeks (Bauman et al., 1988b). If rats were previously fed a 0.5% protein diet for 2 weeks, the initial increase in hepatic GSH concentration was even more pronounced than that in rats fed the 15% protein diet and the peak concentration exceeded the physiological maximum (Taylor et al, 1992). The difference in response to OTC between rats fed low protein diets and rats fed adequate diets is not readily explained, even though hepatic GSH concentration was similar before OTC supplementation and the amount of supplementation was identical for both low and normal protein groups. Other factors, such as the role of hormones in the regulation of GSH homeostatis, need to be considered. Changes in nutritional status, such as malnutrition, have significant effects on hormonal balance, and, as will be discussed under Section IV, various hormones are involved in the regulation of GSH homeostatis.

IV. Regulation of Glutathione by Hormones GSH S5nithesis and efflux from the liver are subject to hormonal control. Although most studies have used in vitro models, it is of particular interest that hormones which are involved in stress responses, such as glucagon and the adrenergic hormones, are potent regulators of GSH homeostasis. Therefore, it seems likely that hormonal responses to disease, infection, and malnutrition will influence GSH synthesis and interorgan metabolism in vivo. The first evidence for hormonal control of hepatic GSH stemmed from work investigating the diurnal rhythm of GSH in the liver (Isaacs and Binkley, 1977; Jaeschke and Wendel, 1985). GSH levels are at their highest after the feeding period and lowest after the nonfeeding period. Isaacs and Binkley (1977) observed that this cycle coincided

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with mobilization of liver glycogen. Since mobilization of glycogen is mediated by increased concentrations of circulating glucagon and consequent increases in liver cAMP, they investigated whether cAMP could directly alter liver GSH concentrations. Intraperitoneal injections of either dibutyryl-cAMP (dbcAMP) or theophylline to increase hepatic cAMP resulted in a corresponding decrease in hepatic GSH (Higashi et al, 1976; Isaacs and Binkley, 1977). Because hepatic GSH is an important source of GSH and cysteine for extrahepatic tissues, a rapid decrease in GSH concentration could be the result of increased release to the circulation. In addition, inhibition of GSH synthesis by hormones could also contribute to decreased hepatic GSH. Data from several laboratories suggest that hormonal control of hepatic GSH is mediated at both the level of efflux from the liver and by the rate of synthesis. GSH is transported across the sinusoidal and canicular membranes of the liver via a carrier-dependent facilitated mechanism (Ookhtens et al.y 1985). Transport at both membranes is of low affinity and is dependent on membrane potential. The transporters at the two poles have different kinetic properties and evidence suggests that transport is mediated by two different proteins (Femandez-Checa et al, 1993). Sies and Graf (1985) found that GSH efflux across the sinusoidal plasma membrane from isolated perfused rat liver is stimulated by hormones such as vasopressin, phenylephrine, and adrenaline which act by binding to specific receptors on the cell membrane and stimulating the hydrolysis of inositol phosphates, the release of Ca^^, and subsequent activation of protein kinase C. These hormones also decreased efflux of GSH into bile (Sies and Graf, 1985). Stimulation of GSH efflux across the sinusoidal membrane and decreased movement into bile can be mimicked by phorbol esters and blocked by inhibitors of protein kinase C, such as sphingosine, staurosporine, and H7 (Raiford et al, 1991). Increased efflux appears to occur independently of changes to the permeability of tight junctions, suggesting that the hormones can specifically regulate efflux of GSH (Raiford et al, 1991). Hormones which act via cAMP-dependent signal transduction pathways also regulate GSH efflux. Treatment of primary cultures of hepatocytes with cholera toxin to increase intracellular cAMP increased the Vmax for efflux, with no effect on K^ (Lu et a/., 1990). In the perfused liver, glucagon and dbcAMP stimulated sinusoidal efflux of GSH, but had no effect on biliary efflux (Lu et al., 1990). Finally, in vivo perfusion with glucagon, at a dose which increased plasma glucose by 80%, doubled the plasma concentration of GSH (Lu et al, 1990). Lu and colleagues suggest that this increase in efflux was attributed to a hyperpolarization of the hepatocyte due to activation of Na^,K^-ATPase by

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cAMP (Lu et aZ., 1990). Interestingly, GSH efflux from hepatocytes has been found to be regulated by cellular thiol status (Lu et al., 1993). Treatment of cultured hepatocytes with dithiothreitol (DTT) stimulated efflux by 400-500%. Stimulation of transport by DTT may be the result of a thiol-reducing action on the transporter (Lu et al., 1993). If m vivo changes in thiol status also regulate transporter activity, then total depletion of hepatic GSH by efflux from the liver into the blood might be prevented. If such a mechanism was active in vivo, as hepatic GSH concentration decreased, and as the cellular thiol disulfide ratio shifted to a more oxidized state, efflux would potentially be decreased. Thus, it is possible that, even after a hormonal signal to increase efflux, movement of GSH across the sinusoidal membrane could still be prevented once the GSH concentration was reduced below a critical level. While most of the work regarding the control of GSH transport has been performed in liver, transport in the rat small intestine also appears to be regulated by a-adrenergic agonists. Treatment of isolated vascularly perfused rat small intestine with epinephrine or norepinephrine increased GSH transport from the lumen into the vasculature independently of any increase in bulk flowthrough paracellular pathways (Hagen et al., 1991). These responses were specific for a-adrenergic agonists. Isoproterenol, a specific /3-adrenergic agonist, had no effect on translocation of GSH (Hagen et al., 1991). The potential significance of hormonal control of intestinal GSH transport in vivo remains to be determined. Hormones which activate cAMP- and Ca^^-dependent signal transduction pathways result in rapid decreases in GSH synthesis in the liver (Estrela et al., 1988; Harbison et al., 1991; Lu et al., 1991). In isolated rat hepatocytes, the rate of GSH synthesis is decreased on activation of oii-adrenergic and Q:2-adrenergic receptors, as well as glucagon receptors (Estrela et al., 1988; Harbison et al., 1991; Lu et al., 1991). Similar effects of hormones were observed in the intact perfused liver and after in vivo administration of hormones (Lu et al., 1991). Thus, hormones interacting via two independent signal transduction pathways can decrease GSH synthesis in the liver. The mechanisms for inhibition of GSH synthesis by hormones have not been well studied. Estrela et al. (1988) suggested that Ca^^-dependent signal transduction pathways could limit the availability of amino acid precursors and thus decrease GSH S3nithesis. However, dbcAMP inhibits GSH synthesis even in a cell-free system in which GSH synthesis is independent of changes in rates of amino acid transport (Lu et al, 1991). Activation of cAMP-dependent protein kinase or protein kinase C in liver cytosolic fractions reduces the activity of y-glutamyl-

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cysteine synthetase (Lu et a/., 1991). This short-term inhibitory control of y-glutamylcysteine synthetase is thought to result from hormonestimulated phosphorylation of the enzyme protein and could account for the decreased rates of GSH synthesis observed both in isolated cells and in the perfused liver. The physiological significance of this short-term inhibitory control of GSH synthesis by hormones is unclear. A decrease in synthesis in response to cAMP- or Ca^^-dependent signaling pathways would appear to counter the effect of stress hormones on increasing the availability of GSH to extrahepatic tissues by stimulating GSH efflux across the sinusoidal membrane. However, inhibition of GSH synthesis in the liver may be a protective response to preserve amino acid precursors for other metabolic functions. Lu et al. (1991) have speculated that decreased synthesis of GSH in response to stress hormones may provide a reservoir of cysteine for synthesis of acute phase proteins. Shortterm fasts in both animals and humans result in a decrease in the responsiveness of hepatocytes to stimulation by glucagon (Soman and Felig, 1978; Fouchereau-Peron et al, 1976). Moreover, recent evidence indicates that, after prolonged malnutrition, hepatocytes are no longer responsive to cAMP-mediated inhibition of GSH synthesis (Goss et aL, 1993). Desensitization of hormonal responsiveness after sustained stimulation may provide a mechanism for limiting hormone-induced inhibition of GSH synthesis. Maintaining an appropriate balance between synthesis and efflux of hepatic GSH may be critical for sustaining both hepatic and extrahepatic functions. It is likely that the alterations in hormonal status observed in response to malnutrition, infection, and other diseases may be a contributing factor to the depletion of cellular GSH concentrations. Hepatic GSH concentrations can also be increased in response to hormones. Biosynthesis of y-glutamylcysteine synthetase protein and amino acid transport can be increased by insulin and glucocorticoids in cultured hepatoc5^es. Insulin and hydrocortisone increase (GSH synthesis in cultured hepatocytes via an induction of y-glutamylcysteine synthetase activity (Lu et al., 1992). These hormones also increase cystine and glutamate transport (Takada and Bannai, 1984; Lu et al., 1992). Together, these increases in substrate availability and biosynthetic capacity are likely to account for the increase in GSH concentrations observed in hepatocytes after treatment with insulin or glucocorticoids. Streptozotocin-induced diabetes or adrenalectomy will decrease hepatic GSH in the intact animal (Loven et al, 1986; Lu et al., 1992), suggesting that hormonal control of y-glutamylcysteine synthetase and/ or transport of GSH precursors may also occur in vivo. It is also possible

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that the insuHn resistance characteristic of malnutrition and infection may Hmit cellular GSH biosynthetic capacity. However, further work investigating the long-term regulation of GSH synthetic enzymes during malnutrition and infection is necessary. Both the murine and the human clones for y-glutamylcysteine sjnithetase have been isolated (Gipp et al, 1992; Yan and Meister, 1990), and availability of these probes should facilitate further research. In addition to hormonal regulation of GSH homeostasis, cellular thiol status or thiol redox state can also modulate hormone function. Thus, hormonal signal transduction pathways and GSH homeostasis appear to interact in a type of feedback cycle (Fig. 2). Many membrane-bound hormone receptors contain thiol groups and disulfide bonds. The cellular thiol status can regulate the interaction of hormones with their receptors and thus produce a hormonal response. For example, glucocorticoid receptor function is dependent on cellular redox state (Grippo et aL, 1983). More recently, physiological concentrations of GSH were found to stimulate autophosphorylation of the isolated insulin receptor (Clark and Konstantopoulous, 1993) and to inhibit binding of ligands Nutritional Status/ Disease

Hormones

Tissue GSH (Plasma GSH)

Nutritional Status/ Disease FIG. 2. Nutritional status and disease influence the interactions between hormones and GSH homeostasis. For example, hormonal regulation of hepatic GSH efflux and GSH synthesis may alter tissue and plasma GSH concentrations. Changes in tissue and plasma GSH concentrations may influence hormone-receptor interactions and the generation of intracellular signals and thus hormonal response. In addition, both hormonal status and tissue GSH concentrations are affected by nutritional status and the presence of disease.

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to solubilized preparations of /x-opioid, substance P, and kainic acid receptors (Liu and Quirion, 1992). Thiol status also modulates generation of intracellular signals in response to hormonal stimuli. For instance, GSSG has been shown to increase the sensitivity of permeabilized heptocytes to inositol 1,4,5-trisphosphate (IP3)-stimulated Ca^* release (Renaud et al, 1992). It is interesting to speculate whether GSH regulation of receptor function occurs in vivo. Most in vitro investigations utilize permeable sulfhydryl reactive agents, such as iV-ethylmaleimide, to alter cellular redox status. These agents can produce more pronounced changes in cellular redox state than may be observed under physiological conditions. Specific in vivo responses to changes in cellular redox state would be dependent on both the degree of change in cellular redox state and the redox potential of the relevant thiol groups. For example, Renaud et al. (1992) predicted that the increased sensitivity of IPs-stimulated Ca^^ release would be more likely to occur under conditions of oxidative stress, rather than from the more moderate changes in thiol status observed after starvation or malnutrition. Thus, the reduction in cellular GSH concentration observed after malnutrition, infection, or disease and consequent changes in cellular redox state could in turn alter cellular signal transduction or gene transcription (Fig. 3). Such changes could accelerate the progression of pathological responses to disease and infection.

V. Glutathione in Vicious Cycle of Disease, Infection, and Malnutrition An understanding of the nutritional and hormonal regulation of GSH homeostasis is important for developing strategies to enhance tissue Protein-Energy Malnutrition

Disease

Drug k Oxygen Therapies

;

^

^ GSH status Immune Defense

fl Antioxidant V Defense

ft'Infection FIG. 3. GSH in the vicious cycle of disease, infection, and malnutrition.

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GSH concentrations and to intervene in the vicious cycle of disease, infection, and malnutrition (Fig. 3). Malnutrition is a common contributing factor to the morbidity and mortality in many diseases. The classic example of wasting malnutrition which affects millions of children in developing countries is protein-energy malnutrition (PEM), also known as kwashiorkor. In affluent countries, a large number and variety of patients suffer PEM secondary to AIDS, cancer, alcoholism, chronic digestive diseases, bums, etc. (Bistrian et al., 1976; Mendenhall etal, 1984; Chlebowski, 1985;Corbuccie^aZ., 1985;Bashire^aZ., 1990). Decreased tissue GSH concentrations have been reported in many of these patient groups (Shi et al,, 1982; Shaw et al., 1983; Rentier and Gelbart, 1985; Burgunder and Lauterburg, 1987; Vendemiale et al., 1989; Eck et al., 1989; Buhl et al., 1989; Staal et al., 1992; de Quay et al., 1992). It has been proposed that the clincal signs of malnutrition result from a weakened defense system unable to detoxify an increased production of free radicals (Golden and Ramdath, 1987). For example, malnourished children who die soon after admission to the hospital have the lowest levels of erythrocyte GSH and GSH peroxidase, plasma vitamin E, and Zn and the highest levels of plasma ferritin and hepatic Fe (Verjee and Behal, 1976; Jackson, 1986; Golden and Ramdath, 1987; Sive et al, 1993). In vitro studies with whole blood from malnourished children suggest that the decreased erythrocyte GSH concentrations are due to increased consumption, not decreased production, of GSH (Golden and Ramdath, 1987). In experimental models of PEM, decreased liver and lung GSH concentrations are associated with an increased susceptibility of xenobiotics and pulmonary oxygen toxicity (Jung, 1985; Deneke et al, 1983, 1985; Taylor et al., 1992). In addition to a weakened antioxidant defense system, individuals with PEM have decreased immune host defense and are more susceptible to opportunistic infections such as Pneumocystis carinii, tuberculosis, Candida, and bacterial diarrhea (Chandra, 1983, 1991). Several factors, including GSH status, are proposed to play a role in the decreased immune response of malnourished individuals. Adequate intracellular concentrations of GSH are required for several immune responses, including the proliferation and activation of T lymphoc3^es, activation of polymorphonuclear leukocytes, production of tumor necrosis factor (TNF) and interleukin-2 by macrophages, and interleukin-2 binding, internalization, and degradation by T cells (Wender et al., 1981; Fidelus et al., 1987; Liang et al., 1989; Suthanthiran et al., 1990; Robinson et al., 1993). During the inflammatory response and respiratory burst, malnourished individuals are exposed to increased amounts of reactive oxygen species which could potentially lead to further reduc-

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tions in tissue GSH concentrations and weaken the antioxidant defense system. Acute infection can precipitate the onset of severe chnical manifestations of disease. This contributes to further malnutrition and wasting in various diseases including kwashiorkor, AIDS, and cancer by reducing appetite and increasing the metabolic requirements to fight the infection (Bhaskaram, 1992; Grunfeld and Feingold, 1992; Singer et a/., 1992). For example, only a small proportion (0.5-2%) of children consuming a diet deficient in protein and energy actually develop kwashiorkor (Golden and Ramdath, 1987). The clinical signs of kwashiorkor, such as edema and fatty liver, are generally precipitated by the presence of infection (e.g., measles, tuberculosis, malaria, and diarrhea), and the disease is characterized by the presence of infection and overgrowth of bacteria in the small intestine (Golden and Ramdath, 1987; Bhaskaram, 1992). HIV-infected individuals can have relatively stable body weight and body cell mass for long periods of time, but the rapid wasting and anorexia observed during repeated secondary infections contributes to the development of AIDS (Singer et a/., 1992; Grunfeld et a/., 1991). Prevention or successful treatment of secondary infection will stop this cycle of malnutrition, infection, and disease. It has been shown that successful treatment of cytomegalovirus infected AIDS patients with ganciclovir prolongs survival and increases body weight, lean body mass, body fat, and serum albumin (Kotler et al, 1989a). Also, the treatment of HIV infection in seropositive individuals with zidovudine (ZDV, AZT) to delay the onset of AIDS has been associated with weight gain during therapy (Singer et al, 1992; Varchoan et a/., 1986). Tissue GSH is intricately involved in the immune response to infection. The production of oxygen-reactive species and release of inflammatory mediators such as TNF are critical for immune defense during infection. This critical inflammatory response, however, can decrease intracellular GSH and sensitize cells to the cytotoxic effects of TNF and reactive oxygen species (Zimmerman et al,, 1989; Yamauchi et al., 1990; Ishii et al, 1992; Roederer et al, 1992). This leads to a cascade effect whereby TNF or oxidative stress (Lowenthal et al, 1989; Osbom et al, 1989; Schreck et al, 1991) can sufficiently alter cellular thiol status to induce the DNA-binding activity of the transcription factor NF-KB to NF-KB-dependent genes such as TNF and other inflammatory C3i:okines (UUman et al, 1990) and stimulate further cytokine production. In the case of HIV infection, TNF stimulates HIV replication via activation of NF-KB (Osbom et al, 1989; Duh et al, 1989). When intracellular thiol concentrations are decreased, activation of NF-KB and HIV replication are enhanced (Staal et al, 1990). AT-Acetylcysteine

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has been used to restore intracellular cysteine and GSH concentrations and to inhibit the activation of N F - K B and HIV replication in several types of m vitro studies (Schreck et ai, 1991; Staal et aL, 1990; Roederer et al., 1990, 1991; Mihm et ai, 1991; Kalebic et aL, 1991; Lioy et al, 1993). The clinical use of ^-acetylcysteine as part of the treatment regimen for HIV and AIDS patients has been proposed (Droge et al, 1992; Droge, 1993; Roederer et al., 1993) and is currently under investigation. Oral administration of a single dose of iV-acetylcysteine has been reported to increase transiently cysteine and GSH concentrations in mononuclear cells of HIV patients (de Quay et a/., 1992). Also, it is tempting to raise the possibility that the regulation of N F - K B activation by TNF, oxygen-reactive species, and intracellular thiol status may have implications for designing therapeutic interventions for other disease states characterized by oxidative stress and inflammation. It has also been postulated that TNF, oxidative stress, and depletion of GSH may be contributing to severe wasting and cachexia observed in malnourished patients with late-stage AIDS or cancer (Roederer et al,, 1992). Many of the metabolic disturbances and wasting of infection and cancer have been attributed to cytokines; however, specific roles of TNF/cachectin and other cytokines (e.g., y-interferon) in wasting and cachexia remain controversial (Grunfeld and Feingold, 1992; Grunfeld and Kotler, 1992). AIDS and cancer patients with the wasting S3nidrome generally do not have elevated levels of circulating TNF unless secondary acute infection is present (Waage et ai, 1986; Socher et al, 1988; Lahdevirta et al, 1988; EUaurie and Rubinstein, 1992). It has been proposed that the effects of TNF in local tissue environments during chronic infection may be more critical (Tracey and Cerami, 1992) or that the synergistic effects of multiple cytokines may be involved (Grunfeld and Kotler, 1992). In addition, it may be that repeated exposure to infection and oxidative stress gradually wears down the body's defenses and ability to recover from subsequent stress (Grunfeld and Feingold, 1992; Singer et al, 1992; Roederer et al, 1992). Does a diminishing tissue GSH status contribute directly to this phenomenon? Although infection and malignancy may contribute to the wasting syndrome, the timing of death in AIDS patients with wasting is directly related to the loss of body cell mass (Kotler et al, 1989b). Thus, it is critical that we develop and test hypotheses to address this perplexing problem of how to intervene in malnutrition and the wasting syndrome. If we take the approach that a weakened antioxidant defense and immune defense are major contributors, and that both of these functions are influenced by intracellular GSH concentrations, then strategies to restore tissue GSH concentrations may have widespread applica-

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tion in various patient groups who develop severe malnutrition and are exposed to oxidative stress. In addition, the treatment of many diseases, even if they are not free radical mediated, requires oxygen and drug therapies, both of which can increase oxidative stress. The lung is often a target of opportunistic infections in malnourished individuals (Hughes et al., 1974; Chandra, 1983; Stover et al, 1985; Tupasi et a/., 1990), and respiratory distress may necessitate the use of supplemental oxygen (hyperoxia) which will further increase the production of oxygen free radicals (Jamieson, 1989). Thus, several factors, including the disease itself, concomitant malnutrition and changes in hormonal responses, or oxygen and drug therapies may contribute to decreased tissue GSH concentrations in various disease states. As a result, several strategies to increase tissue GSH concentrations have been attempted, but a critical examination indicates that many of these approaches have limitations for increasing tissue GSH concentrations in vivo in chronic disease states (Bray and Taylor, 1993). One obvious but often neglected factor is the role of various nutritional states and hormones in the regulation of GSH homeostasis in different tissues. Understanding the role of nutritional factors may contribute to the development of strategies for enhancement of tissue GSH. REFERENCES Abate, C , Patel, L., Rauscher, F. J., and Curran, T. (1990). Science 249, 1157-1161. Adachi, T., Yasutake, A., and Hirayama, K. (1992). Toxicology 72, 17-26. Aw, T. Y., Wierzbicka, G., and Jones, D. P. (1991). Chem.-BioL Interact 80, 89-97. Bashir, Y., Graham, T. R., Torrance, A., Gibson, G. J., and Corns, P. A. (1990). Thorax 45, 183-186. Bauman, P. F., Smith, T. K., and Bray, T. M. (1988a). Can. J. Physiol. Pharmacol. 66, 1048^1052. Bauman, P. F., Smith, T. K., and Bray, T. M. (1988b). J. Nutr. 118, 1048-1054. Beck, L. v., Rieck, V. D., and Duncan, B. (1958). Proc. Soc. Exp. Biol. Med. 97, 229-231. Beutler, E., and Gelbart, T. (1985). J. Lab. Clin. Med. 105, 581-584. Bhaskaram, P. (1992). Indian Pediatr. 29, 805-814. Bistrian, B. R., Blackburn, G. L., Vitale, J., Cochran, D., and Naylor, J. (1976). J. Am. Med. Assoc. 235, 1567-1570. Boebel, K. P., and Baker, D. H. (1983). Proc. Soc. Exp. Biol. Med. 172, 498-501. Bray, T. M., and Taylor, C. G. (1993). Can. J. Physiol. Pharmacol. 71, 746-751. Buhl, R., Holroyd, K. J., Mastrangeh, A., and Cantin, A. M. (1989). Lancet ii, 1294-1298. Burgunder, J.-M., and Lauterburg, B. H. (1987). Eur. J. Clin. Invest. 17, 408-414. Chandra, R. K. (1983). Lancet i, 688-691. Chandra, R. K (1991). Am. J. Clin. Nutr. 53, 1087-1101. Chlebowski, R. T. (1985). Nutr. Cancer 7, 8 5 - 9 1 . Cho, E. S., Johnson, N., and Snider, B. C. F. (1981). J. Nutr. I l l , 914-922. Christensen, H. N. (1990). Physiol. Rev. 70, 43-77. Clark, S., and Konstantopoulos, N. (1993). Biochem. J. 292, 217-223.

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Miura, K., Ishii, T., Sugita, Y., and Bannai, S. (1992). Am. J. Physiol. 262, C50-C58. Ookhtens, M., Hobdy, K., Corvasce, M. C , Aw, T. Y., and Kaplowitz, N. (1985). J. Clin. Invest. 75, 258-265. Osbom, L., Kunkel, 8., and Nabel, G. J. (1989). Proc. Natl. Acad. Sci. U.S.A. 86, 2 3 3 6 2340. Raiford, D. S., Sciuto, A. M., and Mitchell, M. C. (1991). Am. J. Physiol. 261, G578-G584. Renaud, D. C., Seitz, M. B., and Thomas, A. P. (1992). Biochem. J. 284, 507-512. Richman, P. G., and Meister, A. (1975). J. Biol. Chem. 250, 1422-1426. Robinson, M. K., Rodrick, M. L., Jacobs, D. O., Rounds, J. D., Collins, K. H., Saporoschetz, I. B., Mannick, J. A., and Wilmore, D. W. (1993). Arch. Surg. 128, 29-35. Roederer, M., Staal, F. J. T., Raju, P. A., Ela, S. W., Herzenberg, L. A., and Herzenberg, L. A. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 4884-4888. Roederer, M., Raju, P. A., Staal, F. J. T., Herzenberg, L. A., and Herzenberg, L. A. (1991). AIDS Res. Hum. Retroviruses 7, 563-567. Roederer, M., Staal, F. J. T., Raju, P. A., Herzenberg, L. A., and Herzenberg, L. A. (1992). In "Tumor Necrosis Factor: Structure-Function Relationship and Clinical Application" (T. Osawa and B. Bonavida, eds.), pp. 215-229. Karger, Basel. Roederer, M., Staal, F. T. J., Ela, S. W., Herzenberg, L. A., and Herzenberg, L. A. (1993). Pharmacology 46, 121-129. Schreck, P., Rieber, P., and Baeuerle, P. A. (1991). EMBO J. 10, 2247-2258. Shan, X., Aw, T. Y., and Jones, D. P. (1990). Pharmacol. Ther. 47, 6 1 - 7 1 . Shaw, S., Rubin, K. P., and Lieber, C. S. (1983). Dig. Dis. Sci. 28, 585-589. Shi, E. C. P., Fisher, R., McEvoy, M., Vantol, R., Rose, M., and Ham, J. M. (1982). Clin. Sci. 62, 279-283. Sies, H., and Graf, P. (1985). Biochem. J. 226, 545-549. Singer, P., Katz, D. P., Billion, L., Kirvela, O., Lazarus, T., and Askanazi, J. (1992). Am. J. Gastroenterol. 87, 265-273. Sive, A. A., Subotzky, E. F., Malan, H., Dempster, W. S., and Heese, H. D. V. (1993). Ann. Trop. Paediatr. 13, 33-38. Socher, S. H., Martinez, D., Craig, J. B., Kuhn, J. G., and Oliff, A. (1988). J. Natl. Cancer Inst. 80, 595-598. Soman, V., and Felig, P. (1978). Nature (London) 272, 829-832. Staal, F. J. T., Roederer, M., Herzenberg, L. A., and Herzenberg, L. A. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 9943-9947. Staal, F. J. T., Roederer, M., Israelski, D. M., Bubp, J., Mole, L. A., McShane, D., Deresinski, S. C , Ross, W., Sussman, H., Raju, P. A., et al. (1992). AIDS Res. Hum. Retroviruses 8, 305-311. Stover, D. E., White, D. A., Romano, P. A., Gellene, R. A., and Robison, W. A. (1985). Am. J. Med. 78, 429-437. Suthanthiran, M., Anderson, M. E., Sharma, V. K., and Meister, A. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 3343-3347. Takada, A., and Bannai, S. (1984). J. Biol. Chem. 259, 2441-2445. Tateishi, N., Higashi, T., Shinya, S., Naruse, A., and Sakamoto, Y. (1974). J. Biochem. (Tokyo) 75, 9 3 - 1 0 3 . Tateishi, N., Higashi, T., Naruse, A., Nakashima, K., Shiozaki, H., and Sakamoto, Y. (1977). J. Nutr. 107, 51-60. Taylor, C. G., Bauman, P. F., Sikorski, B., and Bray, T. M. (1992). FASEB J. 6,3101-3107. Tracey, K. J., and Cerami, A. (1992). Proc. Soc. Exp. Biol. Med. 200, 233-239. Tupasi, T. E., Mangubat, N. V., Sunico, M. E. S., Magdangal, D. M., Navarro, E. E., Leonor, Z. A., Lupisan, S., Medalla, F., and Lucero, M. G. (1990). Rev. Infect. Dis. 12, S1047-S1054.

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CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 34

Protein Folding and Association: In Vitro Studies for Self-Organization and Targeting in the Cell* RAINER JAENICKE Institut fur Biophysik und Physikalische Biochemie Universitdt Regensburg D-93040 Regensburg, Germany

I. Introduction According to the "central dogma of molecular biology," genes encode proteins based on the colinear relationship defined by the genetic code. The detailed mechanism of the translation of the nucleotide sequence into the corresponding amino acid sequence is well established. Although highly complex, each piece in the puzzle of DNA, mRNA, aminoacyl-tRNAs and the initiation, elongation, and termination factors has been put into its proper place. However, the real importance of a gene is still obscure, and it will remain so until we succeed in cracking the "second half of the genetic code" which determines the translation of the one-dimensional arrangement of its polypeptide chain. If this folding code were known, it would not only aid in engineered changes in protein structure using recombinant gene methodology, but it would also provide a tool to specify hypothetical functions for given polypeptide sequences. Currently, the code of protein folding is still unknown, and there is no hint as to how it might be deciphered, even for small single-chain one-domain proteins. In the case of long polypeptide chains and oligomeric proteins, domain and subunit interactions come into play. Evidently they complicate the topological problem due to ambiguities in the distribution of hydrophobic residues in clusters in the interior core and/or in patches in the intersubunit interfaces. The folding protein solves this problem both in vivo and in vitro. It does so in an amazingly fast reaction and with high fidelity: the formation of the native three-dimensional structure occurs on the time scale of seconds to minutes. Levinthal (1968) was the first to estimate the time required for an average pol5rpeptide chain in exploring the conformational space for the global minimum of potential energy; the result was an astronom* Dedicated to Professor Erwin Chargaff on the occasion of this 90th birthday. 209

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ical period of time. Faced with this paradox, the biological time scale clearly proves that there must be a pathway or a limited number of alternative pathways of protein folding. As shown by X-ray crystallography and nuclear magnetic resonance (NMR) in solution, the folded protein is structurally well defined regarding the atomic coordinates of its residues. Variances are functionally significant since the protein, during its whole lifetime from folding to degradation, requires flexibility of the polypeptide chain. The above tacit assumption that there is a distinct one-to-one relation between a given amino acid sequence and its corresponding unique spatial structure has been generally accepted based on Anson's early observation that "hemoglobin which has been denatured in a variety of ways can be converted back into native protein" (Neurath et ai, 1944; Anson, 1945). Anfinsen, in following up Anson's work, extended this conclusion by reducing and reoxidizing the disulfide bridges in ribonuclease which were previously considered to determine not only the stability but also the three-dimensional structure of a protein (Anfinsen, 1966). Applications of Anfinsen's approach to the recovery of inclusion bodies of recombinant tissue plasminogen activator (tPA, expressed in Escherichis coli) turned out to be successful (Rudolph et aL, 1990a). The reoxidation of the fully reduced and denatured protein yielded the native activator with all its kringles and domains folded and cross-linked correctly, which means that disulfide shuffling leads to just one out of 2.2x10^^ possible combinations of the 35 cysteine residues.* Obviously, the formation of the native structure of the protein is directed by the conformational energy gained in forming the correct fold; the cystine bridges stabilize the native state rather than determine the three-dimensional structure of the protein backbone. tPA represents one example out of a great variety of different proteins— monomeric, oligomeric, and multimeric—in which Anson's early experience has been confirmed again and again. However, there are cases in which all attempts to recover the native state after preceding denaturation failed completely (Miiller and Jaenicke, 1980; Rinas et aL, 1990; Schumann et aL, 1993); in some instances, "helper proteins" were successfully applied to "chaperone" the folding protein to its native state (Horowitz and Simon, 1986; Goloubinoflfe^ aL, 1989a,b; EUis, 1990a,b; Gatenby and Ellis, 1990; Jaenicke and Buchner, 1993). These examples are relevant because they seem to "qualify Anson's observation by suggesting that interactions within and between folding pol5rpeptides * "Assuming that disulfide formation was governed by statistics, the regeneration of 1 ng of active tPA would require the reoxidation of more than 200,000 tons of inclusion body protein" (Rudolph, 1990); in industrial practice, the yield exceeds 60%.

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need to be controlled by pre-existing proteins acting as chaperones" (Ellis and van der Vies, 1991; Hendrick and Hartl, 1993). In recent years, this concept has sometimes been used to disqualify physical principles as the basis of protein self-organization in favor of ideas about the "flexibility of the weak interactions that hold proteins in their functional conformation." However, it has to be stated at this point that, in principle, the acquisition of that native three-dimensional structure of proteins does not necessitate accessory proteins or other cellular components. What is important in the crowded cellular environment is that intra- and intermolecular interactions compete with each other so that "microcompartmentation" may be advantageous in regulating the kinetic competition of folding, association, and aggregation as the fundamental processes involved in protein structure formation (Teipel and Koshland, 1971; Jaenicke, 1974, 1987a; Zettlmeissl et aL, 1979; EUis and van der Vies, 1991; Jaenicke and Buchner, 1993). As mentioned, there are cases in which in vitro refolding has been unsuccessful, even in the presence of chaperones. Hypothetical reasons are (i) the directionality of translation, (ii) the nonuniform rate of translation caused by codon usage and subsequent "interpunctuations" in the folding process, (iii) co- and posttranslational modification of the polypeptide chain, and (iv) "mutual chaperone effects" of folding intermediates. So far, none of these alternatives has unequivocally been shown to be significant in modifying or blocking the folding path. With respect to directionality, protein synthesis in both directions, from the N to the C terminus {in vivo) and vice versa (by Merrifield synthesis) has been proven to generate native polypeptides in their functional state (Jaenicke, 1987a). In the case of codon usage, expression of recombinant proteins in different hosts (with varying codon usage) led to authentic products indistinguishable from the native parent molecule. However, it is interesting to note that, in large proteins, the codon usage seems to reflect the domain structure (Krasheninnikov et ai, 1989; Komar and Jaenicke, 1995); so far the implications of this observation for the kinetics and the final product of folding have not been unraveled in an unambiguous way (A. Komar, unpublished results). Covalent and noncovalent co- or posttranslational modifications such as glycosylation or ligand binding have been shown to affect protein stability without significantly altering the folding characteristics (Kern et aL, 1992, 1993). On the other hand, activation of polypeptide chains by specific proteolysis may have a drastic effect on both the kinetic mechanism and the final three-dimensional structure of the processed protein (Winther and Sorensen, 1991; Baker et aL, 1992a,b; Winther et aL, 1994; Ramos et aL, 1994; Baker and Agard, 1994). Within the

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framework of Anson's and Anfinsen's concept, this is exactly what one would predict, since what is actually compared are two different protein sequences. "Mutual chaperoning" has been found to influence the efficiency of folding by shifting the equilibrium toward the final product of folding and association. For example, domains, domain fragments, or structured apoenzymes may be stabilized by forming "nicked subunits" or holoenzymes (Krebs et ai, 1979; Jaenicke et aL, 1980; Opitz et aL, 1987). The previous examples illustrate the complexity of protein selforganization in the cell. In certain cases, the examples have been successfully modeled by adequate alterations of the solvent conditions in in vitro experiments. No new concepts challenging the classical in vitro approach have emerged from these studies (Jaenicke, 1993a). However, much must be done in order to bridge the gap between the biosynthetic environment and the highly idealized in vitro conditions and to provide a clear idea regarding the quantitative effects of cellular parameters.

II. Hierarchies of Structure, Stability, and Folding In the structural hierarchy of proteins (Scheme 1), the different levels refer to stability as well as folding. Increasing packing density and release of water from hydrophobic residues provide the enthalpic and entropic increments of the free energy of stabilization which accumulate to the marginal difference of the attractive and repulsive forces characteristic for the stability of biological macromolecules in their native state. Considering the numbers involved, 5000 atoms making up an average protein molecule yield a AGstab value of < 60 kJ/mol, i.e., the equivalent Structural levels:

primary - secondary/supersecondary - tertiary quaternary structure

Interactions:

short-range (through chain) - "long-range" (short range through space)

Folding pathway:

next-neighbor interactions - collapse (molten globule), docking of domains - assembly

Intermediates:

kernels/molten globule - subdomains - domains structured monomers

Off-pathway reactions:

misfolding - domain swapping - misassembly - aggregation

SCHEME 1. Hierarchy of protein folding and association.

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of just a few weak interactions (Jaenicke, 1991a). The contributions of the various weak interactions that determine the three-dimensional structure of proteins are still controversial (Kauzmann, 1959; Dill, 1990; Jaenicke, 1991b; Franks, 1995). The distribution of hydrophobic and hydrophilic residues in common globular proteins, as well as water release experiments in connection with endothermic assembly processes, clearly favor the idea that hydrophobic interactions are entropy driven and highly significant (Kauzmann, 1959; Jaenicke and Lauffer, 1969); obviously, cavities in the interior of the protein play a significant role, as shown by the decrease in stability with increasing cavity volume observed in point mutants of phage T4 lysozyme (B. W. Matthews, 1991,1995). On the other hand, high-precision calorimetry has provided transfer energies of nonpolar and polar model compounds and their temperature dependence which seem to indicate that van der Waals interactions, i.e., enthalpy rather than entropy, contribute significantly to the stabilization of the hydrophobic core of globular proteins (Privalov and Gill, 1988). With respect to hydrogen bonds, extensive studies on phage T4 lysozyme have shown that the change from protein-water to water-water interactions in the process of protein folding leads to a compensation of the respective energy increments (Matthews, 1987, 1995); Jaenicke, 1991a). In contrast, in the case of ribonuclease Tl (RNase Tl), subsitution of side chains involved in intramolecular H bonds has shown that there is a net change in stability per H bond of the order of 5 kJ/mol, indicating that there seems to be a difference in bond strength between protein-water and water-water H bonds. Thus, using RNase Tl as an example. Pace and co-workers (Pace, 1990; Pace et al.y 1991; Shirley et al., 1992), in an attempt to make up the balance of the relevant intramolecular interactions involved in the total free energy of stabilization, came up with the following numbers: 2 disulfide bonds, 87 internal H bonds (adding up to -450 kJ/mol), 85% of the nonpolar residues involved in hydrophobic interactions (corresponding to -270 kJ/mol); free energy of stabihzation: 24 kJ/mol (25°C, pH 7.0). Coulomb interactions are well understood in model systems, but they become highly complex in nonhomogeneous environments, such as folded proteins, mainly because of the ill-defined dielectric constant in the immediate surroundings of the charges (Dill, 1990; Sharp and Honig, 1990). Whether ion pairs contribute significantly to protein stability has been questioned since it became clear that most charged groups in globular proteins are exposed to the aqueous solvent. On average, only one ion pair per 150 amino acid residues of a globular protein is buried within the interior core (Barlow and Thornton, 1983).

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Thus, only surface ion pairs are expected to be involved in stabilization. At extremes of pH, the high positive or negative net charge causes denaturation due to charge repulsion. At very low pH, some proteins regain part of their spatial organization ("A state" or other alternative states) as a consequence of increased ionic strength at high activity of the acid. This screens the charge effects, thus modulating Coulombic interactions (Goto and Fink, 1990; Goto et ai, 1990). In addition, effects on water structure and preferential salt binding must be involved. The structures of the A state or alternative states are distinct from those of both the native and denatured states; they are frequently identified as "molten globules," although in certain cases they obviously differ from the collapsed intermediate state by showing well-defined, but nonnative structure (Groto et aL, 1990; Buchner e^ a/., 1991b; Schumann and Jaenicke, 1993). The mechanisms underl3ring the stabilization of proteins that undergo no or relatively slow turnover, such as bovine pancreatic trypsin inhibitor (BPTI) or the eye lens protein y-crystallin, may be totally different from those discussed so far. In many cases disulfide bonds contribute significantly to protein stability (for example, RNase Tl). The fact that reduction of the three cystine bridges in BPTI leads to complete unfolding, even in the absence of denaturants, indicates that in this case folding and stability evidently are coupled to disulfide bond formation (Creighton, 1978, 1988a; Wetzel et a/., 1990; Kemmink and Creighton, 1994). Calorimetric studies on BPTI analogs, selectively modified with respect to the number of disulfide bonds, clearly show the gradual stabilization of the protein with increasing cross-linking. In many cases, the stabilizing effect is much larger than one would expect for the restricted increase in entropy of the denatured state. However, there are exceptions which seem to prove the general observation that proteins are altogether individuals with unpredictable characteristics: (i) the trypsin inhibitor from Erythrina caffra with two disulfide bonds preserves its native structure independent of its state of oxidation; and (ii) for both the oxidized and reduced protein the inhibitor constant is identical, although the stability of the protein is decreased (Lehle et aL, 1994). In the case of protein disulfide-isomerase (PDI or DsbA in Escherichia coli) even this prediction turns out to be wrong: the active site of the enz5rme contains the highly conserved sequence CXYC, which in the catalytic reaction, undergoes an SH/SS exchange reaction. Here the stability of the enzjrme in its reduced state, i.e., with the cystine bridges broken, exceeds the stability of the oxidized protein (Wunderlich and Glockshuber, 1993a,b; Wunderlich et a/., 1993a,b). Mechanistically the enzyme is linked with the closely related thiore-

PROTEIN FOLDING AND ASSOCIATION

215

doxin as a redox pair, with PDI (DsbA) as the weaker reductant. This property may be significant in ensuring that PDI does not reduce correct disulfide bonds that are already stabilized in the nascent native-like protein (Freedman, 1989; Schmid, 1991). y-Crystallin does not contain cystine bridges. Therefore, one has to assume that its anomalous stability (at pH 1-10 and at temperatures up to 75°C, or in the presence of 7 M urea) must be related to its all/3 structure and to specific domain interactions. The imique observation that crystallins do not undergo significant turnover during a person's lifetime still awaits an explanation (Jaenicke, 1994). The biological significance of the common marginal stability of proteins is threefold: (i) optimization of the structure-function relationship in the course of evolution is aimed at flexibility (catalysis and regulation) rather than stability; (ii) under physiological conditions, native globular proteins are commonly at the borderline of denaturation (function versus turnover); (iii) since proteins in their native state occupy states of minimum potential energy, folding intermediates must be even less stable so that misfolding and subsequent kinetic competition of off-pathway reactions are expected to occur (Jaenicke, 1991a,b, 1993a; Jaenicke and Buchner, 1993). There is evidence that protein biosynthesis and folding in the cell do not yield 100% (Hurtley and Helenius, 1989; Helenius et ai, 1992). For example, the tail spike protein of bacteriophage P22, even under optimum growth conditions, yields less than 50%; under unbalanced physiological conditions, only wrong conformers are produced which are continuously removed by proteolysis. The significance of chaperone proteins and the mechanism of inclusion body formation are mentioned here only in passing; both will be discussed in greater detail later. In correlating the free energy of stabilization of proteins with the hierarchy shown in Scheme 1, thermodynamic measurements on point mutants, protein fragments, and homologs differing in their state of association clearly reveal that each structural level makes its own contribution to the overall intrinsic stability (Jaenicke, 1991a). As shown by nuclear magnetic resonance (NMR) and other spectroscopic techniques, oligopeptides may form stable nonrandom conformations (Wright et ai, 1988); at a minimum length of 15 residues, they have been shown to sustain native-like structure (Baldwin, 1991a; Kemmink and Creighton, 1994). Their thermal unfolding/refolding behavior can be quantitatively described by the standard helix-coil theories, even for short peptides, because both the helix nucleation constant and the enthalpy change per mole residue for helix formation turn out to be insensitive to the length of the polypeptide chain. It is obvious that

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in short peptides through-space interactions differ from those in closely packed larger proteins. Thus, it is not surprising that identical sequences in unrelated proteins and short peptides do not necessarily adopt the same conformation. Yet, since next-neighbor interactions are expected to occur cotranslationally, local structures may serve as "seeds" in the folding process (Wright et ai, 1988; Dyson and Wright, 1993; Ilyina and Mayo, 1995). At later stages, close packing of the complete polypeptide chain may modify the initial state so that, for example, reverse turn motifs observed in small peptides do not persist in the final structure (Creighton, 1988a,b). With respect to larger fragments, subdomains and domains have been known for a long time to exhibit high intrinsic stabilities, not far from the free energies observed for the uncleaved parent molecules (Wetlaufer, 1981; Jaenicke, 1987a). Reducing the chain length further, it becomes evident that proteins are cooperative structures showing mutual stabilization of structural elements. In order to find out at which fragment size native-like structure no longer can be formed, thermolysin was used as a model (Table I). Folding/unfolding experiments with a variety of BrCN fragments show that the N-terminal portion of the enzyme stabilizes the all-helical C-terminal domain. This my be shortened drastically, down to the 62-residue three-helix structure, without aggregating or losing much of the stability of native thermolysin; only the C-terminal 20-residue helix is too short to maintain its native structure in aqueous solution (Vita et ai, 1989). Whether the N- and the C-terminal ends of the polypeptide chain are important for protein stability depends on the protein. Taking ribonuclease (RNase A) as an example, it has been shown that the Nterminal end of the protein can be cleaved off without altering the TABLE I PHYSICOCHEMICAL CHARACTERISTICS OF THERMOLYSIN AND ITS FRAGMENTS"

Tn, (denaturation) Sequence

Mcalc

Mobs

Helicity (%)

rc)

AGstab (kJ/mol)

1-316 121-316 206-316 225-316 255-316

34,227 20,904 11,829 9,560 6,630

34,800 23,000 11,900 9,000 < 12,000^

100 96 95 92 100 ± 10

87 74 67 65 64

55 47 31 26 20

" M, molecular mass; helicity from circular dichroism at 222 nm; AGstab, free energy of stabilization. Data from Vita et ai (1989). * At low protein concentration (<10 fiM) monomeric.

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PROTEIN FOLDING AND ASSOCIATION

overall topology; however, the stability is greatly affected. Removing the C terminus alone is sufficient to block the oxidative reshuffling reaction (Teschner and Rudolph, 1989). In the case of lactate dehydrogenase, the N-terminal 10 amino acid residues contribute significantly to the stabihty of the native quaternary structure since this "arm" is involved in the docking of two dimers to form the active tetramer (Rossmann et al, 1975; Opitz et al, 1987) (see below). Mutual stabihzation of structural elements has been observed at all levels of the structural hierarchy. A striking example is the docking of the N- and C-terminal domains of yB-crystaUin (Rudolph et al, 1990b; E.-M. Mayr et al, 1994; Trinkl et a/., 1994) (Fig. 1). The complete molecule shows the expected bimodal equilibrium transition with the second phase close to the unfolding transition of the extremely sta-

0

1

2

3

^

5

6

7

8

010 30 50 70 Temperature (°C)

FIG. 1. Mutual stabilization of domains and subunits: yB-crystallin and lactate dehydrogenase. (A) Urea-dependent equilibrium transitions of yB-crystallin and its N- and Cterminal domains: 0.1 M NaCl/HCl, pH 2.0, 20°C. Upper: denaturation transition monitored by intrinsic fluorescence (O) and sedimentation analysis ( • ) . Lower: denaturation transitions of the N-terminal (O) and C-terminal ( • ) domains monitored by fluorescence (E.-M. Mayr et ai, 1994). (B) Guanidine- and temperature-dependent deactivation of native (tetrameric) lactate dehydrogenase ( • ) and the "proteolytic dimer" lacking the N-terminal decapeptide (O): 0.1 M sodium phosphate, pH 7.6, 1.5 M ammonium sulfate, 20°C. Upper: GdmCl-dependent deactivation; lower: thermal deactivation (Girg et ai, 1981, 1983).

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RAINER JAENICKE

ble isolated N-terminal domain fragment. However, the isolated Cterminal domain is surprisingly unstable, to the extent that, even in the absence of a denaturant, partial unfolding is observed (Fig. lA). Lactate dehydrogenase may serve to illustrate the mutual stabilization of subunits. As mentioned, the tetramer represents a dimer of dimers stabilized by the N-terminal decapeptide. Cleaving off the arm in the course of reconstitution (Girge^ a/., 1981,1983), the "proteolytic dimer" can be obtained, whereas the monomer is exclusively accessible as a short-lived intermediate on the kinetic pathway of folding and association (Herrmann et aL, 1981). Comparing the various structural levels, the stability decreases steadily from the highly stable tetramer down to domain fragments (Fig. IB); the proteolytic dimer requires structuremaking salts to exhibit activity, whereas the structured monomer is inactive and extremely sensitive toward proteolysis. The separate NADand substrate-binding domains are unstable, but still sufficiently structured to recognize each other upon joint reconstitution (Opitz et aL, 1987). Extrinsic factors, such as ions, cofactors, and nonproteinaceous components (e.g., nucleic acids or carbohydrates), apart from contributing to protein stability, may be important in determining both the mechanism of folding and the state of association (Jaenicke et aL, 1980; Jaenicke, 1987a; Krebs et aL, 1979; Risse et aL, 1992a,b; Kern et aL, 1992, 1993).

III. Mechanism of Folding and Association A. Fundamentals of Structure Formation Proteins obey all the stereochemical and physiochemical rules obtained from the study of small organic molecules, especially peptides, containing the characteristic functional groups of the 20 natural amino acids. This implies that the conformational restrictions in a given polypeptide chain can be adequately described by the Ramachandran plot (Ramachandran and Sasisekharan, 1968). Although the barriers of rotation around single bonds are relatively low, the distribution of torsional angles in the amino acid side chains is narrow so that the allowed confirmations of the single residues fit perfectly to known highresolution protein structures (Ponder and Richards, 1987). Evidently, the overall geometry of the covalent bonds clusters around the positions of minimum energy, independent of the sequence and conformational context of a given protein. The previous statement also holds for the denatured state, which, in the present context, stands either for the nascent chain coming off the ribosome or for the protein at zero time of an in vitro refolding

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experiment. The unfolded protein differs from the native one in two ways: first, it represents an astronomically large ensemble of different configurations, and second, it is solvated to a higher extent than the native state. Considering the amount of hydrophobic residues present in most soluble proteins, water is a poor solvent. Structure formation in the cytosol or in aqueous buffer systems is driven by this very fact. Making use of heteropolymers with approximately equal amounts of polar and unpolar residues, nature allows solvation to balance by exposing hydrophilic groups to the aqueous solvent, at the same time minimizing the hydrophobic surface (Richards, 1992). Solubilization of the inner core by mixed solvents or altered temperature leads to imfolding (Privalov, 1992). However, it is obvious from the balance of hydrophilic and hydrophobic amino acids in common proteins that complete solvation cannot be accomplished. For this reason (and as a consequence of excluded volume effects) it is doubtful whether a polypeptide chain will ever be "fully randomized" (Jaenicke, 1987a; Damaschun et al, 1995). Even in the process of translation, the nascent protein is limited in its conformational space because the space-filling properties of the growing polypeptide and its side chains do not allow all /v|i angles in the Ramachandran plot. Because of the high internal flexibility of the molecules, the detailed structural characterization of unfolded or partially unfolded proteins is difficult. However, using proteins enriched in ^^C and particular ^^N isotopes, it has been possible to use multidimensional heteronuclear NMR techniques to enhance the spectral resolution to the extent that assignments can be made. Studies of extensively unfolded states have revealed that considerable residual structure remains, even at high concentrations of urea or guanidinium chloride. The results confirm previous assumptions that local hydrophobic clusters with features of the native state are preserved, at least in equilibrium with lessstructured states (Neri et al, 1992; Logan et al, 1994). As one would predict, there is no evidence for significant long-range structure within the ensemble of such highly denatured states. In contrast to the multitude of denatured states, the native state of a given protein is commonly assumed to be well defined within the limits of the B values of X-ray analysis or the conformational djmamics calculated from multidimensional NMR. It is clear that there must be a certain range of flexibility for the obvious reason that in many cases proteins serve as "machines" evolved, e.g., to bind, transform, and release educts and products of metabolic reactions (Huber, 1988). Whether in this context "conformational substates" (Ansari et ai, 1985, 1986; Frauenfelder et al, 1987) are biologically relevant is not clear.

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The driving forces guiding the folding polypeptide chain to its final structure (either native or denatured) are those discussed before in connection with protein stability: (i) next-neighbor and through-space short-range interactions: H bonds, hydrophobic and Coulomb interactions, and van der Waals forces (Dill, 1990; Jaenicke, 1991a); (ii) optimum packing density connected with minimum hydrophobic cavity space (Richards, 1992; Matthews, 1995); and (iii) entropic effects, e.g., water release in the process of "hydrophobic collapse" and subunit assembly (Kauzmann, 1959; Jaenicke and LaufFer, 1969; Kim and Baldwin, 1990; Dobson, 1992). Along the general "consensus pathway" of protein folding (Goldberg, 1985), next-neighbor interactions will first form fluctuating native or nonnative secondary structural elements. During this step (which is in the millisecond time range) kinetic nuclei gain increasing stability. At the point where the intermolecular interactions surpass the thermal energy, the polypeptide chain collapses into a persistent native-like secondary structure, the "molten globule state," which still lacks the packing and low hydrophobic surface area characteristic of the native tertiary structure. The term "molten globule" has been widely used to describe (i) partly folded early intermediates on the folding pathway that are stable at equilibrium, and (ii) proteins at low pH or medium denaturant concentration (Fink et al., 1994). Its properties may be summarized as follows: high content of secondary structure, overall compactness with highly mobile aromatic side chains, exposure of hydrophobic surface, tendency to aggregate, lack of highly cooperative (thermal) unfolding, and rapid equilibration with the unfolded state (Ptits3m, 1992; Ptitsyn and Uversky, 1994; Dobson, 1994). Evidently, these characteristics are not well defined because observed spectral differences and incomplete assignments of NMR cross-peaks do not allow a clear distinction between native-like structures or irregular "collapsed" parts of the hydrophobic core. Thus, the term "state" is equally inadequate as in the case of the denatured state. The situation is complicated by difficulties and inconsistencies in the treatment of multicomponent systems. In distinguishing between "collapsed unfolded forms" (lacking specific interactions) and "structured molten globules" (with fixed secondary structural elements at defined locations similar to those in the final native state), Baldwin (1991b) made an attempt to clarify the terminology. Both forms show high mobility and tend to aggregate; thus, no detailed structural analysis has yet been reported, except for the clear evidence for persistent secondary structure and a wide variation of the extent of native tertiary fold. The best-characterized system in this context is a-lactalbumin (a-LA) (Dolgikh et ai, 1985; Kuwajima, 1989; Chris-

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tensen & Pain, 1991; Ptitsyn, 1992; Ewbank and Creighton, 1993; Creighton and Ewbank, 1994; Peng et a/., 1995a,b). However, even in this case, the alternative—expanded native-Uke structure versus nonspecific collapsed state—was unresolved until recently, despite a wealth of spectral and hydrodynamic data. The controversy has been clarified by stud3dng the native disulfide pairing of domain fragments. As a result, the molten globule of a-LA is known to consist of two conformationally distinct regions, with the molten-globule properties confined to the native-like a-helical domain, while the )S-sheet domain is largely unstructured. Thus, the molten globule possesses a "bipartite structure" with native-like backbone topology; this does not necessarily encompass the entire polypeptide chain, but can be attained independently by individual domains. By adopting a native-like backbone topology, the molten globule can achieve much of the information transfer of the folding process, providing an approximate solution to the folding problem and thus vastly simplifying the search for the unique folded conformation (Wu et aL, 1995; Peng et aL, 1995a,b). Whether partly folded states in molten globules are generally representative for the natural pathway of protein folding remains to be shown. Therefore, discussions of the role of the molten globule as an intermediate state in protein folding have to be taken with a grain of salt. This holds especially because a variety of "alternatively folded states" with native-like structure and cooperative thermal unfolding transitions have been described (Buchner et aL, 1991b; Schumann and Jaenicke, 1993; Rehaber and Jaenicke, 1993). The detailed discussion of the "thermodynamic puzzle" in the case of apomyoglobin illustrates the complexity of the problem (Goto and Fink, 1990; Barrick and Baldwin, 1993; Jennings and Wright, 1993; Griko and Privalov, 1994). Considering further steps on the pathway of folding and association, the packing of domains as well as the formation of the native tertiary structure are accomplished by water exclusion from the inner core of the molecule and subsequent docking of domains. If the native tertiary fold still leaves excess hydrophobic surface area or charge patterns favoring cooperative Coulombic interactions, higher-order assembly structures will be established, ending up with homologous or heterologous superstructures. Depending on the protein and the solvent conditions, the energetics of quaternary structure formation may be governed by enthalpic and/or entropic contributions; in most multimeric assemblies, the "polymerization" is entropy driven (LauflFer, 1975). B. Folding Intermediates Denaturation/renaturation experiments have been the method of choice in mimicking protein folding in vitro. Experience accumulated

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over more than half a century has proven that proteins may be renatured or, in some cases, even "unboiled," by restoring native solvent conditions after preceding denaturation. As indicated by the previously mentioned consensus pathway, folding seems to follow an ordered compulsory pathway rather than random search. Experimental evidence for the various steps has been gained from folding intermediates differing from the final native state in characteristic spectral [fluorescence emission, circular dichroism (CD)], hydrodynamic [high-performance liquid chromatography (HPLC), gel-permeation chromatography], or electrophoretic properties (gradient electrophoresis, cross-linking, hybridization) (Jaenicke and Rudolph, 1986; Eisenstein and Schachman, 1989; Creighton, 1992a). Because small proteins (or domains as constituents of large ones) commonly fold in a highly cooperative manner within fractions of a second to a few seconds, in most cases intermediate states are populated only at a low level. As a consequence, it is extremely difficult to follow structure formation directly. Solutions to the dilemma could be either altering the solvent conditions or lowering the temperature. However, both approaches may affect the kinetic mechanism by trapping intermediates in local energy minima which must not necessarily be on the regular folding path; what is "regular" in this context is a matter of dispute which will be discussed in connection with the folding of cytochrome c (Section III,C,4). By stepwise lowering the temperature, the decrease in the reaction rate might allow well-defined elementary processes such as proline isomerization, merging of domains, or subunit assembly to be separated along the folding path. However, different activation energies of the single steps may alter the mechanism, even though the final product may remain unaffected. In addition, the temperature coefficients of the various weak interactions involved in protein stabilization and folding are known to differ so that the ranges of stability of the native protein and its folding intermediates may vary with temperature (Jaenicke, 1981; Privalov, 1992). Surprisingly, variation of temperature over a wide range does not necessarily affect the overall mechanism and the final product of folding: in expressing enzymes from hyperthermophilic microorganisms in mesophilic hosts it has been found that temperature differences as large as 60°C have no effect on the structure and function of the recombinant protein (Rehaber and Jaenicke, 1992; Beaucamp et al., 1995). However, in order to be on the safe side and to avoid artifacts, the best approach in analyzing fast steps on the folding path is to use either rapid kinetics or indirect methods such as hydrogen-deuterium (H-D) exchange or ligand exchange kinetics (Creighton, 1992a; Jones et al., 1993; Dobson, 1994). For example, stopped-flow circular dichro-

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ism or fluorescence measurements, as well as pulse labeling of amide protons analyzed by NMR have shown that secondary structure formation precedes slow steps in a sequence of reactions separable on the milliseconds to minutes time scale (Kim and Baldwin, 1982; Schmid, 1992). Based on this kind of evidence it is now widely accepted that defined native-like tertiary interactions are formed at an early stage along the folding pathway. However, the events following the collapse of the polypeptide chain are less general than their earlier counterparts, perhaps in part because of specific characteristics of the folding pathways for different protein families; for the same topology, differences in sequence cause additional specificity due to variations in tertiary interactions (Udgaonkar and Baldwin, 1988; Roder et al., 1988; Staley and Kim, 1990; Bycroft et a/., 1990; C. R. Matthews, 1991; Hardin et al., 1991). Even with the given heterogeneity, common features are obvious: (i) The burst-phase formation of hydrogen bonds is followed by slower phases, reflecting the sequential or parallel (multiple pathway) completion of the tertiary fold, (ii) H-D exchange NMR experiments provide clear evidence that sequestering aromatic side chains from the solvent follows the formation of stable hydrogen bonds (Udgaonkar and Baldwin, 1988; Roder et a/., 1988; Garvey et al, 1989; Bycroft et al, 1990). (iii) Binding sites for specific ligands, either substrates or cofactors, appear before the rate-limiting step in folding. In many cases, the ligands do not affect the amplitude of the CD burst phase, consistent with the idea that the binding sites are formed sequentially, late on the folding path, and not during the early collapse (Frieden, 1990). (iv) The same conclusion has been drawn from the kinetics of appearance of conformational epitopes recognized by monoclonal antibodies (BlondElguindi and Goldberg, 1990; Goldberg et al, 1990; Goldberg and Djavadi-Ohaniance, 1993). All these results, and those obtained with designed peptide models (Oas and Kim, 1988; Wright et al, 1988; Kim and Baldwin, 1990; Dyson and Wright, 1993), suggest that folding intermediates are stabilized by tertiary interactions that resemble those found in the native protein. In this context, elegant "protein dissection studies must be mentioned. Starting from disulfide-linked BPTI, Staley and Kim (1990) were able to show that fragments of the protein are able to form a cooperative structure closely resembling that of native BPTI. The fact that the separate components (without cystine cross-bridge) do not fold stresses the importance of specific contacts between the selected regions; at the same time, the result corroborates Wetlaufer's foldingby-parts hypothesis (Wetlaufer, 1973; Wetlaufer and Ristow, 1973). In extending this approach to a-lactalbumin (the standard molten globule

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system), the isolated a-helical domain of the two-domain protein was shown to form the same overall tertiary fold as that found in the intact molecule. The formation of the native-like structure does not require extensive specific side-chain packing, suggesting that most of the information transfer from one dimension to three dimensions occurs at an early (molten globule) stage of protein folding (Peng and Kim, 1994; Peng et a/., 1995; Wu et ai, 1995). C. Folding of Small Single-Domain Proteins

It is obvious that the various experimental approaches allow the kinetic analysis of conformational changes in certain local environments or around specific groups of the folding polypeptide chain. The elucidation of the folding path of a given protein would require the complete description of the nascent (unfolded) and final (native) states, together with all intermediates along the Un-^ N transition: Un^h-^N

[1]

where Un is the astronomically large number of "unfolded" or "denatured states," // is a series of intermediates in sequential order, and N is the native state. As a consequence of the structural degeneracy of C/„, and due to limited time resolution and the multiple-step and multiplepathway ambiguity, the complete time course of folding has not been elucidated for any protein so far. The most detailed mechanisms which have been worked out in the past refer to small single-chain one-domain proteins: BPTI, ribonuclease (RNase A and RNase Tl), cytochrome c, hen egg-white lysozyme, and bamase. Each of these illustrates a particular problem or methodology. Because recent progress has been discussed in a number of excellent reviews, in the following only a brief summary will be given. For details see Creighton (1978,1992a), Fersht et al (1992), Goldenberg (1992b), Kim and Baldwin (1982, 1990), and Schmid (1992). 1. BOVINE PANCREATIC TRYPSIN INHIBITOR: DISULFIDE SHUFFLING

Experiments following disulfide cross-linking of BPTI to probe the conformational transitions during structure formation resulted in the first successful approach to unravel the folding pathway of a small protein (Creighton, 1978, 1992b) (see Section III,G). The experimental approach was optimized using an acid-quench technique (to block SH/ SS exchange reactions) and HPLC (to separate intermediates) with the general result that there is a consecutive folding pathway containing exclusively correct cystine cross-links, with all but one intermediate showing native-like overall structure (Weissman and Kim, 1991;

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Creighton et ai, 1992; Goldenberg, 1992a). The natural redox system to perform the disulfide exchange reaction is provided by a cysteine residue in the pro-sequence of the BPTI precursor. It takes care of a high local thiol concentration required for the formation of the sterically unfavorable 5-55 cross-link (Weissman and Kim, 1992). Mutant studies on single-disulfide intermediates, with cysteine residues not involved in disulfide bonds replaced by serine, were devised in order to determine the rates and equilibrium constants for the formation of each of the cystine bridges on the folding path (Darby and Creighton, 1993). Further insight into the structure of native and nonnative intermediates with one or two disulfides was gained from extended twodimensional ^NMR and circular dichroism analyses. As a result, there is clear evidence that under conditions in which stable structures are significantly populated, the intermediates exhibit a compact conformation that is structurally very similar to that of the native protein (Darby etai, 1991,1992; vanMierloe^ aZ., 1991,1992,1993,1994). The formation on disulfides is one of the rate-determining steps in the acquisition of the native state of globular proteins. In most cases they have a stabilizing effect on the native state without determining either the folding pathway or the final three-dimensional structure. Because of the required oxidation potential, the reaction takes place only in the endoplasmic reticulum (ER) or in the periplasm. As a consequence, hardly any proteins with disulfide bonds are found in the cytosol. In the cell, the oxidation of SH groups is catalyzed by PDI (in E. coli DsbA), a ubiquitous enzyme abundant in all cells (see Section IV,B). 2 . RiBONUCLEASE: PROLINE ISOMERIZATION

Using RNase as a representative example for a single-chain protein with the size of an average domain, denaturation/reduction and subsequent controlled reoxidation experiments provided the first proof for the one-to-one relationship of the primary and tertiary structure of proteins (Anfinsen, 1973). However, with four disulfide bonds, i.e., 105 possible combinations of SH groups, no folding mechanism could be established. Instead, the oxidized (native) enzyme has been chosen as a paradigm in order to identify rate-determining steps on the folding pathway. Using this system, Garel and Baldwin (1973) discovered that there are populated intermediates during unfolding and folding of proteins. Since then, RNases have been the best-documented examples in the attempt to unravel the kinetics of protein structure formation (Kim and Baldwin, 1990; Schmid, 1992). The primary aim was to explain the apparent discrepancy of thermodynamic two-state behavior

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(U ^ N), on one hand, and kinetic multistate behavior, on the other, which clearly pointed to a kinetic scheme with at least three states: US-UF^N

[2]

where Us and Up stand for slow and fast folding species, and N stands for the enzyme in its native state (Garel and Baldwin, 1973; Garel et aL, 1976). The hypothesis that the isoenergetic cis-^rans-isomerization of proline residues might be involved in the two-state transition (Brandts et aL, 1975) has been confirmed by a wide range of experiments (Schmid, 1992, 1993; Schmid et aL, 1993). In the present context, the essential point is that, due to the high activation energy of the reaction, proline cis-^rans-isomerization, next to disulfide cross-linking, is the second rate-limiting process in the overall folding scheme of singlechain proteins. The reason why the reaction is exceedingly slow is the high activation energy connected with the rotation around the partial C-N double bond of the imino acid proline (J?A = 85 kJ/mol). This rotation is essential in the overall folding reaction because the nascent trans conformation of the peptide bond is isomerized to cis in approximately 7% of all prolyl residues in native proteins. In RNase, two out of four prolyl residues are in cis conformation, giving rise to significant signals in the rate-determining phases of the unfolding/folding reactions. The kinetics were analyzed in great detail with special emphasis (i) on the catalysis of the slow steps by peptidylprolyl cis-^rans-isomerase (PPI), and (ii) on variants with specific proline residues exchanged by site-directed mutagenesis. Both approaches gave clear evidence for the involvement of prolyl crs-^raAis-isomerization in the refolding kinetics of the slow folding species Us (Eq. [2]). Whether peptidylprolyl cJs-^raAis-isomerase plays a significant role in protein folding in vivo is still unresolved (Schmid, 1993; Schmid et aL, 1993; Fischer, 1994). cis-Proline mutants of RNase A (P93A, P93S, P114A, and P114G) show strongly decreased stability compared with the wild-type enzyme (AAG° = 12 kJ/mol). The reason seems to be that, after substitution, the new nonproline peptide bond goes in cis, so that cis-trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state (Schultz and Baldwin, 1992). The double mutant (P93A/P114G) is characterized by one single exponential, i.e., there is no isomerization after unfolding, whereas the single mutants exhibit complex kinetics. Apparently, both P93 and PI 14 are collectively responsible for the formation of Us (Eq.[2]) (Schultz et aL, 1992). For RNase Tl, the assignment of the cis-prolyl residues (S54-P55 and Y38-P39) to the slow kinetic phases allows a native-like intermediate (IN, with one "wrong*' proline residue) to be distinguished from its

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precursor {Ii with two wrong prolines). The detailed mechanism has been worked out in a series of most elegant kinetic studies (Kiefhaber et al., 1990a,b,c). As in the case of RNase A, nonprolyl cis peptide bonds are responsible for slow steps in the folding kinetics; replacing proline39 by alanine, the cis conformation of the 38-39 peptide bond is retained (L. M. Mayr et al,, 1994). Obviously, cis peptide bonds are conserved at positions where trans bonds would destabilize the protein more strongly than nonprolyl peptide bonds in the energetically unfavorable cis conformation. The catalytic activity of the mutant was almost unchanged, while its free energy of stabilization showed a decrease of about 20 kJ/mol (L. M. Mayr et aL, 1993, 1994). Recently, a C25/C10N mutant of RNase Tl was used in order to investigate the interdependence between the acquisition of an ordered native-like conformation and disulfide formation in oxidative protein folding. The single-disulfide model (C6-C103) is unfolded in the presence of urea, but folded when >1.5 M NaCl is present. Thus, the influence of a folded conformation on the individual thiol/disulfide exchange reaction between the protein and glutathion could be studied by altering the urea and NaCl concentrations. The kinetic analysis nicely illustrates the importance of low stability and high flexibility of folded intermediates (Freeh & Schmid, 1995). 3 . RiBONUCLEASE: UNFOLDING INTERMEDIATES

A basic unanswered question about the mechanism of protein folding is whether partially unfolded intermediates, with some hydrogen bonds broken, exist on the unfolding pathway. Due to microscopic reversibility, the answer to this question also reflects late folding events, with the advantage that the analysis starts from the well-defined native state (N) instead of multiple states (Un, Eq. [1]). In the case of RNase A, because of the slow cis-trans isomerization of prolyl peptide bonds, n is expected to be at least 2. Refolding experiments would be unsuited to detecting any intermediates that occur after the rate-limiting step of folding. This space on the energy diagram is only accessible from unfolding experiments. Since folding kinetics are determined by late events on the folding path, close to the native state (Creighton, 1978, 1990), unfolding intermediates should give information about the nature of the rate-limiting step because any unfolding intermediates would occur close to this step. Experiments based on optical probes showed that both the secondary and the tertiary structures unfold with the same kinetics, suggesting that there are no unfolding intermediates (Schmid, 1992). However, molecular dynamics simulations of unfolding indicate that there are

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unfolding intermediates with some hydrogen bonds broken, resembUng the well-estabUshed refolding intermediates. Kiefhaber and Baldwin (1995) approached the problem by comparing the unfolding kinetics of RNase A (monitored by circular dichroism) with the kinetics of destabilization of individual hydrogen bonds involving backbone NH protons (measured by chemical-shift dispersion and pulsed hydrogendeuterium exchange), with the following results: (i) There is a significant difference in the unfolding rates measured by CD and H-D exchange which follows from the fact that CD measures an apparent rate constant that includes microscopic rate constants for unfolding and refolding and for proline isomerization, while H-D exchange is irreversible, monitoring directly the unfolding rate constant, (ii) The unfolding mechanism corresponds to the reverse reaction of Eq. [2], with irreversible side reactions (C/^> U"^), attributable to the H-D exchange processes; the unfolding and refolding rate constants are 1/50 and 1/290 sec"^ for N ^ Up, and 1/60 and 1/300 sec"^ for the prohne cistrans isomerization Up ^ Us. (iii) The results indicate that the entire network of peptide H bonds breaks in the transition state for unfolding, consistent with the experimental observation of unfolding as a twostatereaction. Whether the presence of disulfide bonds is responsible for the high cooperativity or whether proline isomerization affects the mechanism is still unknown, (iv) Chemical-shift dispersion indicates a widespread unlocking reaction at the start of unfolding: when the first spectrum is recorded, the methylene region of the NMR spectrum reflects complete unfolding, while the CD signal at 222 and 275 nm still corresponds to 75% native protein. Obviously, many side chains become free to rotate even though the peptide hydrogen bond network remains intact. The faster exchanging protons correspond to "weak points" in the three-dimensional structure; they are found mainly at the ends of the )3 strands and helices 2 and 3. There is no contradiction between the unlocking reaction and the above two-state considerations because the loosening of the structure does not correspond to the formation of the unfolding transition state: it precedes the slow unfolding reaction monitored by optical probes (Kiefhaber et aL, 1995). 4. CYTOCHROME C AND RIBONUCLEASE. EARLY STEPS OF FOLDING AND DOCKING

Early intermediates on the folding path are difficult to characterize because of their short lifetime. Spectroscopic approaches (UV absorbance, fluorescence, and CD), as well as protein chemical methods (limited proteolysis, antibody binding, and H-D or H-T exchange) are commonly restricted to the analysis of global structural changes with

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a relatively low time resolution, keeping in mind that the rates of helix-coil transition and /3-structure formation are in the microsecond time range. However, initiating the folding reaction by a short light pulse may dramatically improve the time resolution measuring the change in absorption of an intrinsic chromophore to monitor the kinetics. In the case of cytochrome c, Jones et al. (1993) presented an example of an optically triggered folding reaction by using nanosecond photodissociation of the heme-CO complex of reduced cjrtochrome c. The optical trigger is based on the observation that under destabilizing conditions C5d;ochrome c can be unfolded by preferential binding of CO to the covalently attached heme group in the unfolded state. Photodissociation of the CO thus triggers the folding reaction. By monitoring timeresolved absorption spectra at 390-450 nm as a measure of heme ligandation, transient binding of both native and nonnative axial amino acid ligands from the unfolded polypeptide chain is observed before folding begins. This reaction takes place in the /xsec time range. Kinetic modeling suggests that the intramolecular binding of Met-65 and Met80 is faster than that of His-26 and His-33, even though the histidines are closer to the heme. The deconvolution of the recorded spectra allows seven species to be resolved; the definitive assignment of the elementary processes involved during the first millisecond requires further investigation. The exciting prospect of this approach is that folding reactions become accessible to kinetic measurements without the limitations in time resolution. It seems as if no further precursors on the folding pathway become detectable (W. A. Eaton, 1995, personal communication). Thus, the experimental tools for a complete analysis of early kinetic processes are available now, and the question becomes which protein might be best suited to start a detailed investigation. From the mechanistic point of view, the important message in connection with early events in cytochrome c renaturation is that folding may be preceded by the formation of transient loops of the polypeptide chain with nonnative contacts between residues or clusters of residues. This is what has been hypothesized as "collapsed form" of the folding protein which would subsequently relax or shuffle to reach the native tertiary structure. A totally different approach to detect emerging structural elements in the context of a known three-dimensional structure was developed, applying 2-D NMR spectroscopy, combined with the trapping and subsequent identification of exchangeable amide protons. The crucial point for improving the time resolution up to the millisecond time range was the combination of the potential of nuclear magnetic resonance with rapid pulse labeling using stopped-flow multimixing techniques (Ud-

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gaonkar and Baldwin, 1988, 1990; Roder et ai, 1988; Roder and Elove, 1994; Bai et al., 1995). Structure formation is then monitored by the shielding of specific amide protons from the solvent or from hydrogen bonding which gives rise to reduced exchange rates, thus providing a direct measure of secondary structure or core formation upon folding. As one would expect, under quasinative conditions rapid protection of many NH protons from exchange is observed as a consequence of the fast elementary steps of a-helix and /3-sheet formation. A common feature of cytochrome folding in previous studies has been the heterogeneity of the folding kinetics in the sense that different protein fractions fold at different rates. The reason is that there are kinetic barriers along the folding path which slow down the overall process to a time scale of seconds, trapping intermediates by wrong interactions, e.g., between the heme group and nonnative ligands. It has been recognized for a long time that kinetic barriers can be removed by adjusting solvent conditions (Brems and Stellwagen, 1983). Taking this into consideration, the time constant (T) for cytochrome c folding from the fully denatured state to the native protein is reduced to less than 15 msec in a single kinetically imresolved step, i.e., without populating observable intermediates (Sosnick et al, 1994) (Fig. 2). These experiments clearly suggest that elimination of interactions which form in the denatured state of cytochrome c enables the majority of the molecules to fold to

pH 6.2

••^x

] pH 2 -U9 Q003

Q03 Time (sec)

0.3

FIG. 2. Rapid folding of cytochrome c in the presence and in the absence of nonnative interactions. Denaturation at pD 2; fast folding at pH 4.9 compared to slow folding at pH 6.2. Enhancement of folding monitored by Met80 to heme ligation (Aegsmn), secondary (•) and tertiary H bonds (•) using HX/NMR labeling, and fluorescence quench of Trp 59 (Fl). The observed single time constant, T = 7 msec, corresponds to an enhancement of the folding rate by factors of 20 to 10^, compared to the reaction involving wrong hemehistidine ligations (Brems and Stellwagen, 1983; Sosnick et al, 1994).

PROTEIN FOLDING AND ASSOCIATION

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the native state at essentially the same rate as the molecular collapse, in contrast to the view that particularly steps in protein folding (including the supposedly rate-limiting molten globule to native transition) are intrinsically slow. Instead, it appears that folding intermediates may be kinetically trapped by barriers that are optional rather than integral to the folding process. Major barriers may result from misfolding of the polypeptide chain in the initial collapse step. In a recent study, Bai et al, (1995) observed that, at low denaturant concentrations, cytochrome c reveals a sequence of metastable, partially unfolded states that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units within the complete structure of the molecule. The cooperative units are loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even under native conditions and, thus, may define the majority pathway of cytochrome c folding. One might argue that the rapid folding is a specific property of cytochrome c, attributable to the heme group. However, it is highly improbable that the heme could enable the protein to overcome presumed kinetic barriers such as the tight packing of side chains or the squeezing out of water throughout the entire 12.4-kDa protein. Equally, ligand-induced folding, reported for the effects of Ca^^ on the refolding kinetics of parvalbumin or a-lactalbumin (Kuwajima et a/., 1988, 1989), can be excluded for the simple reason that even with the heme present, the folding reaction can be just as slow as that for other (unliganded) proteins. Fast folding is not unique to cytochrome c: chymotrypsin inhibitor2, ubiquitin, RNase A, the Ig-binding domain of streptococcal protein G, and the cold-shock protein from Bacillus subtilis have been shown to fold at similar rates (Udgaonkar and Baldwin, 1990; Jackson and Fersht, 1991; Briggs and Roder, 1992; Kuszewski et aZ., 1994; Houry et al., 1995; Schindler et al, 1995). Whether the fast rate in all these systems depends on the fact that the (re-)folding polypeptide chain, in collapsing to its early intermediate state, avoids nonnative ligations needs further experimental verification (Creighton, 1994). RNase has been the first paradigm in the elucidation of protein folding, applying pulsed hydrogen exchange. Current evidence shows that the ^ sheet is formed rapidly and cooperatively shortly after the start of folding. In contrast to the simple sequential model, its stability increases over the first 0.4 sec so that one must assume that "the early intermediate" actually represents a broad distribution of species which

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RAINER JAENICKE

gradually changes until side-chain interactions lock the fluctuating polypeptide chain in the stable /3-sheet conformation (Udgaonkar and Baldwin, 1990). Similar multiple-pathway behavior has been observed for other proteins (see below). In the case of the refolding of cytochrome c, protection against NH proton exchange shows multiphasic kinetics with at least three phases. Taking the NH exchange rates as a measure, the N- and C-terminal helices are formed within —20 msec, whereas most of the other NH protons follow in the 200 msec time range; the slowest exchange rates (10 sec) are attributable to groups involved in tertiary contacts (Roder et al., 1988). It is important to note that not only the formation of the helices at the N- and C-terminal end is an early event, but also their specific docking. Being the most stable structural elements in the native molecule, this clearly contradicts the idea that cotranslational ("vectorial") folding from the N- to the C-terminal end of the nascent polypeptide chain might be decisive for the folding pathway and/or the final structure of the functional protein. In contrast to helix formation in cytochrome c, in the case of ubiquitin and the cold-shock protein from B. subtilis, )8-structure formation has been shown to be involved in very fast early folding events (Briggs and Roder, 1992). On the other hand, the all-)8 interleukin 1)8 refolds slowly, with a half-time of about 20 min (at 4°C) (Varley et al., 1993). Evidently, there is no clear correlation between either the size or the stability or the structural type of a protein and its folding rate. 5. LYSOZYME AND DIHYDROFOLATE REDUCTASE: EARLY INTERMEDIATES AND M U L T I P L E P A T H W A Y S

Egg lysozyme is similar to RNase regarding its size and cross-linking pattern. However, its folding is not determined by proline isomerization so that one can analyze the folding reaction in the absence of this ratelimiting step. Experiments have concentrated on early folding intermediates and the overall folding pathway of the oxidized protein. Spectroscopic evidence from refolding studies far from equilibrium showed that there is an early folding intermediate with native-like secondary structure, but still lacking native tertiary contacts (Mirankar et al., 1991). The overall unfolding-refolding reaction shows two-state characteristics, although a kinetic intermediate accumulates during folding in the native region. Thus, kinetics allow the two structural lobes of the molecule to be discriminated.t This conclusion is strongly supported t It is a question of definition whether lysozyme is a two-domain protein. Considering the amino acid sequence, the two lobes that constitute the "active cleft" of the enzyme do not represent contiguous stretches of the polypeptide chain. Strong evidence that two independent parts of the enzyme can be differentiated came from X-ray data at elevated pressure (Kundrot and Richards, 1987).

PROTEIN FOLDING AND ASSOCIATION

233

by pulse-labeling H-D exchange experiments. Two-dimensional NMR analysis shows that the enzyme consists of two regions differing in their NH protection. Thus, the kinetic mechanism contains an intermediate in which one lobe has reached its compact native-like state, whereas the other is still unfolded or in the process of folding. In similar experiments, nearly half of the amide hydrogens in the molecule were used as probes for refolding. Stopped-flow CD measurements were performed to show that folding of lysoz3nne not only involves partially structured intermediates, but also multiple pathways (Radford et al., 1992). As indicated by the kinetics of amide protection (applying mass spectroscopic analysis), the enzyme does not fold in a single cooperative event. Instead, different parts of the structure become stabilized with different kinetics. In particular, the a-helical domain folds faster than the j8-sheet domain. Furthermore, different populations of molecules fold by kinetically distinct pathways. An alternative explanation of the observed biphasic kinetics as the result of heterogeneity has been excluded by controls, altering the pulse length and pH, so that the results indicate that folding of oxidized lysozyme is not a simple sequential assembly process but involves parallel alternative pathways, some of which involving substantial reorganization steps. No kinetic scheme for this complex mechanism has been formulated so far (Evans and Radford, 1994). Comparing the kinetics of the reduced polypeptide chain, it has been shown that decreasing the structural constraints leads to a drastic decrease in the folding rate (Goldberg and Guillou, 1994). Thus, the question remains whether the cystine crosslinks may be important determinants in the above kinetic mechanism. In a similar study, dihydrofolate reductase (an a/13 protein with no disulfides and no prosthetic group) has been shown to exhibit two distinct types of transient intermediates: The first forms within 5 ms and has substantial secondary structure but little stability; the second is a set of four species that appear over the course of several hundred ms and have secondary structure, specific tertiary structure, and significant stability (Jennings et al., 1993). A detailed study of specific amide groups involved in a-helices and /3-strands allowed the sequence of protection steps to be analyzed. Only about one half of the molecules exhibit significant protection of amide hydrogens in the sheet and in two of the helices, again indicating parallel pathways at the level of early folding intermediates (Jones and Matthews, 1995). Obviously in both cases, hen egg-white lysozyme and dihydrofolate dehydrogenase, partitioning into multiple pathways refers to the initial development of secondary and/or tertiary structure.

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In summarizing the pulsed H-D exchange experiments and other folding studies of small proteins like cytochrome c or lysozyme, there is clear evidence that folding intermediates with native-like structure accumulate. The fact that they are not well synchronized seems to contradict the classical framework model (Kim and Baldwin, 1990). Theories of the folding process and Monte Carlo simulations of folding suggest that neither the folding pathway nor the set of folding intermediates is unique, and that folding intermediates only accumulate because they are trapped kinetically by partial misfolding (Bryngelson & Wolynes, 1989; Shakhnovich et aL, 1991; Camacho and Thirumalai, 1993; Dill et al, 1993; Shakhnovich, 1994; Abkevich et a/., 1994a,b; Chan and Dill, 1994; Sali et al, 1994). This view resembles the jigsaw puzzle model proposed by Harrison and Durbin (1985). However, experiments give clear evidence that there are folding pathways with successive intermediates and hierarchical order (Schmid, 1992). Further experiments are needed to distinguish between the "classical" and the "new view." As has been pointed out, one important issue in this context is the consideration of secondary structural elements as a major factor determining the folding pathway. One may assume that robust and fast folding pathways have been selected through evolution. Thus, random misfolding in computer simulations may not necessarily apply to the folding behavior of real proteins (Baldwin, 1995). 6. BARNASE: PROTEIN ENGINEERING ANALYSIS OF PROTEIN FOLDING

Bamase is a single-domain extracellular RNase from Bacillus amyloliquefaciens consisting of 110 amino acids. As it contains a helices and an antiparalell (3 sheet, but no cysteine, methionine, and cis-proline, it offers itself as a paradigm for folding studies on a stable cystine-free protein. With bamase as model, Fersht and co-workers developed a "theory of protein engineering analysis of stability and pathway of protein folding" using kinetic and equilibrium unfolding/refolding measurements on numerous mutants to map the formation of structure in transition states and intermediates (Fersht et al, 1992; Serrano et aL, 1992a,b; Matouschek et al, 1992). In order to obtain empirical data with respect to the magnitude of particular interactions and how they affect the kinetics, equilibria, and mechanisms, the following strategy was applied: (i) identification (from the known three-dimensional structure) of an interaction of a side chain that appears to be important in binding or kinetics, (ii) modification of that interaction by "sensible" site-directed mutagenesis, (iii) determination from equilibrium and kinetic measurements of the free-energy profiles for the reaction of mutant and wild-type protein, and (iv) con-

PROTEIN FOLDING AND ASSOCIATION

235

struction of the corresponding difference-energy diagram. The fundamental assumption that the mutations neither perturb the structures of the folded and unfolded states nor the folding pathway, and that the target groups make no additional interactions with new partners in their spatial environment, have been studied in detail by measuring the structure and stability increments of the standard states and the first significant transition states of unfolding of more than 60 mutants. The structure of the transition state (resulting from urea denaturation) is that of the native-like enzyme, with its hydrophobic core weakened and several of the tertiary interactions and loops lost, but with the majority of the secondary structure elements, including tight turns, maintained. This implies that the last events in folding seem to be the consolidation of the hydrophobic core, the closing of loops, and slight rearrangements of tertiary contacts. There is no indication for parallel pathways (Serrano et aL, 1992a). The same approach as that in the case of the transition state for unfolding was applied in order to characterize the structure of an intermediate on the refolding pathway (Matouschek et aL, 1992). This time, the order of events is early formation of the N-terminal a helix, the 13 sheet, part of the core, and docking of the C terminus to this "nucleus"; subsequent steps stabilize the rest of the core and the loops, with the tertiary contacts as the coda. The theory has been confirmed and supplemented by independent evidence taken from H-D exchange NMR studies; in addition, it has been compared with the various folding models developed during the past decade (Serrano et aL, 1992b). The general conclusions are in agreement with the consensus pathway and may be summarized as follows: There is a compulsory pathway of folding which is, at least in part, sequential. Secondary structure formation is driven by the local minimization of hydrophobic surface area; it precedes tertiary structure formation. Tertiary interactions become increasingly defined as water release consolidates the hydrophobic core. D. Folding of Domain Proteins Beyond a certain limiting length of the polypeptide chain, proteins are known to consist of domains. Among their various definitions (Janin and Wodak, 1983), in the present context we may consider domains as descendants of precursor genes fused together to form larger multifunctional proteins with independent folding units ("globules") (Goldberg, 1969; Wetlaufer, 1973, 1981). In the simplest case of two-domain proteins, these may be detectable as separate phases in folding/unfolding equilibrium transitions, according to a three-state model

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RAINER JAENICKE

N^I^U

[3]

where / represents an intermediate with one domain still intact and the other unfolded (Fig. lA). In certain cases the whole molecule may represent one cooperative unit so that the population of / remains undetectable and can be ignored. Using different denaturants, one and the same protein may show different behavior, as in the case of yBcrystallin, in which urea denaturation obeys Eq. [3], whereas guanidinium chloride denaturation shows only one single transition (Rudolph et a/., 1990b; Sharma et al., 1990). As taken from in vitro translation experiments, independent domain folding is no physicochemical artifact, but holds also in the cell: using immunoglobulin and serum albumin, it has been shown that intradomain cystine bridges are formed sequentially during translation (Bergman and Kuehl, 1979a,b,c; Peters and Davidson, 1982). Essentially, folding by parts must be considered a most significant acquisition of evolution for a number of reasons: (i) it enhances the folding rate by synchronous folding at multiple sites along the nascent polypeptide chain, (ii) it is a most efficient way to exclude wrong intramolecular interactions in the case of large protein molecules, (iii) it protects the nascent polypeptide chain from proteolysis, and (iv) it may be considered a simple mechanism to proceed from monomeric to multimeric proteins by domain swapping (Bennett et aL, 1994, 1996) (see below). From the phylogenetic point of view, it has been proposed that domains are the products of exons in the sense that split genes arose from combinations of primordial minigenes (exons) separated by introns as spacers (Darnell, 1978; Doolittle, 1987; Gilbert, 1987). However, attempts to detect significant correspondences between exons and units of protein structure seem to indicate that the exon hypothesis in its original form is not tenable (Stoltzfus et al., 1994). Regarding the folding mechanism, domain proteins may be considered as the sum of their constituent parts; this means that what has been discussed in connection with the sequence of consecutive steps in single-domain proteins holds unchanged. In the overall kinetic scheme, the rate-determining reactions are of first order. Depending on whether the reaction is two state or three state, one or two pairs of rate constants will be detectable. Figure 3 illustrates the situation for yB-crystallin and its N-terminal one-domain fragment: the complete molecule allows all four kinetic constants ki, k-i, ^2, ^-2 (cf. Eq. [3]) to be detected, whereas the single domains show only one single transition characterized by k2 and k-2 (Rudolph et al„ 1990b; E.-M. Mayr et a/., 1994; Jaenicke, 1994; Mayr, 1995).

237

PROTEIN FOLDING AND ASSOCIATION 1

1

lo-'h

1

1

1

I

1

. D-^I -2 I

1

1

1

N-M^

OVD

-i

O g\r-,

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0,0

/^

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/

^5 I-^N > ] / io"^H 1

^ 10-^ ^

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.

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[urea] (M) FIG. 3. Dependence of rate constants of denaturation/renaturation of yB-crystallin on urea concentration: 0.1 M NaCl/HCl, pH 2.0, 20°C. Closed and open symbols refer to denaturation and renaturation, respectively. Fluorescence emission of native protein at 360 nm (•, O) and 320 nm (•, D); fast HPLC gel filtration (•, O) of native protein; fluorescence emission of intermediate I at 360 nm (A, A) and 320 nm (V); fluorescence emission of N-terminal domain at 360 nm (•, O) and 320 nm (•) (Rudolph et al, 1990b).

There are examples in which biological function requires the cooperation of domains, e.g., two domains sharing a common binding site, each contributing amino acid residues to one active center. In such cases, domain pairing is expected to be decisive in acquiring biological function, whereas domain folding may be a precursor reaction, separate on the time scale. Using monomeric octopine dehydrogenase as an example, it has been shown that domain pairing may become rate determining: with increasing viscosity of the solvent medium, reactivation is slowed down, as one would predict from Stokes-Einstein's model of rotational and translational diffusion of rigid bodies (Teschner et aL, 1989). Thus, the overall folding kinetics for a two-domain protein (with domains Di and D2) may contain one more step in addition to the threestate model in Eq. [3], since pairing (docking) and packing may be rate determining in acquiring the native functional state: ^ folding (Di)^^ folding (Da)^^. pairing/packing^^

^^-|

In cases in which proline isomerization or other slow reactions partici-

238

RAINER JAENICKE

pate in the overall mechanism, these steps will lead to a kinetic scheme of even higher complexity (Garel, 1992). Using intrinsic markers (fluorophores, epitopes for monoclonal antibodies, and ligands), sequential folding steps may be resolved on the time scale from a few milliseconds to seconds. Using these approaches, possible artifacts due to the interference of intra- and intermolecular interactions have to be carefully excluded. Considering these precautions, different methods have been shown to be in good agreement, corroborating the sequential folding mechanism (Blond and Goldberg, 1986; Blond-Elguindi and Goldberg, 1990). E. Association

In advancing from domain proteins to protein assemblies, we approach cellular substructures and finally the macroscopic world: surface layers, the microtrabecular lattice, the tubulin-dynein system, flagella, ribosomes, the extracellular matrix, muscle, are all self-assembly systems involving proteins or protein conjugates. From the point of view of the structural hierarchy of proteins, oligomerization corresponds to domain pairing, except that the docking process is dominated by noncovalent interactions. Model reactions simulating quaternary structure formation made use of proteoljrtic fragments. In certain cases they were found to exhibit high specificity of subdomain or domain interactions which allow them to recognize and complement each other. In general, the association process is entropy driven as a consequence of water release from the subunit interfaces. There may be a significant contribution to protein stability from quaternary stucture formation (Richards and Vithayathil, 1959; Gerhart and Pardee, 1962; Gerhart and Schachman, 1965; Jaenicke and LauflFer, 1969; Gerhart, 1970; Schachman, 1974; Opitz et al.y 1987; Jaenicke, 1991a). Complementation requires the correct recognition sites to be preformed; this means that fragments or domains that are expected to trigger the assembly process must fold autonomously (see Section III,D). The overall reconstitution can then be visualized as a sequential folding-association reaction, in which folding provides the correct docking surfaces allowing the consecutive association reaction to take place. At low concentrations, association becomes rate determining (Jaenicke, 1987a). Thus, quaternary structure formation may affect both the stability of proteins and their rate of folding. Both effects can be beautifully illustrated when subunits of oligomeric proteins are fused by short peptide linkers connecting the carboxy termini of subunits to the amino termini of their respective counterparts (Kuchinke and Miiller-Hill, 1985; Liang et al, 1993).

PROTEIN FOLDING AND ASSOCIATION

239

In describing the complete association pathway, the steps preceding subunit docking are the same as those in domain proteins. The overall mechanism consists of three stages: (i) formation of elements of secondary and supersecondary structure; (ii) collapse to subdomains and domains, ending up with structured monomers; and (iii) association to form the correct stoichiometry and geometry of the native quaternary structure. Evidently, the "collision complex" of the structured monomers may still undergo intramolecular rearrangements in order to reach the state of maximum packing density and minimum hydrophobic surface area. Thus, the uni-bi molecular folding/association mechanism may involve further first-order steps belonging to slow shuffling processes at the level of the native-like assembly. Focusing on the ratelimiting steps, in the simplest case for a dimer, the overall reaction would then obey a uni-bi-uni molecular reaction according to 29^~^2M-^M2-^N [5] with !M and M as unfolded and structured monomers, N as the native dimer, and ki and k2 as first- and second-order rate constants (Jaenicke, 1987a; Garel, 1992). At this point, it becomes clear why reconstitution studies have been a powerful tool in elucidating the mechanism of protein folding and association. In contrast to denaturation, where both unfolding and dissociation follow first-order kinetics, renaturation, in many cases, allows for the distinguishing between folding and assembly, making use of the concentration dependence of the association reaction, supposing the assembly of the subunits is not diffusion controlled. Thus, the kinetic analysis of various properties of the refolding polypeptide chain(s) may provide a means to monitor the recovery of secondary tertiary, and quaternary structure, as well as the occurrence of catalytic efficiency, allowing the structure-function relationship in the process of structure formation to be elucidated. Only in rare cases have structured monomers been obtained as stable entities; for example, at low temperature or elevated pressure, dissociation may involve subunits which still exhibit native-like properties (Jaenicke, 1987a), and as kinetic intermediates during reconstitution, they may be accessible to a detailed analysis. How the single steps along the folding/ association pathway can be monitored depends on the specific structurefunction relationship for a given system. In most cases, biological function relates to the native quaternary structure such that the final ratedetermining step can be measured by the regain of activity. Preceding steps may be accessible to spectral analysis, cross-linking, and a wealth of other methods (Jaenicke and Rudolph, 1986, 1989; Eisenstein and Schachman, 1989).

240

RAINER JAENICKE

Evidently, the alternative, which of the two consecutive reactions in Eq. [5] is rate-limiting, depends not only on protein concentration, but also on ki and the specific properties of the system. If association is a rate-determining step, this holds only up to the concentration limit at which the production of structured monomers becomes rate determining. Figure 4A illustrates this case, showing that beyond a certain concentration, the rate of reactivation levels oflfbecause folding becomes rate limiting. In cases in which a slow folding reaction at the level of the monomer determines the overall reaction and association is diffusion controlled, no concentration dependence can be detected despite the bimolecular step. As mentioned, the same holds if the associated protein, Mn, undergoes slow intramolecular rearrangements. Figure 4B shows the reactivation of the 10-MDa multienzjrme complex pyruvate dehydrogenase from Bacillus stearothermophilus as an example (Jaenicke and Perham, 1982). A quite similar behavior has been observed for the protrimer of the tailspike protein of bacteriophage P22 from Salmonella, where folding in vivo and in vitro were shown to obey the

^100

:r 50h 0)

or

0

1 Time

2 (hours)

150

0

1 Time

2 (hours)

3

10

FIG. 4. Rate-limiting reactions in the sequential uni-bi molecular mechanism of folding and association. (A) Reactivation of porcine heart muscle lactate dehydrogenase after deactivation/dissociation at high hydrostatic pressure: 120 MPa (1200 bar), 0.16 M TrisHCl, pH 7.6, 10 mAf dithioerythritol, 20°C. Enzjmie concentration during reactivation: 10.8 (O), 6.0 (•), 1.4 (D), 1.0 (A), and 0.6 /xg/ml ( • ) . Curves calculated according to 4M'

k,

4M

2M2

Af4, with ki = 1.5 X lO-^sec-^ and ki = 3.5 x 10^ M'seC

The recovery of activity and native fluorescence parallel each other (see Jaenicke, 1987a,b). (B) Reactivation of the pyruvate dehydrogenase multienz5rme complex (PDC) from Bacillus stearothermophilus. Denaturation/deactivation in 1 M glycine/H3P04, pH 2.3, 0°C; reactivation in 0.2 M potassium phosphate buffer, pH 7.0, 2 mM dithiothreitol, 5 mM EDTA, 0°C (A), 65°C (D), and 53°C, at varying enzyme concentrations: 160 ( • ) , 80 ( • ) , 40 (open symbols), and (A) 20 /xg/ml (Jaenicke and Perham, 1982).

PROTEIN FOLDING AND ASSOCIATION

241

same mechanism (see below) (Fuchs et al, 1991; Mitraki & King, 1992; Banner and Seckler, 1993; Danner et al, 1993; Beissinger, 1994). Considering the crowding of a great variety of different components in the cell, one important aspect of protein folding and association is the specificity of subunit recognition, i.e., the question of whether or not other proteins may interfere with the formation of the correct native quaternary structure. A qualitative criterion for the fidelity of subunit recognition was gained from renaturation experiments in crude mixtures where reactivation in the presence of excess foreign protein can be considered a direct measure of correct quaternary structure formation. For example, in refolding recombinant antibodies, no significant differences in the yield as well as in the kinetics are observed in the homogenous system compared to the crude mixture obtained upon braking up the E. coli cell (Buchner and Rudolph, 1991). In order to study the specificity of subunit recognition during protein folding in more detail, topologically closely related enzymes were investigated regarding hybrid end products and/or kinetic intermediates. Using dimeric lactate dehydrogenase (from Limulus polyphemus) and mitochondrial malate dehydrogenase under strictly synchronized reactivation conditions as an example, neither hybrid intermediates nor chimeras as end product were detected (Gerl et al,, 1985). As has been mentioned, the same high degree of specificity holds at the level of domains (Wetlaufer, 1981; Opitz et al, 1987). Clear evidence that hybrid formation does occur comes from isoenzymes (e.g. the five forms of heart- and muscle-type lactate dehydrogenase, LDH) or from multifunctional enzymes where certain gene products have been found as subunits in different complexes (e.g., protein disulfide isomerase in prolyl hydroxylase). In the isoenzymes of LDH, the structural homology is extremely high so that complementary subunit surfaces can easily be rationalized. In cases in which isoenzymes in different compartments are involved, both target sequences and different folding mechanisms may contribute to specificity. For example, in the case of dimeric mitochondrial and cytosolic malate dehydrogenases, the second-order subunit assembly of m-MDH is preceded by slow folding (which may even be retarded by the signal sequence), whereas for c-MDH first-order reconstitution indicates diffusion-controlled association (Jaenicke, 1987a). It might be attributed to these mechanistic differences that all attempts to trap hybrid dimers of MDH, either in the process of reconstitution or in equilibrium experiments, failed (R. Jaenicke, 1990, unpublished results). Regarding the evolution of oligomeric or multimeric proteins, advantages attributable to quaternary structure formation can easily be visu-

242

RAINER JAENICKE

alized: economy of the genome, elimination of misfolded polypeptide chains as pieces in the assembly puzzle, stability, allostery, osmotic effects, etc. However, it is much more challenging to understand how oligomers may have evolved. How could random mutations alter the surface of monomeric proteins such that the entropy loss accompanying dimerization or multimer formation is paid by the gain in enthalpy caused by the formation of weak intersubunit bonds? In asking this question, the tacit assumption that monomers preceded oligomers in evolution is generally accepted. As has been pointed out by D. Eisenberg (Bennett et a/., 1996), the gradual accumulation of random mutations that are required to stabilize a dimer of a spherical single-domain "sticky billard ball molecule" and to select for its improved stability cannot be accomplished within biologically feasible time. At this point, the evolution of domains may offer a key to our understanding which adds to their significance in connection with the above folding-by-parts concept. Starting from a monomeric domain protein, "domain swapping'' allows the transition from monomers to assembly structures of any size simply by switching from interdomain to intersubunit interactions (Fig. 5A). Here, the interface is available a priori (perhaps by the duplication of an ancestral minigene), so that now natural selection can improve the stability by additional dimer-stabilizing mutations. Both the low probability to gradually acquire dimer stability by random mutagenesis and the concept of domain swapping may be illustrated using crystallins from the eye lens as examples. In the case of oA-crystallin, the extreme conservation during evolution (1% per 30 million years) suggests a highly specific structure with extreme requirements in terms of intrinsic stability and protein interactions (de Jong et al, 1993; Groenen et a/., 1994). This translates into an extremely slow accumulation of random mutations, rendering significant dimer stabilization most unlikely. jSy-Crystallins form a superfamily of homologous proteins which is possibly related to bacterial spore coat proteins and dormant proteins of primitive eukarya (e.g., Physarum). Their structural data suggest two gene-duplication steps from an ancestral protein folded as a "Greek key" (Fig. 5B). /3-Crystallin forms dimers of mixed aggregates, whereas y-crystallins are exclusively monomeric; both show highly homologous two-domain structures. Comparing the two topologies it is most suggestive to interpret the p dimers as the product of domain swapping and association of a y ancestor (Jaenicke, 1994). An important detail supporting the swapping hypothesis is that twothirds of the residues in the interfaces between the N- and C-terminal

243

PROTEIN FOLDING AND ASSOCIATION

A I Monomers

u Domain-swapped dimer

a:a]=

III Stable domainswapped dimer

FIG. 5. Domain swapping hypothesis (Bennett et al, 1994, 1996). (A) The monomer (I) with its circle and square domains possesses a primary interdomain interface which may participate in interchain interactions, generating domain-swapped dimers (II). Stabihzing mutations in the Unker region (between the monomer domains) allow stable dimers (III) to be formed. (B) jSy-Crystallins as potential products of domain swapping. The ribbon diagrams illustrate the crystal structures of yB- and )8B2-crystallin, the C„ backbones (right) their superposition (yB, bold lines; /3B2, dashed lines), indicating the close structural homology of the two eye lens proteins (see Jaenicke, 1994).

domains in yB-crystallin and between the N- and C-terminal domains in different subunits in /3B2-crystallin are identical or closely conserved (Bax et al, 1990; Jaenicke, 1994; Trinkl et al, 1994; Mayr, 1995). Evidently, domain swapping is not restricted to dimerization: linear and cyclic higher oligomers can arise in both two or three dimensions. However, there are data that do not support the domain-swapping hypothesis: (i) three-dimensional domain swapping cannot explain heterooligomers because the subunits do not share common interdomain interfaces; (ii) there are oligomers in which domains share active sites at subunit junctions—in such cases more complex mechanisms must be involved in the evolution of the oligomeric state; and (iii) in the case of bovine seminal ribonuclease (BS-RNase) the dimeric swapping product of the N-terminal ends coexists, at equilibrium, with another

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RAINER JAENICKE

form in which no swapping occurs. This is the stable end product of oxidative renaturation, indicating that dimerization can occur without domain swapping (D'Alessio, 1995). Obviously, there are stable monomeric proteins such as RNase A, yB-crystallin, and diphtheria toxin in which the domain swapping ability is revealed only in particular circumstances, oligomeric proteins such as j8B2-crystallin in which domain swapping is an established structural feature and, finally, oligomeric proteins such as BS-RNase in which swapping is not the exclusive conformation and coexists in the native state with alternative structures in which no swapping occurs. F. Superstructures In the preceding text, the relationship of folding and association was discussed mainly considering dimers as the simplest possible case. However, successful attempts to reconstitute highly complex biological systems such as phage or giant multienzyme complexes have clearly demonstrated that in transferring from dimeric to multimeric structures no fundamental differences occur (Jaenicke, 1987a). At the highest level of protein organization we reach microscopic or even macroscopic assemblages. Again, there is no demon required in ascending from multimeric proteins to complex structures such as ribosomes or other "molecular machines" involved in signal transduction, polypeptide targeting, locomotion, cell adhesion, etc. Regarding the structural analysis, unpredicted progress in these fields has been accomplished thanks to technical developments in the fundamental methods, i.e., electron microscopy, X-ray analysis, and NMR. The greatest impact in this connection came from cryoelectron microscopy, synchrotron radiation, the perfection of the image plate, and the analysis of tractable structural elements. Within the limits of the reductionist program, "to detect all the nuts and bolts of life and to ascertain how they interact in the hierarchy from atoms to biology" (Erickson and Holmes, 1993), there are good prospects to fit the modules in order to gain insight into the structure-function relationship up to the level of some of the complex macroassemblages. Limited space restricts the following discussion to only a few representative examples: representative insofar as increasing numbers of subunits lead to a wealth of topological variants which might require specific assembly programs for their regulation. In this connection, the "molecular anatomy" of the cell provides us with more or less all platonean bodies as compact core structures. In addition, there are hollow cylindrical or helical rods and shells and, at the other extreme, one- and two-dimensional assemblies, as in the case of acetyl-CoA car-

PROTEIN FOLDING AND ASSOCIATION

245

boxylase and surface layers of bacteria. It would be a hopeless undertaking to present a comprehensive view of known structures. They are innumerable, as far as species and functions are concerned, and may gain additional complexity as a consequence of regulated covalent modifications such as proteolytic cleavage, glycosylation, phosphorylation, etc. For example, in the case of HIV reverse transcriptase, removal of the C-terminal RNase from one of the polypeptide chains transforms the native symmetrical form into a bifunctional asymmetrical form, with poljrmerase activity in the uncleaved precursor and RNase activity in the processed molecule (Kohlstaedt et al., 1992). In bacterial surface layers, chemical modification refers mainly to glycosylation, often followed by esterification of the carbohydrate moiety (Beveridge, 1994). A wide variety of genetic and biophysical techniques have been used to elucidate virus assembly pathways. For example, the use of threedimensional electron microscopy, i.e., the combination of cryoelectron microscopy with image reconstruction, has allowed the visualization of assembly intermediates as well as mature capsids. More detailed structural information has been gained by X-ray crystallography of whole (small) viruses or their parts. On the other hand, the production of virus-like particles and assembly intermediates using recombinant genes and their mutants has been a fruitful approach in investigating virus or phage assemblies in atomic detail (Stewart and Burnett, 1994). An example of this approach will be discussed in connection with the in vivo and in vitro assembly of the tailspike protein of Salmonella bacteriophage P22 (see below). In the following, four examples will be selected: (i) the homomultimeric hollow-shell assembly of horse spleen apoferritin, (ii) the heteromultimeric pyruvate dehydrogenase multienzjrme complex from B. stearothermophilus, (iii) bacterial and eukaryotic two-dimensional surface layers, and (iv) the ribosome as a highly complex conjugated system. 1. APOFERRITIN

Apoferritin, the major iron storage protein in eukaryotic cells, is composed of 24 structurally equivalent subunits in F432 symmetry. In the cage-like shell of the protein (Mr 476,000) each subunit makes contact with five neighbors. The predominant structural units of the rhombic dodecahedron are dyad-related subunits that constitute one rhomb face. The apices of the rhombs touch at the threefold and fourfold axes of the molecule. Interactions around these axes result in two different channels that penetrate the protein shell (Ford et ai, 1984; Crichton, 1990, 1992; Andrews et al, 1992).

246

RAINER JAENICKE

From the crystal structure of the native protein, high stabiHty of the symmetrical dimer has been predicted. Thus, further assembly of the complete protein shell may either proceed by the polymerization of the dimeric protein or it may involve structured monomers, dimers, and multiples of trimers as intermediates. As shown by chemical crosslinking, intrinsic fluorescence, and dichroic absorption, the second alternative is observed in reconstitution experiments starting from guanidine-denatured monomers. Significant assembly intermediates are Mi, M2, M3, and M12, in agreement with predictions based on the crystal structure of the protein (Gerl et al, 1988). The renatured protein is indistinguishable from the native starting material in its structural and functional properties (Gerl and Jaenicke, 1987a,b). 2. PYRUVATE DEHYDROGENASE COMPLEX

To illustrate the concept of superstructures as the sum of structural elements or modules, the pyruvate dehydrogenase multienzyme complex may serve as an example. The enzyme from B. stearothermophilus is one of the largest cell particles currently known (Mr 10^). Therefore, its in vitro reconstitution has been a challenge as a limiting case in which accessory proteins might become indispensable for structure formation. The enzyme consists of ~240 subunits made up of four different polypeptide chains. The acyltransferase (E2, 57 kDa) forms the inner core to which the pyruvate decarboxylase (Ela, 42 kDa, and El)8, 36 kDa) and the dihydrolipoamide dehydrogenase (E3, 54 kDa) are bound as peripheral homodimers. They are linked by highly flexible polypeptide segments of E2 which might be the reason why the complete molecule resists crystallization. Except for El, all constituent parts of the complex have been determined with respect to their structure and catalytic mechanisms. In this connection, E2 is of special interest because it combines at least six functions in a modular arrangement: core formation, binding of Ela, El)3, and E3, coupling of peripheral activities by the swing arm of the lipoyl groups (allowing the respective substrate to "visit" the peripheral catalytic centers) and, flnally, catalysis of the acyl-transfer reaction. Insight into the structure-function relationship was gained from (cryo-) electron microscopy, ultracentrifugation, X-ray analysis, NMR, and other spectroscopies apart from genetic engineering. A variety of homologous enzymes, domains, and fragments were used to "construct" the structure from the established substructures of its modules (Perham, 1991; Mattevi et al, 1992). Given the high degree of complexity, one would expect that the formation of the active multienzyme system needs some kind of cellular machinery. However, complete denaturation of all four components in

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guanidinium chloride and subsequent reconstitution was found to show "intrinsic form determination," i.e., the separate polypeptide chains have the capacity to undergo in vitro folding and association without requiring accessory proteins or morphopoietic factors (Jaenicke and Perham, 1982). In this context, two observations deserve mentioning. First, the quaternary structure of the 2-oxoacid dehydrogenases does not exhibit the regularity commonly observed in protein assemblies: the ratio of E1:E2:E3 is not fixed, i.e., E l and E3 do not precisely correspond to the symmetry of the E2 core. This result, which is clearly detectable by dynamic light scattering and boundary analysis in the analytical ultracentrifuge (R. Jaenicke, 1982, unpublished results), may render the structural requirements for the self-assembly process less stringent than those in other heterologous multimeric systems. Second, in the case of PDH from gram-positive bacteria and eukarya, a small (substoichiometric) amount of an additional component, "protein X," has been found which seems to be essential for the proper assembly and function of the multienzyme complex (de Marcucci and Lindsay, 1985; Jilka et al., 1986). The structure and function of this additional component are still enigmatic, especially considering that the B. stearothermophilus enzyme, with its still higher complexity, obviously can do without protein X. 3. SURFACE LAYERS AND CELL COATS

Microorganisms have developed an enormous variety of surface structures, from simple homomultimeric monolayer lattices to highly complex multilayer structures, often consisting of unusual polymers apart from proteins (Beveridge, 1994). Whatever their symmetry or their chemical and structural principles, microbial surface layers must be amenable to expansion (i.e., self-assembly) in order to allow the cells to grow and divide. The dynamics of the turnover between resident wall constituents and new building blocks are potentiated in cases in which cell motility requires a high degree of flexibility, as in the case of trypanosomes. Because bacteria have no cytoskeleton, the wall must somehow determine the shape and size of the cell. The synthesis of the peripheral proteins occurs in the lumen of the cell so that the turnover altogether involves translation, translocation, and targeting to the final destination where association to the preexisting lattice takes place. This mechanism implies that nucleation events that have been observed in vitro (Jaenicke et al., 1985) are unimportant for the assembly process in situ. The surface layer of the cell envelope of B. stearothermophilus consists of a regular array of one unique glycoprotein (with a helical glycan

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chain with 12 rhamnose residues per turn). Solubihzation in strong denaturants and subsequent dialysis or dilution allow complete reconstitution without the requirement of any specific interaction with a homologous or heterologous support. Thus, the reattachment of a monomer to its neighbor in the growing lattice is determined exclusively by the directionality of the intersubunit interactions without requiring extrinsic factors. The time course of the assembly is multiphasic with a fast initial phase and slow consecutive second-order reactions. The fast precursor reaction may be attributed to the formation of oligomeric intermediates consisting of 12-16 subunits which, during the slow phase, merge and "recrystallize" into the final native S-layer structure (Jaenicke et al, 1985). A similar kinetic mechanism has been observed for the heavily sulfated cell surface glycoprotein from Halobacterium halobium) (Hecht et al., 1986). The variable surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei is arranged as a coat consisting of about 10^ molecules that entirely covers the surface of the trypanosome. This coat shields the parasite from its environment but also triggers a perpetuated immune response of the mammalian host. The VSGs are anchored in the membrane by a dimyristoyl-phosphatidyl-inositol residue that is linked to the C terminus via a glycan moiety and an ethanolamine residue. Upon lysis of trypanosomes, the anchor is cleaved by a membrane-bound phospholipase C that converts the membrane form, mfVSG, to the soluble sVSG protein. sVSG exists in solution as a dimer with a monomer mass of about 60 kDa. The length of the polypeptide chain varies between 450 and 500 amino acids and the carbohydrate content varies between 7 and 17% by weight among different VSG variants. The three-dimensional structure of VSG has been solved to high resolution (for reviews see Blum et a/., 1993; Overath et a/., 1994). In the present context, a number of questions wait for answers: How does the tight packing of the surface coat correlate with the Trypanosoma type of locomotion? What are the intermolecular interactions involved in the localization of the surface protein in the coat? What role does the membrane anchor play in the arrangement of the membrane form of the glycoprotein in the surface coat? In considering the solution properties of the sVSG dimer, no selfassociation can be observed even at protein concentrations corresponding to the local packing density in the coat; thus, the close packing does not interfere with the flexibility of the coat required for locomotion. Applying the Langmuir-Blodget technique, no integration of the sVSG molecule into the lipid-water interface is detectable. Obviously, the two-dimensional arrangement of the protein in situ is determined by

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hydrophobic interactions of the Hpid components rather than proteinHpid interactions. In contrast to sVSG, the membrane form of the protein tends to aggregate so that coat formation cannot be simulated in vitro. The reconstitution of the soluble form follows the sequential uni-bi molecular folding/association mechanism discussed earlier (see Section III,E) (Rehaber et aL, 1990, 1991). From the given examples it becomes clear that form determination in protein assemblies depends exclusively on specific weak interactions between well-defined contact areas of the subunits involved. Polymorphism (often observed in assembly systems) may be related to conformational changes extending from tertiary to quaternary interactions. In complex assembly systems containing various protein species, a whole hierarchy of interactions of varying bond strength is involved in subsequent assembly steps. Evidently, energetics and kinetics become equally important in structure formation, as subunit association may lower the activation energy of subsequent rate-limiting steps. At the same time, previous steps on the pathway may lower the free energy, "guiding" the growing assembly into its free energy minimum. In this context, the tailspike assembly of Salmonella bacteriophage P22 may be considered a well-established model (Mitraki & King, 1992; Seckler and Jaenicke, 1992; Banner et al., 1993; Beissinger, 1994). 4 . RiBOSOMES

The amazing autonomy of structure formation documented by the previous examples cannot be generalized. For conjugated heterologous systems, it has been shown that either morphopoietic gene products or specific sequences aid in accomplishing unperturbed assembly. For example, phages may need scaffolding proteins, and cell organelles such as the ribosome show "assembly gradients" provided by the rRNA during transcription and translation (Franceschi and Nierhaus, 1990; Nierhaus, 1990, 1991). In vitro, these factors can be simulated by specific solvent conditions or reaction sequences comparable to a conveyer belt assembly; in vivo, at this point, both genome organization and accessory proteins play a major role. Attempts to simulate the in vivo self-organization of ribosomal particles by optimizing the interplay of salt conditions, pH, temperature, and protein components clearly showed that intermediate particles are formed along the folding/assembly path. Different classes of proteins were distinguished according to their interactions either with specific sites on the rRNA or with other ribosomal proteins. The sequential incorporation of the various components into the intermediate particles and their combination to the functional ribosome has been summarized

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in "assembly maps," providing an idea how the parts become organized in their specific topographical order. In differentiating primary, secondary, tertiary, etc. binding, it turns out that the sequence of the in vitro assembly (reflected by the increase in the distance of the various proteins from the RNA) is inverse to the sequence in which the proteins are removed by increasing LiCl concentrations. Figure 6 illustrates the mutual interactions of the proteins and the sequence of their integration into the E. coli 508 ribosomal subunit. As indicated by the different arrows in Figure 6, L3 and L24 serve as initiator proteins, thus generating a more stringent pathway along a sequence of decreasing "cooperativity." Apart from L3 and L24, the first intermediate [Rlgg] contains about 20 proteins, only 5 of which are absolutely required for the further conversion and assembly steps. Most of these proteins are clustered near the 5' end of the 23S RNA, suggesting a "cotranscriptional assembly gradient" in the sense that assembly already starts while the ribosomal RNA is still being synthesized. Thus, in vivo the progress of RNA synthesis determines the progress of assembly and its specific pattern (Nierhaus, 1982, 1991; Herold and Nierhaus, 1987). From the

proteins essential fo''Rl5o(1)fbnnation

proteins essential for 5S TRHA integration

FIG. 6. Assembly map of the 508 ribosomal subunit from Escherichia coli. The zones (from botton to top) indicate the protein families which can be split off consecutively with increasing LiCl concentrations (1.3-4.0 M). For details see Nierhaus (1982, 1990, 1991) and Herold and Nierhaus (1987).

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point of view of the conformational entropy, this means that there is an important difference between self-assembly in vivo and in vitro where 32 proteins and the whole RNA chain are involved. This difference may explain the time and temperature requirements of the in vitro approach (90 min, 50°C) compared to the much higher in vivo efficiency (<2 min, 37°C). It is important to note that there is a correlation between the assembly map and the organization of genes for ribosomal proteins within certain operons (Rohl and Nierhaus, 1982). As a consequence, one may assume that transcriptional units lead to specific, genetically determined complexes which are subsequently incorporated into the intermediate particle. As in the case of modular folding, this assembly by parts would accelerate the assembly process and increase its cooperativity. A similar mechanism has been observed for the assembly of bacteriophages and viruses. From the point of view of the three-dimensional structure of the final product of assembly, reconstitution yields the fully functional state of the ribosome. Exploiting cryoelectron microscopy and diffraction-based image reconstruction procedures, as well as crystallographic and NMR data, fascinating progress has been made in the elucidation of its "hollows, voids, gaps and tunnels" (Yonath and Berkovitch-Yellin, 1993). Because the size of the object is far beyond the limits of common diffraction techniques, again the modular approach has to be applied. Despite the exceedingly large number of modules, the declared goal is the complete X-ray crystallography at <3A resolution; considering the technical developments within the foreseeable future, there are good reasons to believe that this goal is not too Utopian (Moore, 1995). As experience with other superstructures shows us, the step from structure to function is another matter. At the descriptive level, the ultraanatomy of molecular assemblies such as the cytoskeleton or myosin and many components of the extracellular matrix or the basal membrane and their respective cellular substructures have been analyzed in great detail. However, when it comes to the energetics, kinetics, and chemical mechanisms involved in their function, what commonly remains are circles, squares, arrows, and (at best) question marks. Actomyosin has the longest history. Its chemical, kinetic, and physiological characteristics have been studied at the molecular level for more than 50 years. However, even after the structures of the components have been determined at high resolution, all that can be done is just "to put forward a hypothesis on how myosin might function as a molecular motor" (Rayment and Holden, 1994). One should use the same caution in discussing the cellular aspects of protein folding.

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G. Off-Pathway Reactions 1. THERMODYNAMICS VERSUS KINETICS

The problem of off-pathway reactions in the process of protein folding is closely related to the long-standing questions, is the folded structure determined by its thermodynamic stability (Anfinsen, 1973) and how is it possible for a polypeptide chain to fold up along a pathway that allows rapid folding and nevertheless arrive at the thermodynamicly most stable structure (Wetlaufer and Ristow, 1973)? The compromise may be a consequence of natural selection, insofar as evolution may have selected sequences t h a t have the ability both to fold rapidly and to arrive at thermodynamically stable structures (Baldwin, 1994; see the Monte Carlo simulations in Sali et al, 1994a,b). However, there are two challenges to this hypothesis: (i) proteins uncapable of reversible unfolding/refolding, and (ii) proregion-dependent folding of (pre-)proproteins or proenzymes. Three examples may illustrate the first point, (i) Certain serpins exist in two different folded conformations in which the biologically active form appears to be the metastable form occurring as a highly populated kinetic on-pathway intermediate (Carrell et al., 1991). (ii) The same kind of competition between different folding pathways has been reported for bacterial luciferase. In this case the folding polypeptide chain can assess more than one conformation corresponding to different energy minima out of which the protein is unable to escape under normal physiological conditions. Whether the biologically active al^ dimer is at the global free energy minimum or whether it is trapped in a local minimum by a high-activation energy barrier cannot be answered. Its formation requires both the a and jS subunits to fold within the same cell at the same time. The reason is t h a t ^ has two options as it folds, formation of the active heterodimer (ajS) or the inactive homodimer (/32); both are separated by an activation barrier sufficiently high to cause kinetic partitioning. Folded a and j8 subunits cannot assemble into active enzyme due to the fact t h a t the stability of ^2 exceeds the stability of a)3 (Sinclair et a/., 1994). (iii) The third example refers to the kinetic partitioning between the assembly and aggregation of the tailspike protein (TSP) of bacteriophage P22 from Salmonella typhimurium. TSP is a homotrimer which is noncovalently bound to the neck of the virus capsid and essential for phage adsorption to the bacterial host. The protein has served as a model system for the folding and assembly of large multisubunit proteins. Its folding pathway comprises subunit folding, followed by the formation of a protrimer in which the chains are stably associated but not fully folded; the final shuffling

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reaction from the protrimer to native TSP is the rate-Hmiting step. Along the whole reaction sequence, off-pathway aggregation competes with proper folding and association. Numerous mutants with altered partition ratios between the two competitive processes have been isolated and characterized, with the general result that the physicochemical properties precisely complement the genetic analysis (Goldenberg and King, 1982; Mitraki et aL, 1991; Fuchs et a/., 1991; Mitraki and King, 1992; Danner c^aZ., 1993; Beissinger, 1994). The high-resolution structure of the N-terminally shortened protein (Steinbacher et aL, 1994) allows both the folding mechanism and the stability of the protein to be explained (Beissinger et aL, 1995). In connection with the effect of the pro region of c3rmogens on the final structure of the processed molecules, recent work has shed some light on the mechanism by which pro regions facilitate folding. As has been shown, protein folding in general involves a kinetic competition between on-pathway reactions leading to the native state and nonproductive pathways leading to aggregation. In this context, pro regions could function either by increasing the rate of the forward folding reaction or by decreasing the rate of aggregation. Chaperones function by suppressing protein aggregation (see below). In contrast, pro regions function by directly increasing the rate of the forward folding reaction. They are required for folding under conditions in which off-pathway reactions are suppressed. In addition, they interact strongly with the product of the folding reaction, i.e., the native state of the processed protein (Ohta et aL, 1991; Winther and Sorensen, 1991; Baker et aL, 1992b). For example, denatured a-lytic protease does not fold to the native state in the absence of the pro region. Instead, upon removal of the denaturant, the protein folds to a stable intermediate with substantial secondary structure but little organized tertiary structure; upon addition of the pro region, the intermediate is rapidly converted to the native state (Baker et aL, 1992a). In this case, because folding competence is maintained for an extended period of time, off-pathway reactions are negligible. Instead, the intermediate appears to be kinetically trapped, and the pro region seems to function by directly reducing the free energy of the rate-limiting barrier which blocks access to the native state. Similar observations have been reported for subtilisin, proteinase A, and carboxypeptidase Y (Eder et aL, 1993; Strausberg et aL, 1993; van den Hazel et aL, 1993; Winther et aL, 1994; for review see Baker and Agard, 1994). 2. PRODUCTIVE VERSUS NONPRODUCTIVE PATHWAYS

As has been mentioned, there are three stages at which side reactions on the folding path may compete with proper folding and association

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of proteins: the hydrophobic collapse, the merging and swapping of domains, and the docking of subunits (Scheme 2).

^

s

Wrong local structures during (re-)folding (Goldenberg, 1992a)

Wrong domain pairing (Jaenicke, 1987a, 1988)

Kinetic partitioning N <- U ^ Aggregates (Goldberg & Zetina, 1980)

SCHEME 2. Off-pathway process competing with correct folding and association.

An example of the first stage was discussed in connection with the initial phase of cytochrome c folding where transient interactions of nonnative and native amino acid ligands to the heme iron were found to precede correct folding; cases illustrating the second and third stages are wrong domain pairing in octopine dehydrogenase, on one hand, and inclusion body formation, on the other. At all three levels, correct folding requires specific substructures to be preformed in order to proceed on the correct folding path. Collapse and domain merging involve intramolecular rearrangements. Due to the high local concentrations of the reacting groups, they are not significantly affected by neighboring molecules, i.e., they obey first-order kinetics with the slowest isomerization reaction determining the overall rate. In the case of domain proteins, the relative stabilities of the domains and the contributions of the domain interactions to the overall stability are crucial. The significance of the linker peptide connecting two well-defined domains has been studied by grafting experiments, e.g., by mutually exchanging the linkers of )S- and y-crystallins (E.-M. Mayr et ai, 1994; Trinkl et al., 1994). In both transplants, domain contacts dominate over subunit contacts. At concentrations up to 0.5 mM, the recombinant separate domains do not interact with each other, which stresses the above local concentration argument. Studies on domain recognition and the folding and association of nicked subunits (i.e., separate domains) made use of octopine dehydrogenase (ODH) and lactate dehydrogenase (Zettlmeissl et al, 1984;

PROTEIN FOLDING AND ASSOCIATION

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Opitz et al, 1987; Teschner et ai, 1989). The fact that after careful optimization in both cases the yield of reactivation is far below 100% illustrates the significance of side reactions. In the case of the monomelic ODH, kinetic partitioning upon reactivation yields 70% native ODH plus 30% inactive protein with native-like secondary structure, but increased hydrophobicity, according to U ^^ IN-^

i

/;

N

(70%) (30%)

[6]

where U, IN, and N are unfolded state, a common intermediate, and native enzyme, whereas IN is irreversibly denatured as a consequence of wrong domain interactions (and aggregation). Repetitive denaturation/ renaturation experiments yield 70% N per cycle so that after two cycles only 49% of the original activity is left (Jaenicke, 1987a, 1988). Obviously, it is kinetic partitioning rather than heterogeneity of N which governs the overall reactivation mechanism. In going from single-chain domain proteins to protein assemblies, kinetic competition of first-order folding and second-order association becomes important as soon as the protein concentration reaches the level where folding becomes rate limiting (Zettlmeissl et al., 1979) (Fig. 7A). The reason for this is that subunit assembly requires the monomers to be close to their proper conformation before they coalesce to form the native quaternary structure. If folding intermediates expose wrong contact sites, they will give rise to aggregation instead of association. Accordingly, not only a shift in the kinetic mechanism but also a decrease in yield of active protein will be observed. The occurrence of inclusion bodies rather than soluble protein in overexpressing recombinant genes illustrates the consequence. The underlying kinetic mechanism n^ —> nM' —> nM —> M„

i

i

[7]

resembles the previously discussed sequential uni-bi molecular mechanism which represented the limiting case at high dilution. With increasing protein concentration, when ki determines the overall rate, the limiting value for ^2 will correspond to diffusion-controlled association. Actually, Kiefhaber e^ al, (1991) were able to demonstrate that aggregation (inclusion body formation) [7M-» A, in the process of protein reconstitution can be quantitatively described by the simple kinetic partitioning equation: N <- UM^ A (Fig. 7B). As indicated, the molecular basis

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LDH FIG. 7. Kinetic competition of reconstitution and aggregation. Porcine muscle lactate dehydrogenase after denaturation in 0.1 M H3PO4, pH2.0, and renaturation at pH 7.0 and 10°C. (A) Partitioning between reactivation ( • ) and aggregation (A). The far-UV circular dichroism 6222 nm (D) reflects the helicity in the transition range (Zettlmeissl et ai, 1979). (B) Concentration dependence of the yield of reactivation (see data in A) calculated based on the competition mechanism A^ according to y ^ D,k2

k.

U

K-^)]

where y is the yield (%), Do is the concentration of denatured protein at zero time, and ki and k2 are the velocity constants of rate-limiting first- and second-order steps (Kiefhaber et aL, 1991).

of the kinetic trapping is simply that the polypeptide chains do not differentiate between intra- and intermolecular interactions. If this explanation is correct, and if the native protein is in a lower energy minimum than the aggregates, it should be possible to shuffle aggregates back to the native state by adding weakly destabilizing agents. In fact, it has been shown that the yield of reconstitution may be improved, e.g., in the presence of moderate concentrations of arginine or urea (Rudolph, 1990) (see below). Considering Eq. [7], three questions need to be answered: (i) what is the committed step in aggregate formation, i.e., at which stage along the sequential reaction are aggregates formed?; (ii) when is the structured monomer committed to end up as the native protein?; (iii) what is known about the structure of aggregates and their constituent polypeptide chains? With respect to the first two questions, commitment to aggrega-

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tion was shown to be a fast reaction, whereas the kinetics of the commitment to renaturation follow precisely the slow kinetics of the overall reaction. This means that there are fast precursor reactions on the folding path (collapsed state) all of which still allow aggregation, whereas, after a certain intermediate has been formed, slow shuffling leads "one way" to the native state (Goldberg et al, 1991). Regarding the structure of aggregates, electron microscopy and circular dichroism indicate that wrong subunit interactions give rise to irregular networks with a broad distribution of highly structured particles at least 10 times the size of the native proteins. They resemble the native protein in its spectral properties, as far as perturbations by turbidity allow a quantitative analysis (Fig. 7 A). Inclusion bodies may represent surprisingly homogeneous precipitates, often with less than a dozen components (including RNA). Whether disulfide bonds contribute to their compactness in the cell or whether they are preparative artifacts is still controversial. For structural characteristics of inclusion bodies, see Mitraki and King (1989), Bowden et al (1991), and Valax and Georgiou (1993). IV. Cellular Aspects A. In Vitro vs in Vivo Issue Aggregation in the cell causes inclusion body formation, probably for the same reasons that are responsible for the reaction in vitro: overexpression yields high local concentrations of folding intermediates which (due to kinetic partitioning) end up in precipitates instead of native protein. From this parallelism one may conclude that there is no difference between folding in vitro and folding in the cell, except that overexpression might be considered unphysiological and, therefore, atypical for the standard cell. In this context, it is important to note that the common assumption that under normal physiological conditions structure formation in vivo yields 100% native protein is incorrect. There is clear evidence that there is misfolding and misassembly in the cell (Pelham, 1989; Mitraki and King, 1989; Helenius et al, 1992). Obviously, secretion or trafficking through the ER and Golgi and subsequent specific degradation provide an inherent quality control, leading to an apparent yield of 100%. Thus, the recovery of refolding does not represent a significant difference between structure formation in vitro and in vivo. The same is true for the directionality of protein biosynthesis from the N- to the C-terminal end in the cell which is in contrast to the integral folding of the complete polypeptide chain in vitro for two reasons: (i) because of nascent chain-binding protein and chaperones, folding in vivo does not necessarily occur cotranslation-

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ally in a vectorial fashion (see below); and (ii) the product of renaturation has been shown to be indistinguishable from the native starting material using all available biochemical and physicochemical criteria, including high-resolution X-ray analysis:!:. Obviously, factors such as local concentrations within specific cell compartments, cytosolic solvent conditions, cotranslational and posttranslational modification, and transcriptional or translational control, do not play significant roles in the folding process. There might be many reasons for this, one being the previously mentioned folding-byparts mechanism which takes care of the concerted folding of the protein in discrete segments of its polypeptide chain. Another reason could be that, although structure formation is assumed to start early during translation, the final search in the configurational space starts only after the release of the polypeptide from the ribosome. There are numerous examples, from small proteins to large multimeric systems, which show that the chain has neither to be complete nor unmodified. Both tertiary structure formation and subunit association tolerate a wide range of variations with respect to sequence and chain connectivities: circular permutations of parts of the sequence, chain extensions, covalent joining of subunits, fragmentation, derivatization, hybridization by peptide interchange, etc. (Jaenicke, 1993a). As has been conclusively shown by Privalov (1994), certain core regions of a protein determine the overall topology, whereas peripheral parts of the protein may be altered or even lacking. The given arguments do not consider the time requirements: nascent proteins acquire their native structure with half-times in the minutes range (at most), whereas refolding rates may vary in a wide range from seconds to days. The reason for this is that optimum conditions regarding the yield of refolding do not necessarily represent optimum conditions for the folding rate (see Section III,C,4). In addition, in the cell, accessory proteins are involved in regulating and/or catalyzing the rates of folding and association. That they assist rather than direct folding is generally accepted (see below). To date, protein folding in the cell has been inaccessible to a detailed analysis of its kinetic mechanism and structural intermediates on its t For this reason, renaturation experiments have been the method of choice to mimic protein folding. Because of the structure-function relationship of proteins, the biological activity has commonly been used to probe "renativated" proteins. That reconstituted enz3nnes often exhibit activities exceeding the activity of the starting material is caused by the fact that the denaturation-renaturation cycle represents an additional purification step. The alternative hypothesis that proteins form multiple stable states with varying functional properties is not supported by sound experiments.

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folding pathway. Thus, it is still unresolved whether refolding polypeptides in vitro proceed via the same pathway as nascent chains released from the ribosome within the cell. In vitro translation experiments in cell-free systems and detailed in vitro refolding studies seem to indicate that, in the case of the tailspike endorhamnosidase from Salmonella bacteriophage P22, the acqusition of the native structure closely resembles the self-organization of the protein in vivo. The rate-determining folding reactions occur with identical rates both in the test tube and in the bacteriophage-infected cell (Fuchs et a/., 1991; Mitraki and King, 1992; Bannered al, 1993; Beissinger, 1994). In the case of firefly luciferase, in vitro translation shows that the ribosome-bound polypeptide chain is essentially inactive; activity appears within a few seconds after release of the enzyme from the ribosome. In contrast, the reactivation of denatured luciferase under comparable solvent conditions occurs within a half-time of 14 min (Kolb et al., 1994). These results support the idea that the nascent polypeptide chain folds cotranslationally, corroborating earlier results for immunoglobulin chains (Bergman and Kuehl, 1979a,b,c; Peters and Davidson, 1982). A beautiful example, illustrating the potential of in vitro studies close to in vivo conditions, has been the investigation of the folding kinetics of two luciferases after synthesis in reticulocjrte ly sates (Kruse et al, 1995). The monomelic and a dimeric bacterial enzymes, Lux AB and Fab2, provide a unique system for studying folding and association in a cell-free translation system because they eliminate the need for the common use of amino acid analogs in order to monitor truncated pol3rpeptide chains. A number of significant observations deserve to be mentioned: (i) synthesis, folding, and association can be separated along the time scale; (ii) due to the bimolecular nature of LuxAb association, the appearance of enzymatic activity is sensitive to dilution; (iii) folding of both enzymes is assisted by molecular chaperones and, thus, ATP dependent; (iv) sensitivity to PPI inhibitors proves proline cis-trans isomerization to be involved in the folding reaction. The latter finding is most important, because it provides direct evidence for the significance of peptidyl prolyl isomerase in protein biogenesis in the eukaryotic cytosol (see below). In summary, the resulting three-dimensional structure is the same in vitro as well as in vivo. Differences refer to the kinetics of folding and association and to the partitioning between folding/association, on one hand, and aggregation on the other. Shifts of the corresponding partition coefficient are caused by the presence of helper proteins or accessory proteins in the cell. They do not affect the "central dogma" that the three-dimensional structure of proteins is determined by their

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amino acid sequence. Thus, the question remains: What are the impHcations of the differences regarding the mechanism of protein folding within and without the cell? To answer this question, three topics need to be discussed: cytosolic solvent parameters, folding catalysts, and molecular chaperones. B. Cytosolic Solvent Parameters

In contrast to the alchemy of solvent variations which has been worked out in order to optimize the in vitro renaturation (renativation) of recombinant proteins, the solvent conditions in common mesophilic cells vary only in a narrow range which is insignificant in connection with protein folding and protein association. On the other hand, in the biosphere, life faces a wide range of extreme conditions which require adaptation of extremophilic organisms to temperatures and pressures up to the boiling point of water and 120 MPa (—1200 atm), as well as water activities as low as 0.6. With respect to proton concentration, the cell works as a pH stat close to neutrality, even in the case of extreme acido- or alkalophiles (Jaenicke, 1991a). From this, two conclusions may be drawn: (i) regarding the structure-function relationship, the entire cell inventory (including precursors on the respective pathways of structure formation) and the balance of the metabolic network have to be adapted to cope with extreme conditions; (ii) in connection with protein folding and association, expressing recombinant proteins from extremophiles in mesophilic hosts, or vice versa, may provide information with respect to the impact of solvent parameters on protein self-organization. They allow limits to be defined on how far conditions may be varied in in vitro experiments. Regarding the intrinsic stability of intermediates, it is obvious that early steps on the folding pathway are most accessible to deleterious solvent effects. Thus, in the case that the native structure can either be expressed in the cell or refolded after preceding denaturation, intermediates and subunits must be stable under the respective in vivo or in vitro conditions. It turns out that alterations in the folding conditions often have surprisingly little effect. A drastic example is the expression of active enzymes from the hyperthermophilic bacterium Thermotoga maritima in E. coli in which the temperature difference between host and guest amounts to ~60°C (Tomschy et al, 1993; Beaucamp et al., 1995). After complete denaturation, e.g., of glyceraldehyde3-phosphate dehydrogenase, renaturation at 5-100°C yields the fully active protein indistinguishable from its initial native state. From this we conclude that all folding intermediates, from the structured monomer and dimer to the tetramer, must be stable over the whole temperature range (Rehaber and Jaenicke, 1992). Interestingly, at 0°C, an

PROTEIN FOLDING AND ASSOCIATION

261

inactive folding intermediate becomes trapped, confirming earlier observations on the moderately thermophilic pyruvate dehydrogenase from J5. stearothermophilus (Jaenicke and Perham, 1982). In both cases, no cold denaturation occurs. Instead, the trapped intermediate is native like, in the case ofThermotoga GAPDH even tetrameric, with its secondary structure fully restored. Increasing the temperature by a few degrees is sufficient to compensate the activation energy necessary to reach the native state (Schultes and Jaenicke, 1991). Little is known with respect to other solvent parameters. As mentioned previously, the cytosolic pH is close to neutrality, even in the case of acidoand alkalophiles. Therefore, in this respect, in vitro refolding conditions do not differ widely from those within the cell. In the case of halophilic proteins, the few proteins that have been investigated in greater detail require salt, not only as stabilizing agent but also for folding and assembly (Hecht and Jaenicke, 1989; Eisenberg et al., 1992; Cendrin et al, 1993; Dym et al., 1995). For nonhalophilic proteins, the ionic strength is not as critical, apart from the well-known stabilizing/destabilizing properties and other Hofmeister effects that may be used to optimize the folding conditions. What may be crucial is the effect of specific structurally or functionally essential ions, either in metalloenzymes or in proteins stabilized by other cations or anions. Such individual requirements can easily be quantified in vitro, thus allowing the cellular situation to be mimicked (Jaenicke, 1987a). Regarding effects of hydrostatic pressure on cellular processes, experiments have focused mainly on the dissociation/association and deactivation/reactivation of oligomeric enzymes (Jaenicke, 1987b). Except for pressure effects on the cis-trans isomerization of proline, no systematic folding experiments have been performed so far (Hauer et aL, 1982). Considering the minute effect (-30% per 100 MPa and proline residue), and the insignificant pressure dependence observed for other biochemical reactions, it is obvious that the adaptive effort to cope with high pressure can be ignored compared to the adaptation to other parameters, e.g., the low temperature prevailing in the deep sea (Gross and Jaenicke, 1994). Given the high cellular concentrations of biopolymers, the cytosol is highly viscous. In order to simulate the frictional characteristics, glycerol or carbohydrates were applied. In connection with protein folding, information is scarce. A decrease in the rate of reactivation of monomelic octopine dehydrogenase was attributed to the deceleration of domain merging (Teschner et ai, 1989). However, the interpretation is ambiguous because polyols not only increase the viscosity but also stabilize proteins; there is no way to alter the viscosity alone. In vivo, the various cellular components known as compatible solutes and pro-

262

RAINER JAENICKE

100<

^
r

0)

0)

o o

_D

0)

cr

50

\^

^O) _J

•u^

•i

n

A

J

Q I OD

A A A

/Lr-fPK

0_ii-M«(?!0VA1 50 60 Temperature (°C)

2_^CaZJ 70

10 c (^ig/ml)

0

20

^0 60 Time (min)

80

100

FIG. 8. Effect of 0-glycosylation on the stability and folding of invertase. Denaturation/ renaturation of internal, core-glycosylated, and external invertase from Saccharomyces cerevisiae, with 0, 34, and 65% carbohydrate content. (A) Thermally induced unfolding of internal ( • ) , core-glycosylated (O, • ) , and external enzyme (A, A): 10 /Ag/ml in 50 mM acetate buffer, pH 5.0, heating rate 0.25°C/min. Open symbols: light scattering at 500 nm (arbitrary units); closed symbols: percentage relative fluorescence at 325 nm (X.exc = 280 nm). (B) Influence of protein concentration on the reactivation and aggregation: 50 mM acetate buffer, pH 5.0, 20°C; symbols as in A. (C) Reconstitution kinetics of core-

PROTEIN FOLDING AND ASSOCIATION

263

tectants may be assumed to exert the same effect. Its physical basis in terms of excluded volume and preferential binding has been most elegantly elucidated by Timasheflfand co-workers (Timasheflf and Arakawa, 1989). Glycoproteins are worth mentioning in connection with the impact of polyols on protein folding. The role of the carbohydrate moiety as a protectant, e.g., against proteolysis, has long been assumed. Evidently, in order to gain importance, a sufficiently high degree of glycosylation is required. In the case of ribonuclease B (the 6% mannosylated form of RNase A), the two forms of the enzyme do not differ either in stability or in the kinetics and mechanism of folding (Krebs et al., 1983). In the case of invertase from yeast, where a wide range of glycosylation levels is available, the "internal," "core-glycosylated," and "external" forms of the enz3mie (with 0, 34, and 65% carbohydrate, respectively) differ significantly in their stability, quaternary structure, and the tendency to aggregate: the glycomoiety as solubilizing component inhibits aggregation, e.g., by heat. However, again there is no effect on the kinetics and mechanism of folding (Fig. 8). In the case of the nonglycosylated, internal form of the enzyme, the lack of the covalently attached carbohydrate can be mimicked by high concentrations of polyols (Kern et al., 1992, 1993). C. Folding Catalysts As has been shown (Section III,C and E), there are three possible rate-determining steps in the self-organization of proteins: disulfide shuffling, proline cis-trans isomerization, and assembly. On the other hand, there are two enz3rmes, localized in the appropriate cellular compartments to catalyze the first two rate-determining steps and there is a whole machinery of chaperones and "cohorts" of helper proteins to assist the third reaction (Georgopoulos, 1992; Gething and Sambrook, 1992; Hendrick and Hartl, 1993; Morimoto et al,, 1994). Faced with a wealth of recent data and claims which seem to result in well-defined reaction cycles, one might assume that the enzyme mechanisms in-

glycosylated invertase (34% carbohydrate) at 20°C, followed by far-UV circular dichroism 0222 nm (deg/cmVdmol~^) (A), fluorescence emission at 325 nm (Xexc = 280 nm) (•) and enzymatic activity (open circles). Renaturation by 1:80 dilution of the 6 M GdmCldenatured enz5rme into citrate/phosphate buffer, pH 7. Subunit concentration during reactivation: 68 (large circles), 17 (medium circles) and 8.5 nM (small circles). Solid lines: simulations of consecutive uni-bimolecular kinetics with ki = 1.6 x lO'^sec"^ and k2 - 1.2 X 10^ Af-^sec-^ (see Kern et al, 1992, 1993).

264

RAINER JAENICKE

volved in folding catalysis, and the regulation of intra- and intermolecular interactions underlying chaperone action, were settled (Ellis and van der Vies, 1991; Ellis, 1992, 1993, 1994a,b,c; Hartl et al, 1992). However, both folding catalysis and chaperone action are far from being understood. Even the biological significance of the two enzymes, PDI (in E. coli DsbA) and PPI, as essential folding helpers has not been unambiguously established apart from the observation that both enZ5anes are abundant and ubiquitous in all organisms. In the case of PDI, the recent isolation of yeast mutants that are severely defective in disulfide-bond formation has confirmed that cystine cross-linking is facilitated in vivo (LaMantia and Lennarz, 1993). Similarly, in prokaryotes, mutations in the dsbA gene show a dramatic decrease in the rate of disulfide-bond formation in secreted proteins (Bardwell et al., 1991). Both findings are clear indications that in vivo disulfide-bond formation is a catalyzed process in both eu- and prokaryotes. Genes of both DsbA and PPIs have been isolated, and the three-dimensional structures of the enz3nnes determined at high resolution (for reviews see Bardwell, 1994; Fischer, 1994); it is to be expected that their mechanism will soon be understood. For chaperones, the prospects are less optimistic for several reasons: (i) some molecular chaperones form exceedingly large complexes; (ii) unlike their Victorian prototype, they act in groups (Pain, 1992); and (iii) their mechanism seems to differ for different protein substrates. However, the crystal structure of GroEL has been solved (Braig et al, 1994). In combination with electron microscopy (Saibil et al., 1993) this allows models to be tested, so that, even for systems the size of GroEL/GroES, a solid basis for mechanistic considerations will soon be available. 1. PROTEIN DISULFIDE-ISOMERASE

Disulfide bridges are essential for the stability of most extracellular proteins, to the extent that reduction of these bonds will, in many cases, cause unfolding. In vitro, disulfide bond formation commonly takes a long time, shifting the time range of renaturation from milliseconds, in the case of cystine-free proteins, to hours or even days (Rudolph, 1990; Sosnick et al., 1994; Gilbert, 1994). Thus, in the cell, the reaction must be catalyzed. The corresponding eukaryotic enz3rme, PDI, has been known for a generation (Goldberger et al., 1963; Venetianer and Straub, 1963; Freedman, 1992). However, only after the prokaryotic homolog, DsbA, and the corresponding dsbA gene became available could recombinant gene techniques overcome many of the experimental problems connected with the complex eukaryotic system (Bardwell et al., 1991).

PROTEIN FOLDING AND ASSOCIATION

265

PDIs catalyze the redox reaction of cystine/cysteine residues in proteins, as well as the reshuffling of disulfide bonds, depending on the accessibility of the target sites, and the redox conditions of the medium. The redox reaction occurs via a mixed disulfide and requires an external electron acceptor (Jaenicke, 1993a,b). Escherichia coli DsbA is only one component of an enzyme machine comprising at least three proteins: DsbA is the key catalyst donating an extremely reactive oxidizing disulfide to substrate proteins, DsbB serves to reoxidize DsbA, and DsbC exhibits reshuffling activity working synergistically with DsbA. The Dsb system is widespread in prokaryotes so that the following characteristics may be generalized to a certain extent. Dsb mutants are generally defective in disulfide bond formation outside the inner membrane. Apart from this common property, they exhibit pleiotropic phenotypes, simply because disulfide-bonded proteins are involved in a wide variety of cellular reactions (Bardwell, 1994). PDI, as well as DsbA show most unusual stability properties. Rather than having the usual stabilizing effect, the highly reactive disulfide destabilizes the structure of the protein (Hawkins et al., 1991; Lundstrom and Holmgren, 1993; Wunderlich and Glockshuber, 1993a,b; Wunderlich et al, 1993a,b; Zapun et aL, 1993, 1994). Both enzymes have strong oxidizing properties, consistent with their ability to form disulfide bonds in proteins. From the three-dimensional structure of DsbA (Martin et al., 1993a,b) it has become clear that the active-site cystine imposes a conformational strain on the structure of the enzyme, thus explaining the high reactivity of the disulfide as well as its destabilizing effect. Comparing the eukaryotic PDI and its prokaryotic homolog, DsbA, a number of similarities and differences are obvious. Corresponding to their origin, the two enzymes are localized in their appropriate compartments, PDI in the endoplasmic reticulum (ER) and DsbA in the periplasm. Both participate in a wide range of exchange reactions in vitro, including redox reactions and isomerization, depending on the redox conditions. Using glutathione concentrations similar to those in the ER, PDI accelerates the oxidative refolding of trypsin inhibitor (BPTI) and its proform (with 13- and 7-residue extensions at its N and C termini) in vitro approximately 40-fold. The rate enhancement in vivo is expected to be considerably higher due to the high local PDI concentration in the ER (Hwang et al, 1992; Creighton et al, 1993). DsbA catalyzes the oxidative refolding of a number of small substrate proteins (Wunderlich et al, 1993a; Kanaya et al., 1994; Zapun and Creighton, 1994). It appears to do so by a rapid and virtually unidirec-

266

RAINER JAENICKE

tional transfer of a disulfide from DsbA to its substrate protein, followed by disulfide isomerization (Eq. [8]). S HS /| \ DsbA I + P

\l

/

S HS

S— S / > DsbA

\

SH

\

/

HS

P

SH S / |\ > DsbA -f I P

\

1/

loj

SH S

As indicated, the transfer of the disulfide from DsbA to the folding pol3^eptide chain occurs via a mixed disulfide intermediate. This transfer is extremely fast due to (i) the extreme reactivity of the DsbA disulfide, (ii) the instability of the mixed disulfide, and (iii) the specific geometry of the active site of the enzyme which stabilizes the folded conformation of the reduced form of the enzjrme (Wunderlich et al, 1993a; Martin et al, 1993a; Zapun et a/., 1994). The reaction rate exceeds that of small disulfide reagents by several orders of magnitude. The reason is the extremely low pif value of ^3.5 observed for the cysteine residue (C30) involved in the first redox reaction. The catalytic efficiency of the enzyme in these experiments is evident: adding catalytic quantities of DsbA leads to rapid refolding under conditions in which no spontaneous refolding can be achieved in its absence. DsbA and PDI share the same active site sequence, C-G-H-C, the essential tetrapeptide involved in the catalytic SH/SS exchange which is also present in thioredoxin, and other bacterial protein disulfide oxidoreductases in bacteria (Loferer and Hennecke, 1994). Interestingly, the catalysis of disulfide formation is still operative in a mutant containing only a single active-site cysteine (Wunderlich et aL, 1993b, 1995). With respect to substrate specificity, inhibition studies with peptides of various lengths and sequences suggest that PDI has a rather broad peptide-binding capacity. The fact that, in the case of DsbA, reduced proteins react faster than the small molecule strong reductant dithiothreitol points in the same direction (Wunderlich et aL, 1993a); the crystal structure of DsbA shows that the active site of the enzyme is surrounded by deep grooves and a hydrophobic patch, features also suggestive of peptide-binding surfaces (Martin et aL, 1993a,b). Apart from the similarities, PDI and DsbA show a number of differences: (i) DsbA has one thioredoxin-like domain per monomer, PDI has two; (ii) in DsbA, the thioredoxin domain is interrupted by an unusual triple-helical domain, while in PDI the thioredoxin-like domains are not interrupted; (iii) DsbA is a monomer of 21 kDa, whereas PDI is a homodimer with subunit molecular masses between 57 (in mammals) and 70 kDa (in yeast). PDI is larger not only because of the duplication of

PROTEIN FOLDING AND ASSOCIATION

267

the thioredoxin domains but also because of the presence of several additional domains that are missing in DsbA. PDI is the f3 subunit of prolyl hydroxylase and is also proposed to be part of the triglyceride transfer complex. The extra domains in PDI may be responsible for these additional roles (Noiva and Lennarz, 1992; Bardwell and Beckwith, 1993). There is clear evidence that DsbA catalyzes protein folding in vitro. It must also act catalytically in the cell since the levels of oxidized substrates can exceed the level of DsbA by almost three orders of magnitude. Being oxidoreductases and isomerases at the same time, the question arises as to how reoxidation of the enzyme occurs. In the case of DsbA, an integral protein of the inner membrane, DsbB, appears to be involved (Bardwell et al, 1993; Missiakas et a/., 1993, 1994). Its four cysteine residues face the periplasmic space and seem to pass oxidizing potential from components of the electron transport chain to DsbA, coupling disulfide bond formation to energy input. Candidates for compounds involved in the reoxidation reaction include oxidized glutathione and selenite (Bardwell and Beckwith, 1993). As for the isomerase activity, catalysis may be operative at two levels, folding of the nascent chain and rescue of misfolded (and aggregated) polypeptides. The latter has been shown to be involved in the dramatic acceleration of the isomerization reactions necessary for the completion of disulfide formation on the folding pathway of BPTI (Weissman and Kim, 1993). Apparently, in certain proteins PDI is capable of gaining access to buried thiol groups after the protein substrate has acquired a substantial percentage of its tertiary structure. In the case of the oxidative renaturation of antibodies, this holds only for the first phase of the reaction (Lilie et al., 1993). If PDI can act late on the refolding pathway, it could help explain why the correct disulfide bonds are made. The proteins have already folded so as to place those cysteines that are to participate in disulfide-bond formation in close proximity, supporting the idea that PDIs are true folding catalysts in the sense that they enhance the rate of folding without determining the final structure. In this context they are involved in both formation and isomerization of disulfide bonds in their specific compartments in the cell. In prokaryotes, the DsbA/DsbB system is supplemented by a third component, the periplasmic disulfide oxidoreductase DsbC. It possesses the active-site sequence C-G-Y-C, which is characteristic of the thioredoxin superfamily, with both cysteine residues being essential for catalysis. The isomerase activity of DsbC exceeds that of DsbA; obviously, both enzymes cooperate in a synergistic manner (Bardwell, 1994). A model illustrating the single steps along the processes of translocation, oxidation, isomerization, and recycling is given in Fig. 9.

268

RAINER JAENICKE

2GSH S

V

GSSG Recycling

j

Cytoplasm

Binding FIG. 9. Model for the catalysis of disulfide bond formation in bacterial periplasmic proteins. Proteins are secreted in a reduced state; DsbA (PDI) binds the unfolded polypeptide and then rapidly transfers its disulfide to the folding protein. Subsequently, reduced DsbA (PDI) is reoxidized by DsbB.

3. PEPTIDYL PROLYL cis-^rans-IsoMERASES PPIs catalyze the rotation around the X-propeptide bond (in vitro) and inhibit signal transduction processes involved in immunosuppression (in vivo) (Fischer, 1994). Translation jdelds the sll-trans configuration of the polypeptide chain. In cases in which the final structure of a protein contains cis residues, the high activation energy of trans-cis isomerization will give rise to slow steps on the folding pathway which may be ratedetermining for the overall reaction. By far the most frequently occurring cis rotamer in known protein structures involves proline residues whose five-membered ring breaks the common secondary structural elements along the polypeptide backbone, resembling a molecular switch with two positions (Scheme 3).

PROTEIN FOLDING AND ASSOCIATION

trans '

^

269

cis SCHEME 3

The activation energy of the isomerization reaction depends on the neighboring groups; in the case of essential prohnes (Levitt, 1981; Schmid et al., 1993) it is of the order of 85 kJ/mol, so that at room temperature and in the absence of a catalyst the reaction becomes rate limiting in the overall folding process. A variety of structurally unrelated families of enzymes capable of catalyzing peptidyl prolyl cis-trans isomerization have been recognized. Two of them, the cyclophilins (CyPs) and the FK 506-binding proteins (FKBP), have been investigated in great detail, whereas new members have only recently been discovered (Rahfeld et aL, 1994a,b). CyPs and FKBPs are abundant and ubiquitous proteins which are known to be involved in a variety of cell biological phenomena. Whether their action is related to their specific catal3rtic function as PPIs is still unresolved. The common denominator of the two major families is the immunosuppressive activity of the two PPI inhibitors, cyclosporin A (CsA) and FK 506, which gave both enzymes their names. They bind with high affinities to their target cells so that their immunosuppressive activity can hardly be related to proline isomerization. Rather, it seems that the PPIs bind the drugs and present them to a target molecule, most probably calcineurin. Site-directed mutagenesis of cyclophilin allowed the separation of PPI activity from CsA binding and calcineurin inhibition, thus proving that PPI activity is not required for calcineurin inhibition (Fischer, 1994). The three-dimensional structures of human CyP18cy and FKBP have been determined; both are structurally unrelated (see Schmid, 1993; Schmid et al, 1993; Fischer, 1994). The structure of the FKBP-FK 506 complex supports the catalysis-by-distortion mechanism. The inhibitor binds in a hydrophobic cleft with its amide bond in planar configuration; however, whereas when free in solution it is in its cis conformation, it is trans in the complex, i.e., the ground state of FK 506 mimics the transition state for the peptidylprolyl cistrans isomerization (van Duyne et al, 1991). In the case of CyP, the active site is located on the outside of the /3 barrel rather than in the

270

RAINER JAENICKE

core of the molecule. Here, the X-Pro peptide bond of the substrate is twisted toward the cis configuration. The position of the CsA-binding site is most exceptional in that solubilization of the water-insoluble drug by CyP causes the complete inversion of the suppressant (Kallen et aL, 1991; Spitzfaden et al., 1992). The catalytic efficiency of the PPIs depends very much on the accessibility of the X-prolyl bonds involved in the isomerization reaction. In some cases in which poor catalysis has been observed, the simplest explanation is that rapid formation of ordered structure renders essential proline residues inaccessible to the isomerase. For small prolyl peptides, the catalytic efficiencies of both CyP and FKBP approach the diffusion-limited second-order rate constant for the association of a protein with a small molecule (Harrison and Stein, 1990; Schmid et al., 1993). Little is known regarding the sequence specificity of proline cjs-^ra^s-isomerases; like protein disulfide isomerase, the substrate specificity seems to be low. The significance of PPI in catalyzing the in vitro refolding of small model proteins containing essential proline residues is well established and has been summarized in a number of lucid reviews (Kim and Baldwin, 1990; Fischer and Schmid, 1990; Schmid et a/., 1993; Schmid, 1992, 1993). The additional activity of PPI or a PPI-related protein as chaperone in the reactivation of carbonic anhydrase II (Freskgard et aL, 1992; Rinfret et aL, 1994) could not be confirmed by Kern et al, (1994). In connection with the cellular aspects of folding catalysts, two further questions need consideration: (i) Are there synergistic effects when PDI and PPI catalysis occurs simultaneously? (ii) Does PPI play a biologically significant role in in vivo protein folding? Regarding the first question, Schonbrunner and Schmid (1992) have shown that PPI accelerates the oxidative folding of reduced RNase Tl; equally, the catalysis of disulfide bond formation by PDI is markedly enhanced in the presence of PPI (Fig. 10). Evidently, the two enzymes act synergistically and according to their specificity. Whether a similar synergism operates in the course of the de novo synthesis and folding in the cell remains to be shown. Concerning the biological significance of PPI catalysis in connection with protein folding, a number of observations seem to indicate that the in vitro results have some bearing on cotranslational or postranslational events in the cell. First, there is striking evolutionary conservation of function, together with the proper location and ubiquity in every organism and subcellular compartment (Schonbrunner et aL, 1991). Second, it has been shown that CyP20 is involved in mitochondrial protein folding in cooperation with molecular chaperones: using either CyP inhibition by CsA or CyP~ mutants of Neuro-

PROTEIN FOLDING AND ASSOCIATION

0

271

1 2 Time (hours)

FIG. 10. Acceleration of the oxidative refolding of RNase T l by PPI and PDI: 2.5 /xM RNase T l in 0.1 M Tris-HCl, pH 7.8, 0.2 M GdmCl, 4 mM reduced/0.4 mM oxidized glutathione, 25°C. Refolding measured by tryptophan fluroescence at 320 nm ( • , O, • , A), double-jump kinetics (monitoring native protein) (A), and native polyacrylamide gelelectrophoresis ( • ) . Reoxidation in the absence of PPI and PDI ( • ) , in the presence of 1.4 yM PPI (O), in the presence of 1.6 /LLM PDI (A), in the presence of 1.4 /iM PPI plus 1.6 /LtAf PDI (A), and in the presence of 10 mAf dithioerythritol (to block disulfide-bond formation (D); the slight decrease in the latter signal is caused by aggregation of the reduced and unfolded protein. Curves calculated for single first-order reactions with time constants T = 4300 ( • ) , 2270 (O), 1500 (A), and 650 sec (A), respectively (Schonbrunner and Schmid, 1992).

spora crassa, a significant delay of the folding of imported proteins in mitochondria was observed. Folding intermediates reversibly accumulated at the molecular chaperones Hsp60 and Hsp70 in the matrix (Rassow et aL, 1995); similar results were reported for yeast mitochondria (Matouschek et aL, 1995). Third, in the case of the maturation of procollagen to form the mature collagen triple helix, CsA was found to have a significant retarding effect. On the other hand, PPI accelerated the rate by a factor of approximately three (Bruckner et aL, 1981; Bachinger, 1987). In transferring this kind of experiment into intact cells, Steinmann et aL (1991) were able to show that on addition of CsA to chicken embryo fibroblasts the time for half-completion of the triple helix increased significantly. Within the framework of the present discussion, the obvious explanation of this result is that the ratelimiting prolyl and hydroxyprolyl isomerizations during the in vivo folding of collagen are catalyzed by PPI. Inhibition of its activity by CsA consequently leads to retarded folding. Fourth, similar results as those for collagen maturation were obtained in connection with the

272

RAINER JAENICKE

maturation of transferrin (Lodish and Kong, 1991). The secretion of the protein from HepG2 cells shows a significant sensitivity to CsA. Undoubtedly, the complex regulatory processes in higher cells do not allow the target molecule to be unambiguously defined. Finally, retroviral Gag protein plays an important role early in the HIV infection of the cell, directing the assembly of HIV-1 particles via specific interactions with CyP A and B. Gyp A is specifically incorporated into HIV-1 virions, whereas CsA efficiently disrupts the interaction, interfering with the HIV-1 life cycle (Luban et al, 1993; Franke et al, 1994; Thah et al, 1994). If in the foregoing experiments CsA indeed turns out to bind exclusively to PPI, the folding mechanism would be the following: The nascent dX{-trans polypeptide chain collapses rapidly to form a compact state with elements of secondary structure and sufficient stability to expel water from its hydrophobic interior; subsequent slow and very slow steps lead to the native-like state which finally undergoes trans -^ cis isomerization of proline residues to reach the biologically active, native conformation. Assuming this mechanism to hold in the cell, extrapolation from model peptides would predict protein folding to be much slower than estimated from in vivo studies. There are three explanations why proline isomerization does not necessarily limit in vivo protein folding: (i) ^rans-prolines may be trapped in their initial configuration; (ii) the occurrence of proline residues in solvent-exposed turns causes their state of isomerization to be of minor functional signifiance; and (iii) constraints of the chain conformation may greatly decrease the energy barrier of proline isomerization, thus speeding up the reaction. These arguments do not consider the idea that PPIs may operate at the site of protein translation or translocation, thus enhancing the trans —^ cis isomerization at defined sites depending on the sequence specificity of the enzyme. This hypothesis illustrates how future in vivo and in vitro folding studies might be tied together in order to solve relevant questions of protein self-organization (Jaenicke, 1993b). D. Chaperones The formation of the native quaternary structure of proteins includes chain folding and oligomerization. Both reactions may be rate limiting (see Section III, E). Evidently, in the assembly process interactive protein surfaces become transiently exposed to the environment. In case folding does not occur fast enough to provide the specific recognition sites for proper docking, aggregation is expected to compete with correct

PROTEIN FOLDING AND ASSOCIATION

273

association. The situation may be quantitatively described by the sequential uni-bi molecular reaction scheme with aggregation as diffusion-controlled side reaction (see Eq. [7]) (Kiefhaber et ai, 1991). In refolding experiments in vitro, the limit at which kinetic competition between folding and association becomes significant is commonly far below the average protein concentrations in vivo (Teipel and Koshland, 1971; Jaenicke, 1974; Orsini and Goldberg, 1978; Zettlmeissl et ai, 1979; Goldberg and Zetina, 1980). Thus, in the cell, some kind of protection must be effective that inhibits unproductive assembly and, at the same time, promotes correct protein folding and association. The accessory proteins involved in this mechanism are the molecular chaperones, a family of unrelated cellular proteins that mediate the correct association, but are not themselves components of the final structures (Ellis and van der Vies, 1991). The requirement for "folding helpers" differs depending on the system; in general, multidomain oligomeric proteins need chaperone assistance for unperturbed assembly. Apart from their involvement in folding, molecular chaperones also limit damage caused by stress conditions such as heat. Accordingly, their cellular level was found to be strongly enhanced under shock conditions; however, not all chaperones are heat-shock proteins (Ellis, 1990a). It has become clear that molecular chaperones play significant roles in a variety of cellular processes such as protein targeting, translocation through membranes, and compartmentation. Correspondingly, the field is expanding extremely fast (for reviews see G^thing and Sambrook, 1992; Morimoto et a/., 1994). Space does not permit the cell biological aspects to be covered in detail. Therefore, the discussion will focus only on five topics: (i) kinetic partitioning, (ii) the occurrence and function of stress proteins, (iii) the GroE system, (iv) the specificity of molecular chaperones, and (v) chaperone assemblies. 1. KINETIC PARTITIONING

Folding is governed by first-order kinetic processes determined by intrinsic properties of a given protein. Early folding steps lead to intermediates with more than average nonpolar groups still exposed to the aqueous solvent. The probability to form intermolecular instead of intramolecular hydrophobic interactions increases with increasing protein concentration. In the case of oligomeric proteins, the reaction order may exceed second order (Zettlmeissl et ai, 1979). As previously mentioned in connection with off-pathway reactions, the time course of folding and aggregation may be expressed by kinetic partitioning according to Eq. [9],

274

RAINER JAENICKE k

^

where C/, N, and A are unfolded states, native state, and aggregates, respectively, kx and k^ are first- and second-order rate constants, and 0 is the partition coefficient. The chaperone concept describes the cellular folding mechanism in terms of the optimization of the yield of N in the A<— U^>N competition, keeping the concentration of aggregationcompetent polypeptide chains at a minimum. It defines molecular chaperones as globular proteins or protein assemblies that assist in the folding and association of nascent polypeptide chains without becoming integral parts of the final native structure. In analogy to their human counterparts, they do so by preventing illegitimate interactions between nonnative polypeptide chains, thus inhibiting aggregation as the major side reaction in protein folding and association, but also preventing folding polypeptide chains from interacting with other cellular components. Despite numerous diagrams, the mechanism of chaperone action is still unresolved. Considering the various classes of chaperones and their functional assemblies, one would predict a wide range of mechanistic differences between the various "chaperone machines" (Georgopoulos, 1992). The substrate polypeptide chain seems to fold in aqueous seclusion, without necessarily being encaged in what has been called an "Anfinsen cage." The challenging problem that still awaits an answer is how can intermolecular protein interactions regulate intramolecular interactions to the result that the intramolecular ones win. 2. STRESS PROTEINS: OCCURENCE AND FUNCTIONS

Cells respond to environmental stresses, e.g., high temperature, high hydrostatic pressure, and high concentrations of heavy metal ions, by the increased synthesis of a number of proteins which are commonly referred to as heat-shock proteins (Hsps) or stress proteins (Morimoto et al, 1990; Nover, 1991; Gething and Sambrook, 1992; Morimoto et al, 1994). Their functional relationship in terms of the general chaperone concept has only recently been discovered so that the nomenclature is all but clear. The majority of the eukaryotic Hsps were discovered as additional bands on electrophoretic gels; accordingly, their names reflect their apparent molecular weights. Homologous proteins in prokaryotes have mostly been identified by genetic techniques; therefore, their names often refer to genes connected with their expression. Their subunit molecular masses range from 10 to >100 kDa, some forming large oligomeric or even multimeric structures.

PROTEIN FOLDING AND ASSOCIATION

275

Based on their subunit molecular weights and similarities in their amino acid sequences, molecular chaperones are grouped in a number of families. The members of the Hsp60 and Hsp70 families have been shown to possess intrinsic ATPase activity (Morimoto et al, 1990; Gething and Sambrook, 1992). However, ATP hydrolysis is not absolutely required for chaperone function; in certain cases, depending on the protein substrate, ATPyS or other nonhydrolyzable ATP analogs can replace ATP, or no trinucleotide is needed at all (Jaenicke, 1993b). It has been demonstrated that Hsps have a protective effect under conditions of heat stress: cells that have been preexposed to elevated temperature for some time tolerate heat-shock conditions more readily than cells grown under normal physiological conditions. Obviously, the observed thermotolerance is correlated with the expression of high levels of Hsps (Li and Laszlo, 1985). The mechanism at the cellular and molecular level is still unresolved, except for three findings. First, at low levels of Hsps, heat shock causes denaturation and subsequent aggregation of proteins (Pelham, 1984; Pinto et ai, 1991; Gragerov et al, 1991). The rpoH E. coli mutant, which has lost the capacity to induce the normal heat-shock response, exhibits anomalous intracellular protein aggregation; this is detectable even at 30°C, proving that Hsps are essential even under normal physiological conditions (Gragerov et al., 1991). Second, using microscopial techniques, Welch and Suhan (1986) were able to show that HspTO binds to damaged ribosomal precursors in the nucleus, and possibly even dissolves them after normal conditions have been restored (Pelham, 1984). Finally, from heat denaturation experiments, both in vivo and in vitro, it is well established that chaperones serve to protect heat-denatured polypeptide chains from coagulation, trapping them over an extended period of time. After the (simulated) heat stress, addition of ATP, and/or supplementation of certain components of the chaperone system, the activity of the substrate protein is fully restored (Holl-Neugebauer et al, 1991; Fisher, 1992). In this connection, one may ask whether thermophilic microorganisms also express chaperone proteins. Evidently, this is the case as Sulfolobus shibatae (maximum growth temperature, T^ax of 105°C) and other archaea contain chaperone-like thermostable ATPases. These enzymes resemble proposed cytosolic eukaryotic chaperones and have the capacity to bind folding intermediates and prevent their irreverible denaturation (J. Buchner, R. Jaenicke, M. Schmidt, H. Sparrer, and K. O. Stetter, 1991, unpublished data); (Phipps et al, 1991; Trent et al, 1991; Waldmann et ai, 1995). The protective effect of molecular chaperones under heat-shock conditions in vitro and the recovery of misfolded or even aggregated polypep-

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tide chains after stress have been explained in terms of chaperones as "unfoldases" capable of rescuing denatured proteins. Whether "resurrection" phenomena are caused by shifts of the equilibrium between intermediate states on the folding path and aggregation as side reaction, or whether "unfolding and refolding into the right shape in an ATPdependent manner" is involved (Pelham, 1986; Skowyra et ai, 1990; Hwang et al., 1990; Parsell et al., 1994) requires further investigation. The above discussion referred only to the universal chaperone function which is "kinetic partitioning" between folding, association, and aggregation in order to protect the nascent polypeptide chain from misfolding and subsequent degradation. A variety of other chaperone functions have been discussed, focusing mainly on Hsp90, Hsp70, Hsp60, and small Hsps, including cohorts and eukaryotic C3rtosolic chaperones (Lindquist and Craig, 1988; Craig and Gross, 1991; Gething and Sambrook, 1992; Yaffe et al., 1992; Lewis et a/., 1992; Gao et ai, 1992; Freedman, 1992; Hendrick and Hartl, 1993; Jaenicke, 1993a,b; Stuart et a/., 1994; Craig et ai, 1994; Feige and PoUa, 1994; Jakob and Buchner, 1994). A few examples may give an idea with respect to the direct or indirect relationships of the observed phenotypes with protein folding and association, (i) Investigating mutants of yeast and Drosophila, Hspl04 was found to be essential for thermotolerance and viability under a variety of stress conditions (Sanchez et al., 1992). (ii) In the case of human Hsp70, retroviral-mediated gene-transfer experiments indicated that the chaperone protects eukaryotic cells, even after deletion of its ATP-binding domain (Li et ai, 1992). (iii) Based on the (weak) sequence homology of movement proteins (involved in the spread of viruses in plants) with members of the Hsp90 family, virus transport through plasmodesmata in plants has been proposed to involve chaperone action (Koonin et al., 1991). (iv) In E. coli, mutant studies have shown that members of the Hsp70 family (DnaK/DnaJ) are of central importance in protein expression and turnover (C. Georgopoulos and M. Zylicz, 1995, personal communication), (v) In eukaryotic cells, Hsp90 is one of the most abundant proteins in the cytosol; homologs have been found in the ER (Grp94, HsplOO) and in E. coli (HtpG). The Hsp90 protein has been shown to be involved in signal transduction, e.g., in the activation of steroid hormone receptors and protein kinases (Jakob and Buchner, 1994). Its abundance both under normal and under stress conditions may either indicate low activity or additional functions of the protein. That Hsp90 may participate in kinetic partitioning has been confirmed in in vitro renaturation experiments (Wiech et al., 1992; Jakob et al., 1995). (vi) In order to target proteins across the E. coli inner membrane or into mitochondria or chloroplasts, their precursors have to maintain a translocation-competent, partially unfolded state

PROTEIN FOLDING AND ASSOCIATION

277

provided by SecB or Hsp70 (Kumamoto and Beckwith, 1985; Randall and Hardy, 1986; Eilers and Schatz, 1986; Crooke and Wickner, 1987; Weiss et al., 1988; Lecker et ai, 1989). (vii) Overproduction of Hsp60 (GroEL/GroES) in bacterial cells lacking a normal heat-shock response protects intracellular proteins from aggregation into insoluble inclusion bodies (Gragerov et al, 1991, 1992). (viii) In mammalian cell cultures, heat shock leads to induction of heat-shock gene transcription, keeping the steady-state level of Hsp70 sufficiently high to avoid depletion: obviously, the chaperone regulates its own stress-induced synthesis (Beckmann et al, 1990, 1992; Baler et a/., 1992). (ix) Small heat-shock proteins (sHsps) have all the characteristics of chaperones: they form complexes with nonnative proteins, suppress aggregation under heatshock conditions (Jakob et al., 1993), and convey thermotolerance to cells (Landry et al, 1989; Knauf e^ al, 1994). (x) a-Crystallin, one of the main components of the eye lens, shows high homology to Hsp25 and shares the properties of sHsps (Horwitz, 1992; Jakob et al., 1993; Merck et al., 1993; Jaenicke and Creighton, 1993). It is tempting to speculate that the protein serves as a molecular chaperone to maintain transparency of the eye lens (Horwitz, 1992; Jaenicke, 1994). The few selected examples clearly show that molecular chaperones display their activity in many different cellular processes. Regarding their substrate specifity, folding helpers such as Hsp60 and Hsp70 are highly unspecific, while others such as PapD show high specificity, exclusively catalyzing pili assembly (Hultgren et al., 1993). The question of what are the preferred sequences and/or conformations involved in the interactions between chaperones and their protein substrates is still unresolved. 3. GROE SYSTEM

GroEL/GroES oiE. coli is currently the best-known chaperone system in terms of its structural and functional characterization. It may serve to illustrate the complexity of the chaperone problem. GroEL is a double disc with seven fold symmetry consisting of 14 identical 60-kDa subunits. It is a weak K^-dependent ATPase whose three-dimensional structure has been solved to 2.8 A resolution (Braig et al., 1994). GroES, the other component of the GroE system, forms rings of 7 identical 10kDa subunits. In the presence of Mg^^-ATP, GroES heptamers bind to GroEL with high affinity. § This interaction results in the inhibition of § Bacteriophage use the E. coli GroE system for folding and assembly. In bacteriophage T4, a phage-coded specialized protein (Gp31) can functionally substitute for GroES in the morphogenesis of phage \ and T5 despite the absence of any sequence similarity (van der Vies et al., 1994).

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RAINER JAENICKE

the intrinsic ATPase activity of GroEL (Chandrasekhar et ai, 1986; Lorimer, 1992). The stoichiometry of the functional GroE complex depends on the ligand, ATP or ADP: in the presence of ATP or its nonhydrolyzable analogs, symmetrical football-shaped GroELi4 (GroES7)2 particles are observed apart from the bullet-shaped asymmetrical GroELnGroES? complex which has previously been considered to be the only functional unit, giving rise to the Anfinsen cage model (Hendrick and Hartl, 1993; Saibil and Wood, 1993; ElHs, 1994a,b,c). However, evidence from chemical cross-linking (Azem et al, 1994) and electronmicroscopy (Schmidt et al, 1994c) seem to suggest that there is successive binding of two GroESy rings to one GroELu double doughnut. Both the symmetrical and the asymmetrical complex seem to be active, i.e., they bind and efficiently assist the refolding of proteins; in the presence of ADP only the asymmetrical form is observed (Fig. 11). The optimal ratio of GroELi4 and GroES? producing symmetrical particles was observed at subimit molar equivalency, consistent with the GroEL^ •(GroES7)2 stoichiometry. Averaging a large number of football particles shows that the binding of two GroES? rings to the opposite end of the GroELi4 cylinder confers a pronounced structural change in GroELu; it looks as if the symmetrical complex has one common seven-fold axis. The statistical evaluation of the particle distribution yields more than 50% symmetrical particles; this amount is unexpectedly high, considering the dynamics of the system in the presence of Mg^^-ATP where respective release and rebinding of GroES? is going on upon ATP binding. It is important to note that binding rather than hydrolysis of ATP is the driving force in the formation of the symmetrical complex; as in the cross-linking experiments (Azem et al, 1994), ATP-yS and AMPPNP lead to the same result as ATP. The given evidence clearly indicates that under conditions in which protein folding is facilitated, i.e., in the presence of ATP, the symmetrical GroELi4(GroES7)2 complex is highly populated in addition to the asymmetrical one; probably both are intermediates in the catalytic cycle (Schmidt et al., 1994c). Faced with the reductionist's necessity to focus on a cytosol, which is far away from reality, the question remains to which extent do the above findings depend on solvent conditions. Analyzing the population of symmetrical vs. asymmetrical complexes at pH 7 and low Mg^^ concentration, the simulation of the cytosolic environment seems to indicate that the GroEL • GroES complex is the predominant species, thus favoring the idea that the substrate protein is bound within the GroEL cylinder (Engel et al, 1995). Correspondingly, Hayer-Hartl et al (1995) proposed a model for the nucleotide-dependent interaction of GroE involving a GroES-dependent increase in the volume of the GroEL

PROTEIN FOLDING AND ASSOCIATION

279

FIG. 11. Electron microscopic imaging of the GroE system of Escherichia coll. Averages of side views of GroEL and GroEL/GroES complexes in 50 mM MgCl2, 50 mM KCl, and 2.5 mM ATP. (A) Average of 580 symmetrical particles representing —60% of the total population. Dimensions (expressed as ellipsoid axes): 25 X 14 nm. (B-E) Averages of side views of classes of families of asymmetrical assemblies: (B), unloaded GroEL; (C-E), closed and open "bullets." Number of particles averaged in the subclasses: B, 48; C, 81; D, 106; E, 68.

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RAINER JAENICKE

cavity. How this model can be reconciled with preliminary X-ray data of the GroE system (P. Sigler, personal communication) has to await further experiments. A number of observations are difficult to explain with the Anfinsen cage model: GroE assists in the folding of high molecular weight protein substrates; nonnative proteins were found to interact with antibodies and could be cross-linked to GroESy without being protected against proteolysis (Martin et al., 1911; Bochkareva and Girshovich, 1992; Ishii et al., 1994). The observation that mammalian mitochondrial Hsp60 is a heptameric single toroid (Viitanen et al., 1992b) seems to be a questionable argument because in its functional state the chaperone might undergo association to a double-ring structure homologous to the E. coli system (P. Viitanen, G. Lorimer, and S. Walke, 1995, personal communication). The intermolecular transfer of substrate protein between independent toroids with different affinities is the basis of the GroE-ATPase cycle which Lorimer and co-workers proposed in putting together single-turnover and quench experiments in a unifying kinetic scheme. Assuming high- and low-affinity sites for the nonnative protein substrate, a two-cycle mechanism allows the GroEL-dependent hydrolysis of ATP in the absence and in the presence of GroES to be summarized (for details, see Todd et al., 1994). In considering the interaction of the complete GroEL/GroES/ATP system with its protein substrate in the context of facilitated protein folding, kinetic partitioning comes into the play in a way which allows the unfolded or kinetically trapped misfolded states of the protein substrate to be repetitively bound and released in the ATP-driven rescue cycle until it has reached the native state (Fig. 12). The overall mechanism would then be equilibrium substrate binding to the chaperone, with a steady decrease of the association constant as the folding polypeptide approaches the native state. This model is in agreement with the idea that GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms proposed by Weissman et al. (1994). The above discussion describes facilitated protein folding by the complete GroE system. As summarized in Table II, the function of GroEL does not necessarily require GroES and ATP; how binding and release of the protein substrate in the absence of GroES and/or ATP are triggered is unknown. Obviously, GroEL binds the nonnative protein, whereas GroES triggers its release in the presence of Mg^^-ATP. By studying the GroE-assisted folding of different substrate proteins, ribulose-l,5-bisphosphate carboxylase (Rubisco), malate dehydrogenase, and citrate

281

PROTEIN FOLDING AND ASSOCIATION

Native

r

7pi

GroEL(L)ADP7

\

. y

/

7

e

Unfolded states

^ Misfolded or kinetically trapped states

GroEL/^\* unfolded protein

7ADP

^''^^'^(H)

7 ATP

/ ^

'^ Aggregated species

FIG. 12. The role of the GroE-ATPase cycle in facilitated protein folding. Unfolded protein partitions to either the native state or an ensemble of misfolded states. The partition factor 6 is equal to the ratio of native protein over the total of native and misfolded protein. Upon quantized hydrolysis of ATP by the chaperone complex, the unfolded protein is released to once again partition between native and misfolded states. Successive rounds of binding, ATP hydrolysis, release, and repartioning ultimately result in the accumulation of the native state. Different proteins are characterized by different values for 6 . For smaller, single-domain proteins, 6 tends toward unity, whereas for larger multidomain proteins, 6 may be small, necessitating many rounds of ATP hydrolysis to reach the native state (Todd et aL, 1994).

synthase, and differently denatured species of an antibody Fab fragment, Schmidt et al. (1994a,b) were able to show that the need for GroES depends on the folding environment: under "nonpermissive conditions," in which nonassisted spontaneous folding does not occur, reactivation to the native state requires the complete GroEL/GroES/Mg^^-ATP system. On the other hand, under "permissive conditions," in which spontaneous folding occurs, GroES is no longer mandatory and ATP is sufficient to release the native enzymes from GroEL; in this case, GroES merely accelerates the rate of the ATP-dependent release. The results clearly show that complex stability, i.e., the association constant of the GroEsubstrate complex, determines the release requirements (Schmidt et al., 1994a). Evidently, association constants involve binding and release, which again stresses that folding assistance corresponds to the steady increase of the off rate of the complex formation between the chaperone and its protein ligand as it approaches the native state. The incompletely folded protein species equilibrating with GroEL, in the absence of GroES, are not necessarily committed to the native state. Similar to the unassisted folding reaction, they still partition

TABLE I1 SUBSTRATE PROTEINS AND REQUIREMENT FORGWESAND ATPOF CHA

Citrate synthase

Antibody fragment (Fab)

Not required, improved yield Not required, improved yield

Essential

Not required

Essential, sufficient without

Sufficient without GroES Essential

Essential

Not required

Requirement for

Dihydrofolate reductase a-Glucosidase

Not required, improved yield, enhanced rate

Not required

ATP

Glutamine synthetase

Not required

GroES

Lactate dehydrogenase

Substrate protein

Pre-P-lactamase

Essential, sufficient without GroES Essential

AMP-PNP,

Essential

Not required, improved yield, enhanced rate Essential

Rhodanese

a

Rubisco

~~

PROTEIN FOLDING AND ASSOCIATION

283

between productive and unproductive folding pathways in an environment-dependent manner. Thus, the relevance of GroES only becomes clear under typically nonpermissive conditions, e.g., at the high local protein concentrations commonly present in the cellular environment, which explains why at high expression levels of recombinant Rubisco in E, coli, active enz5ane can only be found if GroEL and GroES are expressed simultaneously (Goloubinoff e^ ai, 1989a). In following the in vitro reactivation of Rubisco (after preceding denaturation), a lag phase on release of the GroE-bound protein was observed; since only the dimeric enzyme shows activity, association of the released structured monomers must occur in solution rather than on (or within) the chaperone. Thus, it is the unfolded or misfolded, nonnative, monomeric form of the protein which binds to the chaperone (Goloubinoffe^ aL, 1989a; van der Vies et aL, 1992). Reconstitution experiments using E. coli glutamine synthetase (glutamate-ammonia ligase) and bacterial luciferase confirmed these conclusions. In the first case, formation of the native dodecamer was found to occur in solution, after release of the subunits from the binary GroEL complex, providing direct proof against oligomerization of the protein substrate on the chaperone (Fisher, 1993); in the case of the dimeric bacterial luciferase, the recombinant protein was found to bind to GroEL at a stage on the folding path where the subunits form molten globule intermediates incapable of dimerization. Thus, the chaperone facilitates correct association through stabilization of the incompletely folded subunits. What drives the release and what is the conformation of the polypeptide chain in the chaperone-substrate complex still need to be elucidated (see Section IV,D,4). The question whether the GroE system, after having bound its substrate protein, prevents misfolding by catalyzing correct folding, i.e., whether chaperones do or do not alter the folding pathway, was addressed using dimeric citrate synthase (CS) as a substrate. After unfolding in vitro and subsequent dilution of the denaturant, only a small portion ofthe enzyme regains the correct enzymatically active conformation. The major part aggregates, as shown by light scattering (Buchner et al, 1991a). Renaturation in the presence of GroEL alone leads to the formation of a stable binary complex with nonnative CS, thus inhibiting reactivation completely. However, if GroES and ATP are added to this complex, the enzyme is released from GroEL, and recovery of enzyme activity occurs. Under optimum conditions, the yield of reactivation amounts to about 80% compared to <6% in the absence of the chaperone. Experiments in which light scattering was applied to monitor aggregation showed that stoichiometric amounts of GroEL are sufficient to block the side reaction. The slow increase in light scattering observed when GroES

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and ATP are added shows that the released protein is still in a nonnative conformation prone to unspecific aggregation (Fig. 13). It is suggestive to assume that chaperones catalyze folding. Because enzymes that catalyze slow steps in folding had previously been discovered (e.g., PPI and PDI), one could think of energy-dependent "foldases" which use the hydrolysis of ATP to drive the folding reaction toward the native conformation. Under certain conditions, they could also serve as ATP-driven "unfoldases," catalyzing the reverse reaction, e.g., in connection with protein translocation or turnover. For GroEL/GroES this mechanism does not apply: the chaperone is not a catalyst of protein folding. Instead, it is involved in kinetic partitioning between folding, association, and aggregation, without accelerating the reaction. On the contrary, in the case of CS, reactivation is slowed down in the presence of GroE, probably due to the binding and release processes involved in chaperoning the substrate. Thus, what the chaperone in fact is doing, is keeping the concentration of aggregation-competent folding intermediates sufficiently low to allow productive folding and association. It may also cause partial unfolding by destabilizing kinetically trapped species; however, it does not rescue aggregated protein (Fig. 13C) (Goldberg et al, 1991; Kiefhaber et a/., 1991; van der Vies et al., 1992).

Citrate synthase ( n M )

Time

(mini

Time

(min)

FIG. 13. Reconstitution of citrate synthase from porcine heart muscle after preceding denaturation in 6 M guanidinium chloride). (A) Concentration dependence of reactivation (o) and aggregation ( • ) . (B) Reactivation in the presence of GroEL without GroES and ATP ( • ) , with GroES and ATP ( • , and with the addition of GroES and ATP after 30 (V) and 60 min (T), respectively. (C) Aggregation of citrate synthase during reconstitution in the absence of GroE (O), in the presence of GroEL (D), and in the presence of GroEL, GroES, and ATP—addition at zero time of reconstitution ( • ) , after 30 sec ( • ) , and after 4 min (V) (Buchner et al, 1991a).

PROTEIN FOLDING AND ASSOCIATION

285

4. SPECIFICITY OR PROMISCUITY

Target molecules that have been applied as chaperone substrates in in vitro experiments show no detectable similarities at the level of their sequences or their conformations and folding topologies, except that they are misfolded, nonnative, and monomelic. A common feature is their low yield of reactivation in denaturation/renaturation experiments. Because no specific reasons are known why kinetic partitioning for a given protein would favor aggregation instead of folding and association, chaperone affinity can only be attributed to the exposure of hydrophobic residues buried in the inner core of the native protein; there is no detectable affinity to the native state. In the case of GroEL, the substrate promiscuity has been illustrated by adding the chaperone (at a concentration about two orders of magnitude below the in vivo concentration) to the refolding mixture: about 50% of the soluble proteins of E. coli were trapped as nonnative, high-affinity, binary complexes (Viitanen et aL, 1992a).^ They are stable enough to be separable by gel-permeation chromatography. For the folding polypeptide chain, these observations have two consequences: (i) the nascent protein can hardly escape the chaperone when it comes off the ribosome (see below), and (ii) apart from preventing nonproductive aggregation, off the folding path, the formation of the binary complex also arrests the spontaneous folding/refolding of the target protein. In the case of m vitro folding experiments this is a common feature; it keeps the concentration of folding-competent polypeptide chains below the critical concentration at which aggregation takes over, improving the yield of reconstitution (Goldberg et al, 1991; van der Vies et al, 1992). Basically, one may envisage two mechanisms of recognition between chaperones and their target proteins: either the nonnative protein presents some specific unfolding sequence to the chaperone (Blond-Elguindi et al., 1993), or the chaperone recognizes in a more general way parts of the hydrophobic core of the protein (Jaenicke, 1993b, 1995). Features discriminating a correctly folded protein from a misfolded, nonnative polypeptide chain may be solvent-exposed hydrophobic amino acid side chains or parts of the backbone, e.g., in core regions. Studies devised to differentiate between different conformations such as amphiphatic a helices or f3 structures with hydrophobic surfaces showed that GroEL ^ It is difficult to relate this result to the observation that overexpression of GroEL and GroES in E. coli does not affect cell proliferation, leaving the phenotype unchanged (R. Rudolph, 1994, unpublished results). Extremely high levels of GroEL (>50% of total cellular protein) seem to reduce growth at high temperature; overexpression of GroES has an even more significant effect (S. M. van der Vies, 1995, personal communication).

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RAINER JAENICKE

equally recognizes the unfolded (extended) polypeptide chain (Badcoe et aL, 1991), the molten globule state (Groloubinoff e^ al., 1989a; Martin et aL, 1991; van der Vies et aL, 1992; Robinson et aL, 1994), a helices (Landry et aL, 1992), ^ strands (Schmidt and Buchner, 1992), and late highly structured folding intermediates. In the latter case, an antibody Fab fragment was used as a substrate, with the result that the ligand in its denatured, oxidized, and reduced states exhibits different binding properties, supporting the idea that chaperone interactions are determined by the nature of early folding intermediates, i.e., by the kinetics of folding, rather than by a specific type of conformation or even sequence (Lilie and Buchner, 1995). More detailed information was gained from bamase mutants where systematic variations of residues in an exposed region of the enzyme allowed complex formation with GroEL to be correlated with specific charged and hydrophobic stretches of the polypeptide chain (A. R. Fersht, 1995, personal communication). With respect to the ATPase activity and the involvement of GroES, ATP binding alone (in the absence of GroES) is sufficient to trigger the release of the denatured oxidized Fab, whereas the denatured, reduced fragment requires both GroES and ATP (Schmidt et aL, 1994a). Evidently, the different requirements for the release of different GroELbound protein substrates are related to differences in the kinetic partitioning between folding and aggregation. Proteins that refold fast and to high yields are expected to form native-like intermediates early on the folding pathway without requiring folding assistance; slowly folding proteins need chaperones in order to avoid aggregation. The protein released from GroEL is not committed to reach the native state, it can still undergo aggregation (Buchner et aL, 1991a) (Fig. 13B). As previously mentioned, lag phases observed in the formation of oligomeric proteins indicate the release of structured monomers (Goloubinoflf et aL, 1989b; Buchner et aL, 1991a; van der Vies et aL, 1992; Fisher, 1992,1993). Obviously, GroEL protects "aggregation sites" from unspecific interactions by trapping core regions of the nascent or folding polypeptide chain, shifting the partition coefficient toward the productive pathway. In the case of the Hsp70 family, careful biophysical and cell biological studies suggest a recognition mechanism between the chaperone and its target protein that is totally different from the one observed for GroEL. Here, in the case of the heavy-chain binding protein (BiP), the Hsp70 from the ER, a polypeptide stretch of ^ 7 residues was shown to be required in order to induce the ATPase activity of BiP (Flynn et aL, 1991;Rippmane^aZ., 1991;Flajnike^a/., 1991). Based on structural similarities with the major histocompatibility complex (MHC/HLA), a

PROTEIN FOLDING AND ASSOCIATION

287

model was proposed with the substrate accommodated in an extended conformation in a cavity similar to the one known for MHC. Detailed structural information would be required in order to confirm the analogy. Using the direct route, Blond-Elguindi et al. (1993) approached the problem in a systematic way taking advantage of a bacteriophage display system to sample random octapeptides for the potential to be recognized by BiP. Using the panning technique, the authors were able to collect more than 100 octapeptides which, after sequencing, could be compared with the sequences present in the original random library. The pattern that emerged from the statistical analysis allows the conclusion that optimum binding occurs if BiP is offered a heptameric motif with alternating hydrophobic residues; more precisely a pair of residues from the amino acid triple Trp, Phe, Leu, separated by a single amino acid. More detailed scores, as well as reduced abundances, confirm previous results obtained on a trial and error basis (Flynn et aL, 1989). Analyzing synthetic immunoglobulin heptapeptides as potential BiP-binding sites for their ability to stimulate the ATPase activity of BiP yielded a number of authentic BiP-binding sequences. These sequences are not confined to a single domain of the heavy chain. Instead, they invovle residues that participate in contact sites between the heavy and light chains. From this, one may conclude that in vivo BiP assists the folding and assembly of antibody molecules by binding to hydrophobic surfaces on the isolated chains that subsequently participate in interchain contacts (Knarr et al, 1995). The previous data confirm that the substrate oligopeptide binds to the chaperone in an extended conformation, as in the case of DnaK, the Hsp70 homolog in E, coli (Landry et al, 1992). In summary, Hsp70 proteins seem to recognize parts of proteins devoid of secondary structure which are normally buried in the interior of the protein. From in vitro folding studies, it is well established that this conformational state of the polypeptide chain is very short lived and early on the folding pathway. This explains why in in vitro experiments, e.g., with DnaK, a large molar excess of the chaperone is required to compete with protein aggregation (Skow5nra et a/., 1990; Langer et al, 1992). If DnaK binds efficiently only to proteins lacking secondary structure, it must be present in excess over the folding protein in order to influence the kinetic partitioning during the short time span in which this early intermediate state exists. The situation is different in cases in which Hsp70 works as uncoating ATPase on a stable substrate, such as clathrin. Here, only stoichiometric amounts of Hsp70 are required (Rothman and Schmid, 1986). Upon

288

RAINER JAENICKE

clathrin disassembly, the chaperone seems to bind to an exposed loop of the clathrin light chain, thus altering the conformation of the target subunit (DeLuca-Flaherty et aL, 1990; Brodsky et al., 1991). Considering the marginal free energy of stabilization of proteins, it is obvious that the perturbation of just a few interactions is sufficient to disintegrate the triskelion skeleton. Correlating this process with the solubilization of protein aggregates, one has to bear in mind that the interactions responsible for protein aggregation are less specific than those involved in quaternary structure formation. In comparison with the booming activity in the chaperone field, progress in our understanding of facilitated folding of proteins in terms of the detailed mechanisms of molecular recognition, on one hand, and kinetic competition of folding, association, and aggregation, on the other, has been slow (Schmid, 1991; Lorimer, 1992; Jaenicke, 1993b). Despite numerous vague, or even contradicting results along the tedious "we err and err and err, but less and less and less," there are a number of accepted facts which, for sake of clarity, should be summarized. Molecular chaperones do not alter the folding pathway; rather they shift the coefficient of kinetic partitioning toward the native state. Except for nucleoplasmin which binds to native histones, substrate binding to molecular chaperones requires the target protein to be in a nonnative state, suggesting hydrophobic interactions to be involved in recognition. Unscrambling of misassembled or aggregated proteins is the exception rather than the rule; it can be viewed as a shift of the equilibrium between folding intermediates. The selectivity of chaperone action is determined by kinetic partitioning in the sense that rapidly folding proteins (in the case of SecB, without a leader sequence) escape chaperone binding, whereas proteins (with a leader sequence) which fold more slowly and expose nonnative conformation are trapped (Hardy and Randall, 1991). In considering in vitro refolding reactions, there is a wide range of variations regarding ATP involvement, complex formation with other chaperones, and target specificity; very few hard facts are known in this context. However, model studies allow certain characteristics of chaperone-target interactions to be defined. A deeper understanding must await the complete determination of the threedimensional structure of the binary complexes. From the current binding data, various chaperones (or chaperone families) seem to have qualitatively different characteristics. For example, Hsp60s and Hsp70s seem to exhibit distinctly different specificities. Despite its cage-like structure, facilitated folding by the GroE system cannot be explained by "threading," "channeling," "seclusion in a cage."

PROTEIN FOLDING AND ASSOCIATION

289

One phenomenon that has only been mentioned in passing so far, is the observation that in vivo chaperones cooperate with each other in a sequential manner, giving rise to chaperone machines or chaperone cohorts (Georgopoulos, 1992). To avoid the association of the upperclass Victorian institution with the industrial revolution or Roman military, the final section will briefly discuss chaperone assemblies. 5. CHAPERONE ASSEMBLIES

The cooperation between chaperone components has been discussed in some detail in connection with the GroE system. In this special case, the assembly is exceedingly stable so that the single components, GroEL and GroES, can be reversibly dissociated and reassembled in vitro; they have been analyzed by X-ray diffraction as separate entities; their assembly structure has been investigated in detail by electron microscopy and image reconstitution; finally, their cooperative effects during the ATP cycle have been rigorously determined (Section IV,D,3). From this point of view, the phenomena give the impression that chaperone assembly is comparable to other heteromultimeric assembly structures such as other ATPases, aspartate transcarbamoylase, or pyruvate dehydrogenase. In the present context, the assembly has an impact at higher levels of cellular organization, e.g., the bacteriophage growth cycle, translocation through biological membranes, protein secretion, etc. The corresponding level of complexity renders a physiochemical analysis difficult if not impossible so that only a short perspective will be attempted. For details, a number of reviews are recommended (Georgopoulos, 1992; Kirchhausen, 1993; Jakob and Buchner, 1994; Stuart e^ al., 1994; Rothman and Warren, 1994; Kiibrich et al., 1995; Hammond and Helenius, 1995). Geneticists and molecular biologists interested in the development of E. coli bacteriophage \ very early discovered a number of host or bacteriophage genes coding for a variety of functions summarized in Table III. It contains the GroE system, and at this point it must be mentioned that the name stands tor growth and E gene because mutations in either the groEL or the groES gene blocked the growth of bacteriophage \ . In the case of DnaK (which corresponds to the eukaryotic Hsp70 family), the DnaK/DnaJ/GrpE system is involved in the disassembly of the \0-XP-DnaB helicase complex at the origin of X DNA replication, thus liberating the activity of DnaB in the growth cycle of \. A remarkable additional facet of the DnaK/DnaJ/GrpE example is the wide variety of functions these assemblies or their components may exert. In most cases they were discovered and explored in surprisingly

hsp90

Eukaryotic

Main chaperone Prokaryotic" HlpG

hsp60

hsp70 BiP

GroEL

DnaK

TFSS SecB

Tcp 1 SRP/docking protein, hsp70

?

Bind to nascent polypeptide localize them to the memb

Bind to and maintain the un globular structure?) and re Role similar to GroEUGroES

Bind to and maintain the un them following ATP hydro disaggregate protein aggre

Bind unfolded polypeptides a receptor protein), help them proper compartment

TABLE I11 VARIOUS CLASSES OF MOLECULAR CHAPERONE ASSEMBL

hsp56 50 kDa(?) 23 kDa(?) Sec63 Scl 1 Ydll (Mas5) Sisl GroE-like cpnlO

Eukaryotic

Cochaperone Prokaryotic

DnaJ

GrpE GroES GroES-like(?) SecA (PrlD) SecY (PrlA) See (PrlG)

GroES-like(?) ? Sec61 ? Sec62 Sec63

Except for TFSS, the nomenclature for the prokaryotic chaperons refers to E. coli.

PROTEIN FOLDING AND ASSOCIATION

291

distant fields and converged only after many years of highly specialized research. In the case of the DnaK/DnaJ/GrpE system, well-established functions are (i) regulation of the DNA replication of both bacteriophage \ and its host, heat-shock and other stress responses, and their autoregulation (Craig and Gross, 1991; Georgopoulos, 1992); and (ii) chaperoning of nonnative proteins, in certain cases, disassembly of misfolded protein aggregates. Being involved in such divergent and essential cellular processes, it is not surprising that DnaK, like the other chaperone assemblies and their components, is a ubiquitous and abundant protein in all cells and cell compartments, not only under stress but equally under normal physiological conditions. In connection with Hsp90, members of this chaperone family have invariably been found in stable association with conserved partner proteins from yeast to man. Interestingly, some of these partners, like Hsp70 and peptidyl prolyl cis-^ra^is-isomerase, are well-established accessory proteins in protein folding. Their presence is essential for the Hsp90 action in the maturation of certain kinases and the activation of steroid receptors by hormones (Pratt, 1993; Smith et aL, 1993; Jakob and Buchner, 1994; Chang and Lindquist, 1994). However, the abundance of the complex suggests that it may have a more general function as "foldosome," or "transportosome," or both. At present, our understanding of the function of either single components or the whole assembly is only rudimentary. It is difficult to obtain a clear-cut answer to the question of when during the lifetime of a protein, from translation to degradation, chaperone assemblies start interacting with their target molecule for at least three reasons: (i) the concentration of nascent polypeptides is low, (ii) they are usually heterogeneous with respect to their chain length, and (iii) analytical methods may interfere with the chaperone-target interactions. Previous work indicated cotranslational folding by parts (Bergman and Kuehl, 1979a,b,c; Peters and Davidson, 1982). Domains of —100 amino acid residues have about double the size which DnaJ requires for recognition of its substrate (Hendrick et aL, 1993). How the binding of the Hsp70 chaperone assembly correlates with translation has been studied in a mammalian translation system using truncated luciferase mRNA (lacking the codons for 11 C-terminal amino acids). This allows preventing chain release form the ribosome and yields a homogeneous population of the truncated enzyme. After release from the ribosome by puromycin, the enzyme shows some activity, confirming cotranslational folding. Upon addition of ATP, the activity is strongly enhanced, indicating the release of folded enzyme from chaperones. Actually, washed ribosomal pellets are found to contain

292

RAINER JAENICKE

Hsp70, Hsp40, and Trie C (TCPl) which, altogether, seem to form a stable 1200-kDa chaperone assembly. Obviously, all three components of the assembly are required; the sequence of events and the geometrical arrangement of the assembly has only been touched, so that it is premature to propose a model. This holds especially true because of recent observations which clearly demonstrated that further components and still earlier processes are involved in the cotranslational folding of the nascent polypeptide chain (Wiedmann et ai, 1994). It is well established that, in the case of proteins targeted to the ER, complex formation with the signal recognition particle (SRP) starts when the polypeptide is just leaving the ribosome (Hann and Walter, 1991; Rapoport, 1992; Gorhch and Rapoport, 1993; Hartmann et al., 1994). Obviously, there is at least one more component involved in targeting, which again raises the old question of whether the nascent chain leaves the ribosome through a channel or a cleft. The nascent polypeptide associated complex (NAG) is a heterodimeric protein which binds exclusively to the nascent polypeptide chain, shielding nonsignal peptide regions from promiscuous interactions with the signal recognition particle. As shown by Wiedmann et al. (1994), in the absence of NAG, proteins lacking the signal peptide are recognized and mistargeted by the SRP. Addition of NAG restores the specificity of SRP binding, as well as correct targeting and translocation. Obviously, NAG binds to signal peptides which are only partially exposed from the ribosome, serving as an adaptor between the translation complex and the cellular folding and transport machineries, thereby protecting nascent chains from premature and inappropriate interactions. Thus, the nascent chain is tunnelled through a groove between the ribosome and NAG. Gorrespondingly, the length of the amino acid sequence accessible to the cytosol is unexpectedly short. Using truncated mRNA as a means to produce fixed peptides of constant length, it turns out that at no more than ~15 amino acid residues away from the ribosomal peptidyltransferase site does the growing polypeptide become the target of chaperone-like components regulating their interactions (M. Wiedmann, 1994, personal communication). The further fate of nascent proteins on their way to organelles, the cell membrane, the periplasmatic space, or the outside medium cannot be covered here. The field has been mainly the domain of cell biologists, and physical biochemists only entered it quite recently. V. Practical Aspects A. Aggregation and inclusion Body Formation As previously mentioned, aggregation in vitro and inclusion body formation in the cell correspond to each other; overexpression leads to

PROTEIN FOLDING AND ASSOCIATION

293

high local concentrations of folding yielding precipitates instead of native protein. There are various strategies to cope with the problem. Making use of weaker promoters, one may reduce the concentration; since the aggregation reaction is of > second order, this will drastically reduce the local level of folding intermediates, thus promoting correct folding and association. However, there are two reasons why this approach is of questionable practical use: (i) the overall yield of the recombinant protein per gram cell mass is drastically decreased; and (ii) its purification requires a full-scale separation of the guest molecule from the bulk of the host proteins, whereas inclusion bodies, with their characteristic low heterogeneity, allow highly simplified purification procedures for their fractionation. Thus, starting from inclusion bodies is often the method of choice; in this case, optimization has to focus on the in vitro reconstitution of the mixture after solubilization (and denaturation), e.g., in guanidinium chloride or urea. A discontinuous "pulse dilution technique" has been devised in order to perform the dilution/reconcentration cycle in an economical way: a certain amount of the protein is subjected to dilution and reactivated at c < 1 fiM; after folding has proceeded to a point where aggregation is no longer limiting, a new portion of the concentrated solution of the denatured protein is added. This process continues until the whole batch is transferred. The method has two advantages: (i) the actual concentration of the folding intermediate never exceeds the critical concentration of aggregation; and (ii) the increasing level of the renatured protein exerts a stabilizing effect on the folding intermediate, comparable to the one used routinely by adding, for example, serum albumin. Additives such as arginine may strongly increase the 3deld by shuffling aggregates back on the productive folding path (Rudolph, 1990). Little is known about specific groups involved in the aggregation reaction. Early systematic experiments suggested hydrophobic interactions to be of major importance apart from covalent disulfide linkages (Jaenicke, 1967). In vitro and in vivo studies confirm this result (Mitraki and King, 1989; Hurtley and Helenius, 1989; Rudolph, 1990). A test case has been the monomeric two-domain enzyme rhodanese which is inaccessible to reactivation because of its strong tendency to form aggregates; competition for the hydrophobic aggregation sites by detergents allowed successful renaturation (Tandon and Horowitz, 1986). More detailed insight came from mutant studies in which an extension of the hydrophobic surface in the case of bovine growth hormone was shown to result in enhanced aggregation (Brems et al, 1988). The partitioning between folding and aggregation has been most intensively studied for the tailspike endorhamnosidase from Salmonella phage P22 and the numerous mutants of this protein which either

294

RAINER JAENICKE

increase or suppress aggregation (Mitraki and King, 1992). The wild type trimer is highly stable and shows a close similarity in its in vivo and in vitro folding behavior (Fuchs et al, 1991). Upon release from the ribosome or dilution from denaturant solutions, the polypeptides fold into a conformation sufficiently structured for proper assembly; the bulk of the )3 sheet secondary structure and the packing of the aromatic amino acid side chains are close to the native state, but even as protrimers they are still highly unstable. The anomalous stability is only acquired in a slow rearrangement reaction when the intertwined parallel j8 helices merge to form the native trimer. Correspondingly, a significant part of the tailspike folding reaction occurs after subunit association. During both self-assembly in vivo and refolding in vitro, the fraction of chains capable of maturing to the native form decreases with increasing temperature, the remaining polypeptides accumulating as aggregates (Haase-Pettingell and King, 1988; Mitraki et al, 1993). The aggregates are formed from partially folded intermediates that can chase either into native tailspike or into aggregates. Temperaturesensitive folding {tsf) point mutations reduce the folding yield at elevated temperatures, while second-site suppressor mutations {su) improve folding under such conditions (Mitraki and King, 1992). Both types of mutations act by altering the stability of folding intermediates, tsf substitutions destabilize it and su substitutions stabilize it; in the native structure, the denaturation of which is kinetically controlled, the effects of the mutations are masked by the complicated unfolding pathway (Banner and Seckler, 1993; Banner et ai, 1993; Beissinger, 1994; Beissinger et al, 1995). B. Reconstitution in Presence of Accessory Proteins As previously mentioned, inclusion bodies are the product of intracellular aggregation. They differ from aggregates formed in the test tube by their high packing density and large size, which may sometimes span the entire diameter of the cell (Valax and Georgiou, 1993). As a consequence, they can easily be harvested and washed by fractionated centrifugation which favors their use in the downstream processing of recombinant proteins. The isolation of the desired protein follows exactly the same routine of in vitro denaturation-renaturation described previously. Betailed guidelines have been worked out by Jaenicke and Rudolph (1986, 1989) and Rudolph (1990). The repertoire of methods has been extended by attempts to mimic in vivo conditions with respect to folding catalysts and chaperone proteins. Two examples may serve to illustrate the results. The first refers to the reactivation of a denatured and reduced immunotoxin [B3(Fo)-

295

PROTEIN FOLDING AND ASSOCIATION

PE38KDEL] composed of the VH region of a carcinoma-specific antibody and connected by a flexible linker to the corresponding VL chain, which is in turn fused to truncated Pseudomonas exotoxin. The chimeric protein contains three disulfide bonds, one in each antibody domain, and one in the toxin part. Upon renaturation, aggregation of nonnative polypeptide chains and the formation of incorrect disulfide linkages lead to >90% inactive molecules. Attempts to improve the yield by adding the E. coli chaperones GroE and DnaK, as well as PDI, were successful. As illustrated in Fig. 14, both GroE and DnaK have been found to influence the reaction: GroEL alone inhibits reactivation, whereas the complete GroEL system significantly increases the yield of active protein. DnaK exhibits the same effect in both free and immobilized forms which allows the chaperone to be reused in the downstream

100

1 Ratio

•G

80

£

60

2 3 GroE:lmmunotoxin

re

*> r I AO"F

/•—•

100

without DnaK 60

o

o

20

20 ^0 60 80 100 Ratio DnaK: Immunotoxin ( • ) Ratio BSA

without ATP

A ^V

o

O

"=

FD

80

20

L

J

__J

1

2 4 6 8 Ratio PDI:lmmunotoxin

\

1

10

Immunotoxin (O)

FIG. 14. Renaturation of a single-chain immunotoxin facilitated by chaperones and disulfide isomerase. (A) GroE-facilitated renaturation of the GdmCl-denatured reduced protein. Optimum renaturation at equimolar GroE/immunotoxin ratio. GroEL alone has no effect; GroEIVGroES in the absence of ATP traps folding intermediates. (B) Effect of Mg2^-ATP on the yield of renaturation. (C) DnaK-facilitated renaturation after GdmCl denaturation. Bovine serum albumin or DnaK, heated to 100°C, have no effect. (D) PDImediated oxidation of denatured and reduced immunotoxin in the presence of 60-fold excess DnaK. As indicated by the arrows, PDI and DnaK show synergistic effects (Buchner et al, 1992).

296

RAINER JAENICKE

processing of the protein. PDI synergistically stimulates reactivation. Under optimum conditions, reactivation yields are doubled compared to those of nonenzymatic disulfide bond formation (Buchner et al,, 1992). The second example deals with the renaturation, purification, and downstream processing of antibody fragments. As in the case of the immunotoxin, cytoplasmic expression of murine antibody chains (MAK 33) in E, coli results in the formation of inclusion bodies. Rudolph, Buchner, and co-workers designed a renaturation protocol which allows the production of microbially expressed authentic Fab fragments at yields up to 40% of the total amount of recombinant protein. Faced with a system which is known to show assisted folding in the cell (Haas, 1991, 1994), a whole set of solvent parameters (temperature, protein concentration, redox buffer, and labilizing components) had to be varied in order to mimic the in vivo conditions (Buchner and Rudolph, 1991). Lilie et al. (1993,1994) have included folding catalysts in the investigation, showing that, in the case of the oxidized Fab fragment, PPIs significantly accelerate the refolding reaction. Obviously, proline cistrans isomerization is involved in the folding reaction. However, apart from acting as a folding catalyst, PPI also stabilizes folding intermediates similar to serum albumin or to the increasing concentration of native protein in the previously mentioned pulse renaturation approach. PDI has no chaperone effect on the renaturation of oxidized Fab. Instead, the enzyme increases the yield of reactivation, and at the same time shifts the redox dependence from a GSHVGSSG ratio around 10 mM to <1 mM observed for the spontaneous reaction. Again, there is kinetic competition; this time between domain folding and the interaction of PDI with its target cysteine residues (Lilie et al., 1994). C. Reverse Micelles and Immobilized Proteins From the foregoing results one might assume that overexpression of a specific protein together with chaperones and folding catalysts from the same plasmid would finally yield 100% of the desired protein in its native state. Active research toward this goal has so far been unsuccessful. Therefore, it might be worthwhile to devise other concepts allowing off-pathway reactions to be eliminated. Two alternatives may be considered: (i) reconstitution in reverse micelles, and (ii) folding of polypeptide chains bound to solid matrices. Considering the properties of proteins in reverse micelles, it has been found that at low water content (<60%) proteins show enhanced stability (Luisi and Magid, 1986; Garza-Ramos et al. 1992a,b; Femandez-Velasco et al., 1995). By mixing a denatured protein (e.g., in 6 M

PROTEIN FOLDING AND ASSOCIATION

297

guanidinium chloride) with micelle-forming compounds at a sufficiently low protein/micelle ratio, a more or less monodisperse system may be established in which, at most, one monomer per micelle is present (Luisi and Magid, 1986). Since the micelles contain renaturation buffer, "caged" subunits will form structured monomers in separate micelles. Only after merging of micelles or protein transfer can assembly occur. Using this approach, preliminary reconstitution experiments with oligomeric enzymes resulted in high yields of reactivation without significant side reactions (A. Gromez-Puyou, R. Jaenicke, and co-workers, 1994, unpublished results). Reconstitution experiments using matrix-bound polypeptides go back to studies designed to determine the catalytic properties of isolated subunits of oligomeric enzymes (Chan, 1970; Gottschalk and Jaenicke, 1991). By using low levels of activation and low protein concentrations, W. W.-C. Chan succeeded in fixing oligomers only at one or very few sites so that after denaturation/dissociation and subsequent washing only monomers were covalently bound to the matrix. Their renaturation led to unexpectedly high yields which could easily be quantitated by hybridization. Expanding this idea, more recent folding studies on immobilized enzymes have shown that the properties of the matrix (solid or gel, cross-linking, porosity, polarity, etc.) and the linker connecting the protein to the matrix need careful consideration (Gottschalk and Jaenicke, 1987, 1991). It is obvious that the approach may be adapted to technological applications, using polyionic N- or C-terminal tails to immobilize the protein to an ion-exchange resin in a reversible fashion. The outcome of a-glucosidase is most encouraging: the enzyme with an Arge tail shows long-term stability over weeks, its reactivation yield after preceding guanidine denaturation is increased at least five-fold, and the upper limit of protein concentration at which maximum reactivation without aggregation can still be accomplished is shifted from -lOfjLg/ml to - 5 mg/ml (Fig. 15) (G. Stempfer et ai, 1995a,b).

VI. Conclusions The "protein folding problem" started as a surprise, if not a provocation, when proteins as biomolecules were found to undergo reversible denaturation in the same manner as crystals undergo cycles of recrystallization (Perutz, 1940). Were biochemists in the days of Anson and Mirsky (1925) and Wu (1931) still confronted with relicts of vitahsm? After Neurath et al, (1944), Anson (1945), and Anfinsen (1966) had paved the way, there remained no doubt: the laws of thermodynamics

298

RAINER JAENICKE

B

0.001

0.01

0.1

1

10

100

Enzyme loading (mg/mll

FIG. 15. Renaturation/reactivation of soluble and immobilized a-glucosidase-Arge after denaturation in 6 Af GdmCl. (A) Kinetics of reactivation at 0.1 mg/ml a-glucosidase concentration. (B) Profiles of the kinetic competition of folding and aggregation for free (O) and immobilized a-glucosidase (•). The immobilized enzyme allows reconstitution up to 5 mg/ml (G. Stempfer et al, 1995a,b).

hold also for protein molecules. The accepted view almost swung back to Jaques Loeb (1906) who considered living organisms as chemical machines which will sooner or later be produced by engineers. Protein chemists began looking for the algorithm which would allow the calculation of protein structures ab initio based merely on the amino acid sequence (Scheraga, 1980; see also Fasman, 1989; Merz and Le Grand, 1994). However, in the 1980s this optimism had vanished again for a couple of reasons: (i) the ab initio approach was unsuccessful, because statistical trials to determine the correlation between a given primary structure failed for lack of data (Rooman and Wodak, 1988); (ii) based on physical reasoning, theoreticians came to the conclusion that there can be no protein folding code and that perceived correlations between sequence or composition and three-dimensional structure are more likely to be an artifact of a limited data base than a real result (Thomas, 1992). With the advent of chaperones, physicochemical reasoning became old fashioned in some people's minds, while vitalism returned through the backdoor. In fact, after 20 years of in vitro protein folding, the practical application of denaturation/renaturation cycles in the downstream processing of recombinant proteins forced researchers in the field to ask questions

PROTEIN FOLDING AND ASSOCIATION

299

which focused on the cellular aspects of folding. C. B. Anfinsen anticipated them at both levels—catalysis of folding (shuffling enzyme) and folding on a template, today called chaperone-assisted folding (Epstein et ai, 1963). However, before approaching the problem at the cellular level of complexity, he and his followers had to solve the problem "under ideal conditions," i.e., in dilute solution in vitro, varying all relevant parameters. In summary, the outcome of their studies led to the mechanistic concept of multiple-pathway sequential folding with very fast early events (hydrophobic collapse), middle events (local shuffling toward the native tertiary structure), and late events determining the rate of the overall reaction; the latter are proline cis-trans isomerization, disulfide cross-bridge formation, and subunit assembly. As has been discussed in detail, all three are facilitated by accessory proteins, either enzymes or chaperones. From their ubiquity and abundance in all organisms, cells, and cell compartments, the two isomerases may be assumed to be essential for the formation of the native structure of proteins in the cell. In the case of the chaperones, the fundamental importance, beyond the stress response, has been clearly shown (i) for the kinetic partioning between folding, association, and aggregation of oligomeric proteins; (ii) for protein targeting, e.g., translocation of mitochondrial proteins across membranes; and (iii) for processes involved in morphogenesis, such as growth and self-assembly of phages. The elucidation of the detailed folding mechanism of a given protein would require the complete description of the nascent (unfolded) and final native states, together with all intermediates along the multistep folding path. Obviously, to date this information has not been collected for any protein or model system. The main obstacle has been the elusive nature of the folding polypeptide chain. Traditional spectroscopic techniques still do not allow the early elementary processes to be analyzed in detail. New developments, such as nanosecond laser pulse techniques (Jones et al, 1993), provide hope for future progress. Even the analysis of the slow (late) steps (proline isomerization, merging of domains, and association of subunits) commonly does not go beyond a global inspection. Again, recent improvements in stopped-flow and NMR-mass spectroscopy techniques are highly promising for providing insight into folding events along the whole pathway. In the case of chaperones and chaperone functions, the previously defined maximalist's goal would be absurd: even extremists among the photorealistic painters leave out one or the other hair of their model. Obviously, the next steps are the elucidation of the three-dimensional structures of chaperones and their target-protein complexes at the atomic level. Equally important will be the kinetic and thermodynamic

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RAINER JAENICKE

analysis of the steps involved in the correlation of the ATPase function of chaperones, on one hand, and folding of their substrate protein on the other. At the next higher level of complexity, chaperone assemblies need further examination regarding topology, stoichiometry, sequence of binding, involvement of ligands, etc. In this exciting field in which the combined effort of cell biologists, biochemists, and biophysicists could be most fruitful, it is desirable for the accumulation of sound quantitative data to get a higher priority than drawing cartoons of models, or priority per se. Models are indispensable in planning the next experiment, but unfortunately, today they have a tendency to slip into elementary textbooks as settled facts before the necessary control experiments have been performed. This way, they inhibit our understanding more than they stimulate it. Perhaps we should follow Horace's advice: nonumque prematur in annum [Horatius Flaccus, De arte poetica, verse 388 (10 BC)]. ACKNOWLEDGMENTS This review was written during a stay at the National Institutes of Health, Bethesda, Maryland. I t h a n k the Fogarty International Center for Advanced Studies for generous support and hospitality. Work performed in the author's laboratory over the years was supported by the German Science Foundation, the Fonds der Chemischen Industrie, the Max Planck Gtesellschaft, the Alexander von Humboldt Stiftung, and the European Community. Fruitful and most enjoyable discussions with Drs. R. L. Baldwin, J. Bardwell, J. Buchner, R. Glockshuber, M. E. Goldberg, T. Kiefhaber, G. Lorimer, P. L. Privalov, R. Rudolph, F. X. Schmid, R. Seckler, S. M. van der Vies, P. V. Viitanen, and D. B. Wetlaufer are gratefully acknowledged.

REFERENCES Abkevich, V. I., Gutin, A. M., and Shakhnovich, E. I. (1994a). Biochemistry 33, 1002610036. Abkevich, V. I., Gutin, A. M., and Shakhnovich, E. I. (1994b). J. Chem. Phys. 101, 6 0 5 2 6062. Andrews, S. C , Arosio, P., Bottke, W., Briat, J. F., von Darl, M., Harrison, P. M., Laulhere, J. P., Levi, S., Lobreaux, S., and Yewdall, S. J. (1992). J. Inorg. Biochem. 47, 161-174. Anfinsen, C. B. (1966). Harvey Lect 61, 95-116. Anfinsen, C. B. (1973). Science 181, 223-230. Ansari, A., Berendzen, J., Bowne, S. F., Frauenfelder, H., Iben, I. E. T., Sauke, T. B., Shyamsunder, E., and Young, R. D. (1985). Proc. Natl. Acad. Sci. U.S.A. 82,5000-5004. Ansari, A., Di lorio, Dlott, D. D., Frauenfelder, H., Iben, I. E. T., Langer, P., Roder, H., Sauke, T. B., and Shyamsunder, E. (1986). Biochemistry 25, 3139-3146. Anson, M. L. (1945). Adv. Protein Chem. 2, 361-384. Anson, M. L., and Mirsky, A. E. (1925). J. Gen. Physiol. 9, 169-179. Azem, A., Kessel, M., and Goloubinoff, P. (1994). Science 265, 653-656. Badcoe, I. G., Smith, C. J., Wood, S., Halsall, D. J., Holbrook, J. J., Lund, P., and Clarke, A. R. (1991). Biochemistry 30, 9195-9200. Bachinger, H.-P. (1987). J. Biol. Chem. 262, 17144-17148. Baker, D., and Agard, D. A. (1994). Biochemistry 3 3 , 7505-7509.

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Index

Accessory proteins, see also Chaperones multienz)rme system formation, 246-247 preventing aggregation, 259 reconstitution in presence of, 294-296 in ribosome assembly, 249 Acetate-CoA ligase in CheY acetylation, 150, 151 Acetylation, CheY-switch interaction regulation by, 150-151, 153 AT-Acetylcysteine, 202-203 Aconitase, RNOS inhibition of, 167 Acquired immunodeficiency syndrome glutathione levels in elevated plasma glutamate levels, 193-194 with iV-acetylcysteine administration, 194 loss of body mass/death from, 203 PEM secondary to, 201 secondary infections, 202 Actin affinity for calponin, 41 caldesmon binding to, 58 isolation of calponin binding to, 35 Actin-activated Mg^^-ATPase. see also Actomyosin caldesmon inhibiting, 59 calponin dephosphorylation of, 55 inhibiting, 43-45 in myosin competition test, 34 Activation of acetate, 150 of CheY protein, 147-148, 153^ of guanylate cyclase in vivo, 167 of multicatalytic protease by proteins, 14-16 by small molecules, 18 of TrpRS gene by interferon, 125 o f t y p e l P D E s , 64 Actomyosin. see also Actin-activated Mg^^-ATPase; Myosin calponin-induced inhibition of ATPase activity calponin phosphorylation and, 4 9 - 5 1

regulation of, 45-57 superstructure versus function of, 251 Actophosphorylated heavy meromosin, 44-45 Adaptation in signal transduction, 138 Adenosine 3',5'-cyclic monophosphate feedback-regulation of, 72 hepatic GSH concentrations and, 195-196, 197 in hormonal regulation of PDEs, 75-78 insulin inhibition of lipolysis and, 78-80 as second messenger in stimulus response, 6 3 - 6 4 Adenosine diphosphate, GroEL and, 278 Adenosine triphosphate adipocyte particulate cAMP PDE activation by insulin and, 80 GroE system and, 277-285 myosin molecule hydrolysis of, 33 proteolysis, stimulating, 17 Adenylyl cyclase activation in adipocytes, 76, 77 cAMP generation, 63 feed-back regulation of, 72 insulin action in lipolysis and, 78, 80, 88-89 Adipocytes type III cGI PDE activity in, increased, 72 adenylyl cyclase activation in, 76, 77 hormonal regulation, 75-78, 8 8 - 9 1 and insulin action in, 78-80 molecular cloning of, from rat, 74-75 phosphorylation and, 80-81 purification from, 72-73 Adrenergic agonists, GSH transport and, 197 Adrenergic hormones regulating GSH homeostasis, 195 Aggregation, see also Inclusion bodies folding steps committing to, 256-257 GroEl blocking, 283-284, 286 insufficient folding speed, 272-273 kinetic partitioning, see Kinetic partitioning 315

316 nonproductive folding pathways, 253 practical aspects of, 293-294 Air pollution nitric oxide as constituent of, 160 nitrogen dioxide in, 181 RNOS formed from NO/O2 reaction, 165 Allosteric effectors, cGMP as, 6 4 - 6 6 Amino acid sequences ArgRS N-terminal, 118 calponin, 3 9 - 4 1 coresponding spatial structure for, 210 ofPDEs purified, 6 structural domain pattern in, 74 ValRS, 123 Amino acid transport mechanisms, glutathione S3mthesis and, 193 Amino acids cognate tRNAs and, 101 GSH regulation by dietary sulfur, 192 NOx reactions with sulfhydrylcontaining, 170 RS classifications and, 126-127 in synthetases, 104, 111 Aminoacyl-tRNA AspRS synthetase interaction with, 114-116 E F l a , channeling to, 109, 116-117 Aminoacyl-tRNA synthetases, see also entries for specific synthetases amino acid classfications and, 126-127 functional significance, 111-112 in interpretation of genetic code, 101 structure of, 102-105 Aminoacylation by free form of ArgRS, 118 free versus complexed synthetases in, 112 LysRS complex versus free forms, 119 as recognition signal in protein degradation, 128 structural bases in synthetases in heterologous, 130 5-Aminosalicylic acid, 165 Amphipathic helices, AspRS highly charged face of, 112 N-terminal peptides, 116

INDEX Amphiphilic helices ArgRS N-terminal sequence regions, 118 LysRS, 119-120 ANF. see Atrial natriuretic factor Antibodies autoantibodies mammalian synthetases and, 127-128 ThrRS reaction with PL-7, 126 identifying possible multicatalytic protease, 20 Antigens MCP identification and, 20 RSs as nuclear and cytoplasmic, 127 Antioxidants nitric oxide as, 176-178 PEM decreasing levels of, 201 Apoferritin, 245-246 ApppA nucleotide, 128 Archea, MCP structure in, 3 Arginyl-tRNA synthetase cDNA sequence, 107 cross-linkage to 38K protein, 104 description, 118-119 dissociation from sjmthetase complex, 105-106 purification and characterization of, 106 Ascorbate as NOx intermediate scavenger, 166, 182 Aspartyl-tRNA synthetase association, regulation, and subcellular localization of, 117-118 bacterially-expressed human, 113 cDNA sequence, 107 interaction with aminoacyl-tRNA, 114-116 N-terminal domain amphipathic helix and, 112-113 catalysis, effects of, 113-114 discovery of, 109 functional significance in synthetase complex, 116-117 synthetic peptides of, 116 Aspartylation of mammalian tRNA, 114-116 Association of AspRS to synthetase complex, 117

INDEX cystolic solvent parameters and, 260-263 of GluProRS with synthetase complex, 121 of LysRS with other synthetases, 119 of proteins hierarchy of, 212-218 kinetic competition with first-order folding, 255 quatenary structure formation, 238-244 and RNAs in protein biosynthesis, 129 of superstructures, folding and, 244-251 of ValRS complex, 123 in vitro versus in vivo, 257-260 ATP. see Adenosine triphosphate ATP-dependent degradation of N-terminal modified proteins, 128 by ubiquitin, 5-7 ATPase complex, see also Actin-activated Mg2^-ATPase in 26S protease formation, 2 calponin's inhibitory effect on, 4 5 - 5 7 GroES and, 286 Hsp60/Hsp70 famiUes and, 275 S4-like subfamily subunits coiled coil regions in, potential, 25-26 function of, 7 localization in nucleus, 20 p97 protein and, 2, 16 substrate selection, influencing, 14 subunit recognition of ubiquitin or poly(Ub) chains, 2 4 - 2 5 Ub-conjugate degradation and, 14-15 Atrial natriuretic factor, inhibition of cAMP-induced steroidogenesis by, 69-70 Attractants bacterial swimming patterns and, 137-138 rotational bias in absence of CheZ, 142 Aurintricarboxylic acid, 11 Autoantibodies to mammalian synthetases, 127-128 ThrRS reaction with PL-7, 126 Autoimmune diseases, sjmthetases and, 127

317 Autophosphorylation of CheA, 139, 140 Autoxidation of nitric oxide, 164-167

Bacillus amyloliquefaciens, 234 Bacillus stearothermophilus pyruvate dehydrogenase complex, 246-247 surface layer of cell envelope, 247-248 Bacteria RS synthetases catalytic domains, 108 three-dimensional structures in, 101 surface layers and cell coats, 247-249 swimming patterns, 137-138. see also Bacterial chemotaxis; CheY-switch interaction Bacterial chemotaxis signal transduction in, 138-140 swimming pattern modulation, 137-138 Bacteriophage \, E. coU, 289-290 B a m a s e in protein engineering analysis, 234-235 Binding properties AspRS N-terminal domain and, 113-114 ofCheY magnesium ion to acid pocket on, 141 by switch proteins, 144 of CheZ to Chey~P, 148 of factors in protein biosynthesis, 129 of ValRS, 124 Biochemical properties of calponin amino acid sequence, 3 9 - 4 1 protein-protein interactions, 4 1 - 4 3 size and distribution, 36-39 Biosynthesis, protein channeling of amino acids and aminoacyl-tRNA to E F l , 127 SerRS and, 126 synthetases in, 129-130 in vitro versus in vivo, 257-260 BiP. see Heavy-chain binding protein Bovine pancreatic trypsin inhibitor disulfide shuffling, 224-225 stabilization of, 214 structural resemblance of fragments, 223

318 BPTI. see Bovine pancreatic trypsin inhibitor

C ring, flagellar motor, 143 C-terminal domains, see also Domains; N-terminal domains of yB-crystallin, 217-218 ofProRS, 120-121 protein stability and, 216-218 Calcium, see also Type I calciumcalmodulin phosphodiesterase Ca^^ ion in caldesmon interactions, 58 in calponin interactions ATPase inhibition, 45-47 with calmodulin, 42-43 with tropomyosin, 41 in glutathione efflux from liver, 196 in physiological role of calponin, 56-57 regulation of CheY-switch interaction by, 149 in thick filament-linked regulatory mechanism, 33 in thin filament-linked regulatory mechanisms, 34-35 Ca^^-CaM complexes refolding kinetics, 231 type I PDE activation by, 64 Caldesmon isolation of, in thin filaments, 35 properties shared with calponin, 58-59 Calmodulin, see also Type I calciumcalmodulin phosphodiesterase in ATPase inhibition, 46-47 caldesmon binding to, 58 calponin interaction with, 4 2 - 4 3 Calpain activation of, 12-13 as cytoplasmic endoprotease, 1 in degradation of oxidized proteins, 23 Calponin biochemical properties amino acid sequence, 3 9 - 4 1 protein-protein interactions, 4 1 - 4 3 size and distribution, 36-39 functional properties actin-activated myosin Mg^^-ATPase inhibition, 43-45

INDEX regulating ATPase inhibitory effect, 45-57 isolation of, in thin filaments, 35-36 properties shared with caldesmon, 58-59 structure-function relations, 57-58 Calponin phosphatase, 51-55, 57 CAMP, see Adenosine 3',5'-cyclic monophosphate CAMP-specific PDEs. see Type IV phosphodiesterase Cancer cells, see Malignant cells Carbohydrate metabolism, insulin regulation of, 88 Casein kinase II, 11 Catabolism, protein, see Proteases; Proteolysis Catalysis of aminocylation, 113-114 chaperones and folding, 263-264, 284-285 Catalysts folding chaperones as, investigation of, 284-285 description, 263-272 multicatal)i:ic protease as, 3 - 5 Catalytic domains, see also Domains of PDE gene family sequences, 67 of synthetases AspRS N- and C-terminal, 114 GluProRS, spacer sequence in, 121 mammalian versus other, 108 Cell coats, 247-249 Cell proliferation, kinase cascades and, 85-88 Cell regulation, see also Regulation MCP vs. other complexes, 2f by posttranslational modification, proteases and, 10, 11-14 of smooth muscle contractile state, 33-36 Cells, see also Eukaryotes; Prokaryotes; Tissue damage to. see Cytotoxicity differentiation and dediflferentiation of, calponin expression during, 3 8 - 3 9 malignant multicatalytic proteases and, 9, 18

INDEX NO and radiation therapy of, 180-181, 182 protein biosynthetic machinery in, 129 protein folding and assembly in. see Association; Protein folding redox status of. see Thiol redox status surface layers and cell coats, 247-249 thermotolerance, 274-277 TrpRS in ruminant pancreas, 124 types of multicatalytic protease levels versus, 8^ 9-10 PDE inhibition versus, 68 CGMP. see Guanosine 3',5'monophosphate CGMP-inhibited PDEs. see Type III cGI phosphodiesterases CGMP-specific PDEs. see Type V phosphodiesterase CGS PDEs. see Type II phosphodiesterase Channeling, aminoacyl-tRNA presorting of amino acids, 127 in vivo translation studies, 109 Chaperones components of, as assemblies, 289-292 in failed versus successful attempts to refold proteins, 210-211 folding catalysis and, 263-264, 284-285 GroE system, 277-285 kinetic partitioning and, 273-274 mechanism of, prospects for understanding, 264 reconstitution and, 294-296 specificity versus promiscuity, 285-289 as stress proteins, 274-277 suppressing protein aggregation cymogen pro regions versus, 253 requirement for, 272-273 Characterization of aminoacyl-tRNA synthetases, 105-106 of type III cGI PDEs, 70-74 CheA kinase CheZ interaction with short form of, 149 description, 139 as sensor for CheY protein, 141 Chemotaxis proteins, 139, 152

319 Chemotaxis. see Bacterial chemotaxis; Chemotaxis proteins CheW protein, 139 CheY protein chemical modifications and factors activating, 153^ description, 140-142 interaction with flagellar motor, 138 mutations in, 143-144 phosphorylation of by CheA, 139 description, 141-142 regulation by, at switch, 144-146 CheY-switch interaction, see also Switch complex CheZ in termination of, 142 regulation of by acetylation, 150-151 by calcium ion, 149 by CheZ, 148-149 CW generation, additional requirements for, 146-147, 147-148 experimental observations, 143-144 forms of, 153 by fumarate level, 151-152 by phosphorylation, 144-146 by protonmotive force, 152 CheZ protein binding to CheY, 141 description, 142 interaction with switch, 149 regulation of CheY-switch interaction by, 148-149 Cleavage, peptide bond, see also Peptidase; Proteolysis active enzyme conversion, 12-14 of glutathione, 192 by multicatalytic protease, 3 Clockwise flagellar motor rotation attractants/repellents and, 137-138 CheY aceltylation of, 150, 151 overproduction of, 144 phosphorylation of, 145 as signaling molecule, 138 cytoplasmic constituents required, 146-147 switching from CCW to, 147-148 termination of, by CheZ, 140

320 Cloning process energy dependence of 26S subunits, 7 o f G l u P r o R S , 122 of mammalian synthetases, 107-108 ArgRS, 118 LysRS, 119 of multicatalytic protease subunit genes, 3 ofProRS, 120-121 ofSerRS, 126 of switch proteins, 144 of TrpRS, 124-125 of type III cGI PDE subfamilies, 74-75 of ValRS, 123 Clp protease 26S and Lon protease similarities to, 7 - 8 , 13 N-terminal residues, missing, 13 Codon usage, rates of translation and, 211 Constitutive nitric-oxide synthase, 161, 182 Counterclockwise flagellar motor rotation attractants/repellents and, 137-138 CheZ in presence of CheY, 142 flagella of gutted strains defaulting to, 144 PMF level reduction and, 152 switching to CW from, 147-148 y-Crystallin, stabilization of, 214-215 yB-Crystallin denaturants changing behavior of, 236 stabilization of, 217-218 Crystallins dimer stability and domain swapping in, 242-243 productive versus nonproductive pathways, 254 CW. see Clockwise flagellar motor rotation Cyclases, see Adenylyl cyclase; Guanylate cyclase Cyclic nucleotide phosphodiesterase cyclic nucleotide concentrations, 64 gene families, 6 4 - 7 0 ion channels gated by, 63 Type III cGMP-inhibited activation in insulin action, 78-80 adipocyte regulation mechanisms, 88-91

INDEX cloning of subfamilies of, 74-75 hormonal regulation of, 75-78, 80-84 insulin, kinase cascades and, 8 5 - 8 8 purification and characterization, 70-74 Cyclic nucleotides PDEs contribution to hydrolysis in, 68 determining concentration of, 64 protein kinase mediation of effects of, 63 Cyclins, required destruction of, 1 Cyclophilins, 269-272 Cyclosporin A in folding catalysis, 269-262 Cymogen pro regions, 253 CysRS synthetase, sequence, 107 Cysteine in glutathione synthesis, 191, 193 increasing in HIV patients, 203 Cysteine prodrug bypassing intestinal digestion, 192 hepatic GSH concentrations, 194 Cysteine protease, 3 - 5 Cysteine residues in disulfide shuffling of BPTI, 225 NO modification of, 167, 168 Cystine glutamate inhibition of, 193 hormones increasing transport of, 198 redox reactions and disulfide shuffling, 265 Cystine bridges formation during translation of interdomain, 236 lack of, in y-crystallin, 215 reduction leading to BPTI unfolding, 214 Cytochrome c in early folding and docking, 228-232 productive versus nonproductive pathways and, 254 Cytoplasm, muscle, see Sarcoplasm Cytoplasmic endoproteases activation of, 12 types identified, 1 Cytosolic solvent parameters description, 260-263 GroE system activity and, 278

INDEX Cytotoxicity GSH depletion sensitizing cells to, 202 nitric oxide and metal, enhancement of, 171 physiological role versus, 160 ROS, effect on chemically generated, 176-178 enzymatically generated, 178-179 in ionizing radiation, 180-181, 182 NO, and ROS-induced, 181-182 D Degradation, protein, see Proteolysis Deoxyribonucleic acid nitric oxide-caused damage to nitrosative deamination and strand breaks, 171-172 repair protein inhibition, 170-171 as toxic agent, 160 peroxide-caused damage prevention by nitric oxide, 179 thiol redox status and, 189-190 type III cGI PDEs encoded by, 74-75 Dephosphorylation of calponin calponin phosphatase activity, 5 1 - 5 5 reassociation with thin filaments, 57 of lipase, insulin and, 89 of myosin, 33 DIFP protease inhibitor in ProRS dissociation, 120 in ValRS purification, 123 Dihydrofolate reductase, 232-234 Dihydrorhodamine oxidation, 178-179 Dimerization, domain swapping and, 242-243 Diseases autoimmune, mammalian synthetases and, 127 malnutrition and low GSH levels in morbidity and mortality from, 200-201 in vivo GSH homeostasis, 190-191 NO as causative agent, 160, 176, 183 Dissociation of Arginyl-tRNA synthetase, 119 of Asp-tRNA from AspRSA32, 114 of chaperone assemblies, 289 of CheY from quaternary complex, 141

321 of ProRS from synthetase complex, 120 of RS complex, 105-107 of ValRS complex, 123 Disulfide bonds in aggregation reactions, 293-294 PDI and correct formation of, 267 protein stability and, 214 shuffling of of BPTI, 224-225 enzymes catalyzing, 263-266 in vitro formation of, 264 DNA. see Deoxyribonucleic acid DNA-interacting proteins, nitric oxide inhibition of, 170-171 DnaK chaperone, 287, 289-229 Domain proteins, folding of, 235-238 Domains, see also C-terminal domains; Catalytic domains; N-terminal domains docking of cytochrome c and ribonuclease in early steps of, 228-232, 232 in stabilization process, 217-218 evolution of, 242-244 folding proteins with single b a m a s e , 234-235 " BPTI, disulfide shuffling in, 224-225 cytochrome c and ribonuclease, 228-232 lysozyme and dihydrofolate reductase, 232-234 ribonuclease proline isomerization, 225-227 ribonuclease unfolding intermediates, 227-228 lysozyme structural lobes versus, 232n packing of, 221 pairing of in folding kinetics of two-domain proteins, 237-238 a-LA molten globule properties, 221 PDI versus DsbA, 266-267 in productive versus nonproductive folding pathways, 254-255 stabilization of, 216-217 Drosophila GluProRS catalytic domain spacer sequences, 121

322 proteases absence of MCP from nuclei, 18 expression of MCP and 26S subunits, 10 MCP concentration in embryonic tissue, 9 oocyte /x-particle and 26S, 15 phosphorylation of MCP subunits, 11 Drugs increading oxidative stress, 204 inhibiting PDE gene famihes, 68-69 type III cGI PDEs, 66, 70 Dsb enzyme system, 264-268 DsbA enzyme, 264-268. see also Protein disulfide-isomerase DsbB enzyme, 265 DsbC enzyme, 265 E Effector, cGMP as allosteric, 64-66 18S S5nithetase complex dissociation, 106 Elongation factor l a aminoacyl-tRNA channeling to, 109, 116-117 Asp-tRNA dissociation from AspRS, 114-116 mammalian synthetase sequences homologous to, 109 ValRS N-terminal domain and, 123 phosphorylation revealing interactions between, 124 Embryonic development calponin expression during, 38-39 MCP peptidase activities during, 21 26S peptidase activities during, 21-22 Embryonic tissue Drosophila MCP concentrations in, 9 MCP subunit expression, changes in, 10 subcellular distribution of MCP in, 18 Endoproteases, cytoplasmic, 1 Enthalpy in globular protein stabilization, 213 in quaternary structure formation, 221 Entropy globular protein stabilization and, 213 in quaternary structure formation, 221

INDEX Enzymatic properties of 26S protease, 5 - 8 of MCP, 2 - 5 Enzymes, see also Isoenzymes; Multicatalytic protease; 26S ATP/ ubiquitin-dependent protease; entries for specific enzymes biosynthetic components and, 129 catalyzing folding rate-determining steps description, 263-264 peptidyl prolyl cis-trans-isomerases, 268-272 protein disulfide-isomerase, 264-268 in CheY acetylation, 150 fumarate level, affecting cellular, 152 glutathione synthetic diet and nutritional status effects, 194 locational specificity of, 192 in multienzyine superstructure, 246-247 NO and RNOS inhibition of, 167-171, 182 rapidly degraded, 22 regulated by, thiol:disulfide exchange, 189-190 steady-state concentration of, 9 synthesis of, by peptide bond cleavage, 12-14 synthetases in multienzyme complexes, 104-105 occuring as free soluble, 102 ERKs. see Mitogen-activated kinase cascades Erythrocytes multicatalytic protease inhibitor in, 17 oxyhemoglobin and NO concentrations, 172-173 PEM reducing GSH concentrations in, 201 Escherichia coli. see also Bacteria AspRS expression in, human, 113 flagella per cell, 140. see also Bacterial chemotaxis fumarate as switching factor, 151-152 GroE system in bacteriophage X, 289-290 GroE system of, 277-285 Lon and Clp proteases, 7 - 8

INDEX RS structures resolved in, 101 temperature and refolding of Thermotaga maritima in, 260-261 Eukaryotes. see also Prokaryotes apoferritin, 245-246 chaperone assemblies, classes of, 290t c)d:opasmic endoproteases, 1 MCP and 26S distribution, 18-21 protease subunits in expression of MCP and 26S, 10 MCP cloning, 3 Excitation in signal transduction, 138 Exons domains as phylogenetic products of, 236 Glu and ProRS, encoding, 122 Extensions, mammalian synthetase N and C-terminal, 108-111. see also C-terminal domains; Domains; N-terminal domains Extracellular regulators, PDE gene family, 65 Extracellular signal-regulated kinases. see Mitogen-activated kinase cascades

F-actin, 41 Fab2 enzyme, 259 Feedback regulation ofcAMP, 72 of glutathione synthesis, 191, 193 FK 506-binding proteins, 269-272 Flagella bacterial versus eukaryotic, 140 nomenclature for gene products of bacterial, 143n Flagellar motor C ring, switch proteins on, 143 CW and CCW states of, 147-148 in excitation process, 138 in signal transduction pathway, 140 Flagellar rotation CheZ and, 142 in gutted strains, 144 PMF regulatory effect on, 152 proteins involved in, 140 restoration to imstimulated mode, 138 swimming modes resulting from, 137-138

323 switch proteins and, 143 switching from CCW to CW, 147-148 FUG protein, 143-144 FliM protein binding to wild-type, 145 mutations and, 143-144 FUN protein, 143 Fluorogenic peptides MCP in complexes affecting, 3 proteins activating hydrolysis of, 15-16 Folding catalysts chaperones as, 284-285 description, 263-272 Folding intermediates, see Intermediates, protein folding Formamidopyrimidine-DNA glycoslase, 170-171 Fumarate, 151-152 G Gene transcription, see also Transcription factors ATPase subcellular location and, 20 thiol redox status and, 189-190, 200 Genes calponin a and /3 sequences, 39 cytoplasmic and mitochondrial synthetases encoded by same, 128 DsbA and PPI, isolation of, 264 GluProRS, 122 PDE gene families as products of related, 67 protein folding, encoding, 209 regulator proteins and stimulus response by, 141 ValIRS, location of, 123 GlnRS synthetase cDNA sequence, 107 initial identification of ProRS cDNA as, 120-121 Globular proteins disulfide formation as rate-determining step in refolding, 225 stability of, enthalpy versus entropy in, 213 Glucagon cGI PDE activity in hepatocytes, 76-77

324 glutathione homeostasis regulation, 195 GluPro-tRNA synthetase cDNA sequence, 107 description, 120-122 GluRS synthetase, 120-121 Glutamate in glutathione synthesis, 191 hormones increasing transport of, 198 inhibition of cystine by, 193 Glutamine, 193 y-Glutamylcysteine synthetase biosynthesis of, 198-199 in glutathione synthesis feedback inhibition of, 191, 193 protein kinase activation inhibiting, 197 substrates for, 191 y-Glutamytranspeptidase, 191 Glutathione in disease, infection, malnutrition cycle, 200-204 homeostasis of, interorgan, 191-192 as NOx scavenger, 167, 182 range of tissue concentrations, 190 regulation of by diet and nutritional status, 192-195 by hormones, 195-200 synthesis of, 191 therapies decreasing levels of, 190-191 to enhance tissue concentrations, 191 thiol redox status regulation, 189-190 Glutathione disulfide, 190 Glutathione reductase, 190 Glutathione S-transferase, 113 Glutathione synthetase, 191 Glycogen, glutathione and mobilization of, 195 Glycogen synthase, 88 Glycogenolysis, cGI PDE activity and, 78-80 Glycoproteins protein folding, impact on, 263 variable surface, 248-249 Glycosylation invertase folding and stability, 262-263/"

INDEX multicatalytic protease regulation, 11-12 GlyRS synthetase autoantibodies to, 127 core catalytic domains, 108 GroE system, 277-285 GroEL, 264, 285-286. see also GroE system GSH. see Glutathione GSSG. see Glutathione disulfide GST. see Glutathione S-transferase GTP-binding proteins, 86. see also Ras protein Guanidinium chloride denaturation renaturation without accessory proteins, 246-247 single transition in, 26 Guanosine 3',5'-monophosphate calcium-calmodulin-sensitive PDE affinity for, 6 4 - 6 6 in mediating stimulus response, 6 3 - 6 4 Guanosine 5'-triphosphate in Asp-tRNA dissociation from AspRS, 114 calponin function regulation and, 47-48 Guanylate cyclase activation in vivo, 167 cAMP generation, 63 nitric oxide activation of, 167, 182 H Halobacterium salinarium, 151-152 Heat-shock proteins assemblies of, 291-292 chaperones as, 274-277. see also Chaperones target protein recognition by, 286-289 Heavy-chain binding protein, 286-287 HeLa cells MOP a subunit production, 13 polypeptides in 26S protease from, 10-11 Helices algorithm predicting occurence of a, 25 amphiphilic in h u m a n AspRS, 112, 116 in synthetase N-terminal domains, 109-111 fast formation of, 232

INDEX Heme proteins lipid peroxidation and cytotoxicity, 177-178, 181 nitric oxide reactivity with, 162 Hemoglobin degradation of oxidized, 22-23 nitric oxide controlling concentrations of, 172-173 reaction with dioxygen heme complexes, 162-163 Hepatic glutathione, see also Glutathione regulation of, 192-193 release into bile, 191 sulfur amino acid supplementation, 194-195 Hepatocytes hormonal regulation of decreased glutathione synthesis by, 198 glutathione efflux from, 196-197 type III cGI PDE feed-back regulation of cAMP content, 72, 76-77 insulin or glucagon and, 76-77, 78-80 High Mr synthetases mammalian synthetase studies and, 102 Valyl-tRNA synthetase as, 102 HisRS synthetase autoantibodies to, 127 N-terminal domain in yeast, 108 Histidine, 4 - 5 Homeostasis, glutathione interogan, 191-192 regulation of tissue concentrations, 190 Hormonal regulation of glutathione description, 195-200 factors altering metabolism, 190-191 nutritional status and, 195 PDE gene family regulators, 66^ oftypelllcGIPDEs cAMP increases, 72 experimental results, 75-78 mechanisms for, 80-84, 8 8 - 9 1 Hsps. see Heat-shock proteins Human immunodeficiency virus infections delaying onset of AIDS in, 202

325 glutathione synthesis inhibition, 193-194 Hydrogen peroxide, nitric oxide and, 163, 176 Hydrolysis of ATP on myosin molecules, 33 ofcAMPandcGMP to 5' mononucleotides, 64-67 insulin increasing, 75-76 of NOx species to nitrite, 165 peptide bond by MOP activators of, 14-16 inhibitors of, 17-18 while inhibiting cleavage of others, 3 Hydrophobic interactions in aggregation reactions, 293-294 in association of synthetases, 105 ofGluProRS, 121 of LysRS extension, 119 Hydrophobic residues, chaperone affinity for, 285 Hypoxanthine/xanthine oxidase, 178-179

IleRS synthetase autoantibodies to, 127 cDNA sequence, 107 dissociation from synthetase complex, 105 purification and characterization, 106 Immune system GSH levels in malnutrition, 201-204 mammalian synthetases and, 127 NOx and antiviral activity of, 171 PPIs inhibiting immunosuppression, 268 in ROS and NO formation, 175 Immunoreactivity of calponin, 3 7 - 3 8 Inclusion bodies, see also Aggregation in aggregation versus association, 255 formation of, practical aspects in, 293-294 isolation of, 294-295 recovery of tPA, 210 Inducible nitric-oxide synthase, 161, 182 Infections, GSH depletion and, 201-204 Inhibition of actin-activated myosin Mg2^-ATPase, 4 3 - 4 5

326 by cGMP of cAMP hydrolysis, 66 of chemotaxis, 149 of cystine by glutamate, 193 of enzymes, by NO and RNOS, 167-171 of y-glutamylcysteine synthetase, 191, 193, 194 of glutathione efflux of, from liver, 196 synthesis, by hormones, 197-198 of insulin action by wortmanin, 88 of leukoc5rte and neutrophil adhesion, 180 of lipolysis by insulin, 78-80 of multicatalytic proteases, 3 - 5 , 18 of nitrosation by NO-superoxide reaction, 178 of proteins by NOx, 181 of signal transduction by GSH depletion, 189-190 oftypelllcGIPDE by cGMP, 72 by drugs, 66, 70-72 by phosphotase, 81 Inhibitors of multicatalytic protease, protein, 17-18 PDE gene families inhibitor versus family, 65t, 66 physiological effects of, 68-69 ofPPI, 269 protease DIFP, 120, 123 MCP peptide cleavage and, 3, 5 PDE purification and, 72 in synthetase purification, 103 of RSs, anti-JOl antibodies, 127 of Ub-conjugate degradation, 26S ATPase complex phosphorylation, 11 INOS. see Inducible nitric-oxide synthase Inositol phosphates in glutathione efflux from liver, 196 second messengers of insulin action, 79-80 Insulin hepatic GSH concentrations and, 198 type III PDEs and in adipose tissue and liver, 75-76 inhibition of lipolysis by, 78-80, 89-91

INDEX initiating reactions activating, 70 kinase cascades in cell metabolism/ proliferation regulated by, 85-88 serine phosphorylation and, 80 Insulin receptor substrate mediation of Ras activation, 86-87 in type III cGI PDE activation by insulin, 90 Insulin-stimulated protein kinase, 88 Insulinase, 23 y-Interferon MCP subunits and antigen presentation, 10 TrpRS synthesis, stimulating, 125, 128 Interferons stimulating TrpRS synthesis, 128 TrpRS gene activation by, 125 Intermediates protein folding aggregation versus association, 255 description, 221-224 dihydrofolate reductase, 232-234 in disulfide shuffling of BPTI, 225 fast precursor reaction of oligomeric, 248 kinetically-trapped, 253 lysozyme and dihydrofolate reductase early, 232-234 optically triggered folding reactions, 228-229 ribosomal, 249-251 Un-*N transition, 224 protein unfolding, 227-228 Intestine, glutathione and, 191, 197 Intracellular regulators, PDE gene families, 65^ Invertase, 262-263/* Ion pairs in protein stabilization, 213-214 Ionizing radiation, NO and, 180-181, 182 Iron, nitric oxide reactivity with, 162 Ischemic reperfusion injury, NO and, 176, 180 Isoenzymes in cAMP and cGMP hydrolysis, 64 Isoforms of calponin amino acid sequences, 3 9 - 4 0 cysteine residues of a and )3, 57

INDEX cis-trans Isomerization, proline residue, 225-227, 259 Isopeptide bonds, proteolysis of, 24-25 Isoproterenol activation of type III cGI PDEs, 76, 77,80 inhibition of lipolysis stimulated by, 79-80 K Kinase cascades, 8 5 - 8 8 Kinases, see also Kinase cascades; Myosin light-chain kinase bacterial protein regulators and, 141 cAMP-dependent hormone-sensitive lipase, 8 8 - 8 9 PDE and insulin action, 76-78 serine phosphorylation, 80-84 cyclic nucelotide-mediated biological responses and, 63 insulin-sensitive, 88, 90 phosphorylation by noncAMPdependent, insulin-sensitive, 82-84 protein activated by insulin, 90 associated subunits of, 22 calponin in smooth muscle, 56, 57 as substrate for, 49 cAMP-dependent, 76, 77-78 MCP and 26S protease regulation by, 11 Kinetic barriers in cytochrome c folding path, 230-231 Kinetic partitioning chaperones description, 273-274 determining selectivity of, 288 DnaK binding, 287 in TSP assembly, 252-253 Uni-Bi molecular mechanism, 255-256 Kinetics protein folding of domain proteins, 236-238 intermediate identification by altering, 222 late events in, 227-228 lysozyme two-state characterisics, 232-233

327 measurement of early steps, 229 in off-pathway reactions, 252-253 productive versus nonproductive pathways, 255-257 RNases and structure formation, 225-227 structure-function relationship, 239 of tRNA aspartylation, 114-116 Kwashiokor GSH tissue concentrations in, 201 infections precipitating clinical signs of, 202

a-lactalbumin a-helical domain tertiary fold resemblance to intact molecule, 224 molten globule properties, 220-221 Lactate dehydrogenase, 218 Leukocyte adhesion, nitric oxide and, 180 LeuRS synthetase dissociation from synthetase complex, 105 purification and characterization, 106 Light, cGMP hydrolysis in response to, 66 Lipase, activation of hormone-sensitive, 8 8 - 8 9 phospholipase C, 79 Lipids, NO in peroxidation of chain termination, 163 LDL oxidation and, 179-180 Lipolysis, type III cGI PDE activity and insulin inhibition of, 78-80, 8 9 - 9 1 stimulation of, 76, 88-89 Liver, efflux of hepatic GSH from, 191, 195-196 Localization of AspRS, subcellular, 117-118 Lon protease, 7 - 8 Luciferase folding pathways in bacterial, 252 in protein folding studies, 259 Lux AB enzyme, 259 Lysine-109, 147-148 Lysosomal cathepsins abundance in eukaryotic cells, 1 activation of, 12

328 Lysozymes early intermediates for, 232-234 phage T4, stabilization of, 213 Lysyl-tRNA synthetase, 119-120 cDNA sequence, 107 dissociation from synthetase complex, 105 purification and characterization, 106 M Magnesium Mg^^ ion binding with CheY, 141 in myosin competition test, 34 Major histocompatibility complex BiP ATPase activity analogous to, 286-287 genes for MCP-like subunits in, 10-11 Malignant cells MCP concentration in, 18 MCP subunit synthesis in human, 9 NO and increased radiosensitization of hypoxic, 180-181, 182 Malnutrition GSH levels in cyst(e)ine uptake in wasting, 193-194 drugs and oxygen therapies decreasing, 190 insulin resistance and, 198 GSH synthetic enzyme levels, 194 Mammalian aminoacyl-tRNA synthetases amino acid classifications and, 126-127 arginyl-tRNA synthetase, 118-119 aspartyl-tRNA synthetase, 112-118 autoantibodies to, 127-128 classification of, 102 dissociation and organization of, 105-107 functional significance of, 111-112 gluPro-tRNA synthetase, 120-122 lysyl-tRNA synthetase, 119-120 as multifunctional proteins, 128-129 N-terminal domains, 108-111 prospects for future research, 129-130 protein biosynthetic machinery and organization of, 129 research of, overview of progress in, 101-102

INDEX seryl-tRNA synthetase, 125-126 structures of general, 102-105 primary, 107-108 threonyl-tRNA synthetase, 126 tryptophanyl-tRNA synthetase, 124-125 valyl-tRNA synthetase complex, 122-124 Mammalian P450 enzymes, 168-170 MAPK. see Mitogen-activated kinase cascades Marking hypothesis, protein, 6 - 7 Matrix-bound polypeptides, reconstituting, 296-297 MCP. see Methyl-accepting chemotaxis protein; Multicatalytic protease Meiosis, insulin stimulation of, 78-79 Membrane receptors, chemical stimuli interactions with, 138 Meromyosin, heavy, 4 4 - 4 5 Messenger ribonucleic acid calponin, detection of, 40 ofGluProRS, 122 polyanion binding, 129 oftype III c G I P D E s , 74-75 Metabolism, cellular kinase cascades in regulation of, 85-88 nitric oxide in hemoglobin concentrations, 172 mitochondrial respiration and, 173-175 Metal complexes, NO reactivity with, 161-163 Metalloprotease, 1 Metalloproteins, 162-163 Metallothionein, metals released from, 171, 181 Metals, nitric oxide and enhancement of toxicity of, 171 labilization of, 167, 181, 182 Methyl-accepting chemotaxis protein, 138-140 MetRS synthetase derepression of, in CHO cells, 104 dissociation from synthetase complex, 105 endoplasmic reticulum and, 112 purification and characterization, 106

INDEX Mg2+-ATPase inhibition of actin-activated myosin caldesmon and, 59 by calponin fragments, 57-58 calponin phosphorylation on, effect of, 4 9 - 5 1 , 55 description, 43-45 MHC. see Major histocompatibility complex Micelles, reverse, 296-297 Microbial surface structures, 247-249 Microorganisms, see Bacteria; Viruses Microsomes, MCP-generated peptides in, 11 Mitochondria NOx-caused dysfunction of, 181 respiration of, NO and, 173-175 Mitogen-activated kinase cascades insulin activation of, 85-88 type III cGI PDE activation, 90 Mitosis, MCP subcellular distribution during, 18 MLCK. see Myosin light-chain kinase Molecules activation/inhibition of MCP by small, 18 inhibiting MCP-induced peptide bond hydrolysis, 17-18 Molten globule state information transfer from one to three dimensions, 224 native and denatured states versus, 214 properties of, 220-221 M.O.P. complex, 23. see also Multicatalytic protease MotA and B proteins, 140 Motor, flagellar, see Flagellar motor Multicatalytic protease as cytoplasmic endoprotease, 1 during development, cell cycle, and after physiological stress, 21-22 protein complexes, interactions with, 2f regulation by, 2 2 - 2 4 regulation of by associated proteins, 14-18 expression of subunits, 10-11 by posttranslational modification, 11-14 protease levels, 8-10

329 structural and enzymatic properties, 2-5 subcellular distribution of, 18-21 subunits 26S protease, proposed as parts of, 7 expression of specific, 10-11 Muscle MCP peptidase activities in fasting rat, 22 smooth calcium in sarcoplasm of resting, 56 calponin expression in, 37-39 mechanisms regulating contractile state of, 33-36 Mutagenesis calponin structure-function relations and, 57-58 in oligomeric/multimeric protein evolution, 242 protein folding interactions and site-directed, 234-235 Mutations in protein folding/unfolding pathways, 234-235 in regulation of CheY-switch interaction, 143-144 temperature-sensitive folding point and second-site suppressor, 294 Myosin actomyosin ATPase activity inhibition, 43-45 caldesmon interaction with, 58 smooth muscle contractile state and, 33 Myosin competition test, 34-36 Myosin light-chain kinase, 33, 56 Myositis, ThrRS reaction with autoantibody in, 126 N N-terminal domains Arginyl-tRNA synthetase, 118-119 Aspartyle-tRNA synthetase, 112-113 in association to synthetase complex, 117 peptides in, synthetic, 116 in synthetase-aminoacyl-tRNA interaction, 114-116 yB-crystallin, versus stability of C-terminal, 217-218 Lysyl-tRNA synthetase, 119-120

330 mammalian synthetases characteristics of, 108-111 GluProRS catalytic domain as template for, 121 protein stability and, 216-218 ValRS complex, mediating association of, 123 N-terminal modified proteins, 128 Neutrophils, NO prevention of adhesion of, 180 Nitric oxide biochemical targets for DNA, 171-172 enzymes, inhibition of, 167-171 chemistry of metal complexes, reactivity with, 161-163 peroxynitrite, formation of, 164 radicals, reactivity with, 163 RNOS from NO/O2 reaction, 164-167 types of reactions involved in, 161, 173f direct versus indirect effects compared, 160-161 description, 182-183 extracellular and intracellular metabolism of hemoglobin in NO concentration control, 172-173 lifetime of, 165, 172-173 mitochondrial respiration, 173-175 oxidative stress and cytotoxicity by chemically-generated ROS, 176-178 formation of ROS with, 175-176 ionizing radiation and, 180-181 on lipid peroxidation, effect of, 179-180 NOx and ROS-induced cytotoxicity, 181-182 ROS, effect on biologically-generated, 180 enzymatically-generated, 178179 regulation of physiological processes, 159-160 as toxin versus physiological functions of, 160 Nitric-oxide synthases, 161, 182

INDEX Nitrite NOx hydrolysis to, 165-166 oxidation to nitrogen dioxide, 181-182 2-Nitro-5-thiocyanobenzoic acid, 5 7 - 5 8 Nitrogen dioxide, NO autoxidation and, 165, 181 Nitrogen oxide, reactive species of in nitric oxide autoxidation, 165 ROS-induced cytotoxicity and, 181-182 toxic effects of, 160 Nitrosation, inhibition of, 178 S-Nitrosothiol adducts, 166-167 Nitrosyl complexes, 162-163 Nitrovasodilators NONOates and, 176-177 prostaglandin effects on platelets and, 70 NOx- see Nitrogen oxide, reactive species of NONOates, 176-177 NTCB. see 2-Nitro-5-thiocyanobenzoic acid Nucleotides, synthesis of signal, 128. see also Cyclic nucleotides Nucleus, presence of MCP in cell, 18-21 O ODC. see Ornithine decarboxylase Off-pathway protein folding reactions productive versus nonproductive pathways, 253-257 thermodynamics versus kinetics, 252-253 Oligomeric proteins assembly of, requirement for chaperones in, 273 complicating genetic code for folding, 209 evolution of, 241-242 Oligopeptides, unfolding/folding behavior, 215-216 Ornithine decarboxylase, 24 Oxidants, peroxynitrite as, 164 Oxidation of dihydrohodamine by peroxynitrite, 178-179 DsbA reoxidation, 267 nitric oxide and autoxidation of, 164-167 halflife in mitochondrial respiration versus, 173

INDEX P450 enzyme inhibition, 168-170 as protection against peroxidemediated, 176-178 of nitrite to nitrogen dioxide, 181-182 Oxidative stress glutathione levels decreased by, 190, 202 nitric oxide in cytotoxicity by chemically-generated ROS, 176-178 formation of ROS and, 175-176 ionizing radiation and, 180-181 lipid peroxidation and, 179-180 NOx and ROS-induced cjdotoxicity, 181-182 ROS, effect on biologically-generated, 180 enzymatically-generated, 178-179 oxygen and drug therapies increasing, 204 in severe wasting and cachexia, 203 2-Oxoacid dehydrogenases, quaternary structure of, 247 Oxygen, see Reactive oxygen species Oxyhemoglobin erythrocyte NO levels and, 172-173 nitric oxide reactivity with, 162-163, 167 Oxymyoglobin, NO reactivity with, 162-163, 167

P97 protein-MCP interaction, 2, 16 P450 enzymes, 168-170 PAGE patterns, MCP and other subunit, 4/* PDE. see Cyclic nucleotide phosphodiesterase PDH. see Pjrruvate dehydrogenase complex PDI. see Protein disulfide-isomerase PEM. see Protein-energy malnutrition Peptidase, 21, 22. see also Cleavage, peptide bond Peptides, see also Pol)rpeptide chains affinity of sulhydryl-containing for NO,, 166 fluorogenic, MCP and, 3 Glu- and ProRS into single, fusion of, 121

331 MCP cleavage of, 3 in microsomes, y-IFN-induced MCP subunits, 11 synthetic of N-terminal domain in AspRS, 116 PDE phosphorylation sites and, 81-82 ValRS, 124 Peptidyl prolyl cis-trans isomerases, 226, 268-272 Peroxides in nitrate to nitrogen dioxide oxidation, 181 NO protection against Fenton-typemediated toxicity, 177 Peroxy radicals, NO reactivity with, 163 Peroxynitrite aconitase inactivation, mediating, 167 nitric oxide reactivity with superoxide, 164 NO concentration required to form, 174 NO-XO interactions and, 178-179 PH levels protein denaturation and, 214 in vitro versus in vivo folding conditions, 261 PheRS synthetase, 112 Phosphatase, cells containing insulinsensitive PDE and, 88 Phospholipase C, 79 Phosphorylation activated kinases catalyzing, 63 of bacterial protein regulators, 141 of calponin actomyosin ATPase activity inhibition, 4 9 - 5 1 , 55-57 in resting smooth muscle, 56 ofCheY in acetylation of, 150-151, 153 by CheA, 139 CheZ binding and, 148 description, 141-142 CheY-switch interaction regulation by, 144-146 insulin-initiated kinase cascades, 85, 86-87 of MCP and 26S proteases, 11 of myosin experimental measurement of, 43-45

332 regulating muscle contractile state, 33 in resting smooth muscle, 56 type III cGI PDE activation via serine, 80-84 of ValRS complex, 124 Phosphotase inhibitors, 81 Photoreceptors, 66 Phototaxis of H. salinarium, 151-152 Physiological role of calponin, 56-57 of glutathione synthesis inhibition, 198 of nitric oxide, 160 Physiological stress, MCP and 26S activities after, 2 1 - 2 2 PI-3 kinase activation, 87 type III cGI PDE ISK activation, 90 Wortmanin as inhibitor of, 88 Platelets, type III cGI PDEs and activity in, increased, 72 insulin and, 77 potentiation of prostaglandin inhibitory effects on, 70 purification of, from human, 70 serine phosphorylation and, 81 PMF, see Protonmotive force Pole cells, MCP accumulation in, 9 Polyanions in protein biosynthesis, 129 Polymorphism in tertiary to quaternary interactions, 249 Polyols, 261 Pol5rpeptide chains, in 26S protease, y-IFN-treated HeLa cells, 10-11 Polypeptide chains, see also Peptides in 26S protease, MCP subunits and, 7 cheA gene, encoded by, 139 coagulation of heat-denatured, 275 complicating genetic code for folding, 209 conformational restrictions of folded/ unfolded, 218-219 disulfide transfer to folding, 265-266 exit from ribosomes, 292 GroEl interaction with hydrophobic portions of, 286 inhibitor forming multimers as, 17 matrix-bound, reconstitution of, 296-297

INDEX in MCP complexes, y-IFN-treated HeLa cells, 10-11 misfolded, rescue of, 267. see also Chaperones stability of, N-terminal/C-terminal ends and, 216-218 synthetase complex identification of, 103-104 ValRS, 122 in vivo versus in vitro folding, 257-260 Posttranslational modification, see also Phosphorylation MCP and 26S proteases embryonic subunit synthesis, 10 glycosylation, 11-12 phosphorylation, 11 proteolytic processing, 12-14 in protein folding, 211 PPIs. see Peptidyl prolyl cis-trans isomerases Pressure as cytosolic solvent parameter, 261 Primary structure of proteins, 225 Pro region, cymogen, 253 Prokaryotes. see also Eukaryotes chaperone assemblies in, 290^ Dsb enzyme system characteristics in, 265 dsbA gene mutations, 264 MCP-like protease in, 3 Proline endopeptidase, 1 Proline residues cis-trans isomerization of, 225-227, 259 enzymes catalyzing, 263 Properties binding AspRS N-terminal domain and, 113-114 ofCheY, 141, 144 o f C h e Z t o C h e y ~ P , 148 of factors in protein biosynthesis, 129 of ValRS, 124 biochemical, calponin, 36-43 PDE gene families, 65^ of proteins in reverse micelles, 296-297

INDEX structural of 26S protease, 5 - 8 of aminoacyl-tRNA synthetase complex, 102-105 of apoferritin, 245-246 of calponin versus function, 57-58 E. coli ATP-dependent proteases, 7-8 of eukaryotic heat-shock proteins, 274 ofMCP, 2 - 5 of PDE isoenz5nnes, 67 of unfolded/partially unfolded proteins, 218-221 ProRS synthetase, 121. see also GluPro-tRNA synthetase Prosomes, equivalence to MCP of certain, 9 Prostaglandins, platelets and, 70 Proteases, 2. see also Multicatalytic protease; 26S ATP/ubiquitindependent protease Protein biosynthesis amino acids and aminoacyl-tRNA channeling, 127 SerRS and, 126 synthetases in, 129-130 in vitro versus in vivo, 257-260 Protein disulfide-isomerase. see also DsbA enzyme as folding catalyst, 264-268 PPI catalysis and, 270 significance as folding helper in vivo, 264 stability in reduced versus oxidized state, 214-215 Protein engineering, analysis of protein folding and, 234-235 Protein folding cellular aspects of chaperones, 272-292 cytosolic solvent parameters, 260-263 folding catalysts, 263-272 in vitro versus in vivo issue, 257-260 description, 209-212 difficulties in elucidating mechanisms of, 299-300 genetic coding of, 209

333 hierarchies of structure, stability and folding, 212-218 history of research on, 297-299 mechanisms of association, 238-244 domain proteins, 235-238 folding intermediates, 221-224 off-pathway reactions, 252-257 small, single-domain proteins, 224-235 structure formation, 218-221 superstructures, 244-251 pathways for biological time scale requiring, 209-210 BPTI, elucidation of, 224-225 chaperone alteration of, 283-284 multiple, 233-234 protein engineering analysis of, 234-235 in vivo versus in vitro, 258-259 practical aspects aggregation and inclusion body formation, 293-294 reconstitution with accessory proteins, 294-296 reverse micelles and immobilized proteins, 296-297 propects for future research, 300 stabilization of, 212-218 structure formation of, 218-221 hierarchies of, 212-218 time-scale resolution intrisic markers in, 238 limitations of, overcoming, 228-232 Protein kinase C in glutathione efflux from liver, 196 inhibition of, 167-168 Protein-energy malnutrition GSH tissue concentrations, 201 infections and, 202 Protein-protein interactions calponin in ATPase inhibition, types responsible for, 4 9 - 5 1 with caldesmon, 5 8 - 5 9 investigations of, 4 1 - 4 3 MCP, 2f

334 Proteins, see also Protein folding; entries for specific proteins accessory multienzyme system formation and, 246-247 in ribosome assembly, 249 aminoacyl-tRNA synthetases as, 128-129 biosynthetic machinery for, 129 calponin sequence homology, sharing, 40-41 in CheY-switch interaction CheY, 140-142 CheZ, 142 in switch complex, 143 coiled coil regions in, 25-27 contractile, 43 flagellar motor rotation, 140. see also Chemotaxis proteins glutathione S-transferase-AspRS fusion, 113 GTP-binding, insulin and, 86 immobihzed, 296-297 marking for destruction, by ubiquitin, 6 methyl-accepting chemotaxis, 138-140 molten globule state, see Molten globule state nitric oxide inhibiting, 170-171 interaction with, 167-171 NO, inhibition of, 181 nonconjugated, 24 proteases removing degraded, 1. see also Proteolysis receptor/effector, 6 3 - 6 4 reconstitution with accessory, 2 9 4 296 regulating MCP activators, 14-16 inhibitors, 17-18 small molecule, 18 regulatory isolated from chicken gizzard, 43 short half life of, 22 stability in cytoplasm versus nucleus, 21 structural hierarchies, 212-218 sulfhydryl oxidation state of, 189 switch complex, 143

INDEX Proteolysis ATP/Ub-mediated in fasted rat muscle, 22 marking proteins for destruction, 5-7 multimer-forming inhibitor, 17 N-terminal modified proteins marked for, 128 of calponin, 57-58 enz)niies from inactive precursors, 12-14 of MCP subunits, 13-14 nuclear location of selective, 20-21 of oxidized hemoglobin by insulinase, 23 of oxidized proteins, 23 of oxygen radical-damaged proteins, 22-23 in protein folding, 211-212 as regulatory mechanism, 1 synthetases dissociation of synthetase complex by controlled, 105-106 lipids and, 107 susceptibility to, 102, 103 type III cGI sensitivity to, 72, 73-74 of ubiquitinated proteins, 7 by ubiquitins, 5-7 Proteolytic complexes, see also Multicatalytic protease; 26S ATP/ubiquitin-dependent protease generated by MCP and ATPase subunits, 1 PAGE patterns compared, 4f Proteolytic processing of MCP and 26S proteases, 12-14 Protonmotive force CheY-switch interaction regulation by, 152 driving flagellar motors, 140 Purification of inclusion bodies, 293 of synthetases ArgRS, intact free, 119 not specified in synthetase complex, 102 RS complex, 102-103 SerRS, 125-126

INDEX ThrRS, 126 ValRS, 122-123 of type III cGI PDEs, 70-74 Pyruvate dehydrogenase complex as protein superstructure, 246-247 reactivation of, in folding of, 240-241 Q Quaternary structure of proteins of 2-oxoacid dehydrogenases, 247 enthalpy and entropy in forming, 221 formation of, in association, 238-244 R Radiation, nitric oxide and ionizing, 180-181, 182 Radicals nitric oxide as molecular, 161 nitric oxide reactivity with, superoxide and peroxy, 163 Raf-1 kinase, 85-86 Ramachandran plots, conformational restrictions of polypeptide chains, 218-219 Ras proteins activation by insulin, 86 by Raf-1 kinase, 85 CheY as structural homologue to, 141 Reactive nitrogen oxide species formed from NO/O2 reactions, 164-167 NO concentration required to form, 174-175 Reactive oxygen species cytotoxicity from NOx, 181-182 peroxynitrite and, 164 decreasing GSH concentrations, 201, 202 nitric oxide effect on biologically generated, 180 cytotoxicity of chemically generated, 176-178 enzymatically generated, 178-179 Reactivity of nitric oxide with metal complexes, 161-163 as molecular radical, 161 with radicals, 163 Recombinant proteins, see Protein folding

335 Recombinant tissue plasminogen activator, 210 Reconstitution accessory proteins, in presence of, 294-296 in reverse micelles and of immobilized proteins, 296-297 Regulation, see also Cell regulation of AspRS synthesis, 117 of ATPase inhibitory effect of calponin, 45-57 of CheY-switch interaction by acetylation, 150-151 by calcium ion, 149 by CheZ, 148-149 description, 143-144 forms of, 153 by fumarate level, 151-152 by phosphorylation, 144-146 by protonmotive force, 152 requirements for CW generation, 146-148 of GluProRS, 122 of GSH tissue concentration levels, 190, 192-195 hormonal, see Hormonal regulation of physiological processes by NO, 160 of protein bios)mthesis, 129 Regulators bacterial proteins as, 140-141 PDE gene families as regulators, 69-70 regulators of, 65^ response, phosphorylation of, 141-142 Regulatory domains of PDE gene family sequences, 67 type III PDEs, 74, 81 Repellents, bacterial swimming patterns and, 137-138 Residues cis-prolyl, in slow kinetic phases of protein folding, 226-227 Clp protease N-terminal, missing from, 13 cysteine in disulfide shuffling of BPTI, 225 in isoforms of calponin, 57 NO modification of, 167, 168 hydrophobic, chaperone affinity for, 285

336 proline, 225-227 serine in calponin phosphorylation studies, 49 as phosphate-accepting, 11 solvation of, 219 threonine as phosphate-accepting, 11 Response regulators, phosphorylation of, 141-142 Retroviruses, tRNAs and, 128 Reverse micelles, 296-297 Rhodopsin, 66 Ribonuclease b a m a s e as single-domain, 234-235 cytochrome c and, 228-232 glycoproteins and, 263 proline isomerization, 225-227 side chain substititution, 213 unfolding intermediates, 227-228 Ribonucleic acids 5S, 107 polyanion binding, 129 ribosomal, 249-251 viral aminoacylation by synthetases, 128 autoantibody etiology involving, 128 Ribonucleotide reductase, NO inhibition of, 167 Ribosomal S6 kinase substrates, 85 Ribosomes exit of nascent chains from, 292 MCP values versus number of, 8 as protein superstructures, 249-251 RNase. see Ribonuclease RNOS. see Reactive nitrogen oxide species ROS. see Reactive oxygen species Rotation, flagellar, see Flagellar rotation RSKs. see Ribosomal S6 kinase substrates RSs. see Aminoacyl-tRNA S5rnthetases S Salmonella typhimurium bacteriophage P22 TSP kinetic partitioning, 252-253, 294 reaching free energy minimum in TSP assembly, 249

INDEX flagella per cell, 140. see also Bacterial chemotaxis fumarate as switching factor, 151-152 Sarcoplasm Ca^* entry into, 33 free calcium in resting muscle cell, 56 Scavengers of NO, 5-aminosalicylic acid as, 165 ascorbate as, 166, 182 glutathione as, 167, 182 NO of reactive oxygen species, 182 Second messengers cAMP and cGMP as, 63-64 inositol phosphate glycan as, 79-80 PDEs and, 69 Secondary protein structure in determining folding pathways, 234 kinetics of unfolding and, 227-228 monitoring formation of, 230 Self-digestion by MCP, 13-14 Sequences amino acid, of TrpRS, 125 ArgRS N-terminal, 118 calponin amino acid, 3 9 - 4 1 cDNA calponin a and ^8, 39 GluPro RS, 120-121 mammalian synthetase, 107-108 of ValRS, 123-124 EFla aminoacyl-tRNA channeling and, 109, 116-117 Asp-tRNA dissociation from AspRS, 114-116 GluProRS genomic, 122 PDE amino acid, 6, 74 Serine activation of type III cGI PDE, 80 in disulfide shuffling of BPTI, 225 as phosphate-accepting residue, 11, 49 Serine proteases, 3 - 5 Serpins, 252 Seryl-tRNA synthetase, 125-126 Signal transduction in bacterial chemotaxis, 138-140 GSH depletion inhibiting, 189-190, 200 pathways of glutathione and, 196, 197

INDEX PMF circumvention of, 152 Ras protein in regulation of growth and differentiation, 86 regulating pp70 and pp90 RSKs, 85 PPIs inhibiting immunosuppression, 268 Solvation balancing hydrophilic groups and hydrophobic surfaces, 219 in identifjdng folding intermediates, 222 Solvents, parameters of cystolic, 260-263 Stability, protein cytosolic viscosity and, 261 of dimers in protein evolution, 242 disulfide bridges and extracellular, 264 folded structure thermodynamic, 252-253 hierarchy of, 212-218 of incompletely folded subunits, 283 protein engineering analysis of, 234-235 Starvation, see Malnutrition Stimuli cAMP and cGMP mediation of response to, 6 3 - 6 4 chemotactic, 137, 138-140 Stress proteins, see Heat-shock proteins Structural properties of 26S protease, 5 - 8 aminoacyl-tRNA synthetase complex, 102-105 of apoferritin, 245-246 of calponin versus function, 57-58 E. coli ATP-dependent proteases, 7 - 8 of eukaryotic heat-shock proteins, 274 ofMCP, 2 - 5 of PDE isoenzymes, 67 PDI versus DsbA, 266-267 of unfolded/partially unfolded proteins, 218-221 Substrates cGMP as, 64 chaperone GroE system, 280 Hsp versus others, 277 specificity versus promiscuity, 285-289 hepatic GSH concentration, 192-193

337 limited access to multifunctional synthetases, 128-129 for MCP and 26S proteases, natural, 22-24 PDI, specificity of, 266 peroxynitrite oxidation and nitration of, 164 polypeptide, MCP and degradation of, 3 protease location and access to, 2 of ribosomal S6 kinases, 85 synthetase organization and, 106-107 Sulfhydryls in amino acids, NO reactions with, 170 in peroxynitrite detoxification, 164 redox status and cell processes, 189-190 Sulfur amino acids, GSH concentrations and, 192-193, 194-195 Superoxide radicals, NO reactivity with, 163 Superoxides NO lifetime in vivo, 172-173 reaction with NO inhibiting nitrosation, 178 Superstructures, protein apoferritin, 245-246 complexity and diversity of, 244-245 pyruvate dehydrogenase complex, 246-247 ribosomes, 249-251 surface layers and cell coats of, 247-249 Surface layers, 247-249 Swimming patterns, bacterial, 137-138 Switch complex, see also CheY-switch interaction description, 143 in signal transduction pathway, 140 Synthesis, see also Protein biosynthesis of glutathione diet and nutritional status in, 192-195 feedback inhibition of, 191, 193 hormonal regulation of, 195-200 MCP, tissue types versus, 8-10 Synthetases acetyl-coA, 150 complexed form, 111-112 enzymes, occuring as free soluble, 102

338 high M„ 104 proteolysis, susceptibihty to, 102, 103

T lymphocytes GSH levels and proliferation of, 201 signal transduction inhibition in, 189-190 Tailspike protein free energy minimum, 249 kinetic partitioning, 252 Target selection molecular mechanisms for, 24-26 of proteins for destruction, 5 - 7 Targets for nitric oxide direct effects of, 182 DNA, 171-172 inhibition of enzymes, 167-171 Temperature in protein folding, see also Heat-shock proteins as cystolic solvent parameter, 260-261 identifying folding intermediates by altering, 222 Temperature-sensitive folding point mutations, 294 Tertiary structure of proteins folding intermediates and, 223 kinetics of unfolding and, 227-228 relationship with primary structure, 225 water exclusion from molecular core, 221 Therapeutic agents inhibiting PDEs, 66, 68-69 Thermodynamics in off-pathway folding reactions, 252-253 Thermolysin, intrinsic stability of, 216 Thermophilus enzyme MCP subunits location of )3, 3 processing of )3, 13 Thermophilus thermophilus, SerRS structure and, 101 Thermostability of ArgRS, 119 AspRS N-terminal domain and, 113-114 Thermotoga maritima, refolding of, 260 Thermotolerance, cellular, 274-277 Thick filament-linked regulation, 3 3 - 3 4

INDEX Thin filaments caldesmon in situ presence on, 58 calponin as bona fide protein in situ, 4 1 - 4 2 deassociation of, 56 reassociation with, 57 myosin competition test and, 34-36 Thiolidisulfide exchange GSH efflux from liver, 196-197 sulfhydryl oxidation state of proteins, 189-190 Thiol groups, see also Glutathione S-nitrosothiol adducts in nitric oxide oxidation, 166-167 preventing P450 enzyme degradation, 169-170 thiol-nitrosyl adducts, 167 Thiol redox status DNA and gene transcription, 189-190 hormones and, 199-200 Thiol-nitrosyl adducts, 167 Threonine, 11 Threonyl-tRNA synthetase autoantibodies to, 127 cDNA sequence, 107 description, 126 Tissue, see also Cells calponin expression estimated value of, 41 versus types of, 37-39 CaM PDE concentrations in, 64 glutathione concentration in in extrahepatic, 194 intestinal tract absorption and, 191 in malnutrition and disease, 200-204 narrow physiological range of, 190 MCP and levels of, 8 - 1 0 subcellular distribution, 18 NO and destruction versus protection of, 176, 180 PDE gene families hormonal regulation of type III cGI PDEs in adipose, 75-78 type III cGI purification from, 70-74 types expressed in, 65^ Toxicity, see C)^toxicity Toxins, NO as environmental, 160

INDEX TPA. see Recombinant tissue plasminogen activator Transcription factors, see also Gene transcription leucine repeats in, ArgRS similarity, 119 as rapidly degraded proteins, 22 as substrates for pp90 RSKs, 85 sulfhydryl group redox state and interactions with DNA, 189 TrpRS, 125 Transducin, 66 Transfer ribonucleic acids differentiating human from yeast and bacterial, 108 free versus complexed sjrnthetases in lysylation of, 112 mammalian aspartylation by AspRS, 114-116 E F l a binding, 116-117 as primers for reverse transcriptase in retroviruses, 128 RSs and recognition of, 101 serylation of, 126 Translation aminoacyl-tRNA channeling and, 109 organization of machinery of, 129 polypeptide aW-trans configuration, 268 in vitro versus in vivo, 259 Tropomyosin caldesmon binding to, 58 calponin binding with actin-tropomyosin, 35 Ca2^ and, 41 purified paracrystalline form, 42 in GTPyS binding to calponin, 48 similarity of calponin tissue content to, 36 Trjrpanosoma brucei, 248-249 Tryptophanyl-tRNA synthetase, 124-125 TSP. see Tailspike protein Tumbling Ca^^ in E. coH cells, 149 PMF effect on, 152 swimming patterns leading to, 137 Tumor cells, see Malignant cells Tumor necrosis factor low GSH levels sensitizing cells to, 202 in severe wasting and cachexia, 203 24S synthetase complex, 106

339 26S ATP/ubiquitin-dependent protease argRS stimulation of, 118 as cytoplasmic endoprotease, 1 regulation by molecular mechanisms for target selection, 2 4 - 2 5 natural substrates for, 2 2 - 2 4 regulation of expression of MCP subunits and, 10 by posttranslational modification, 11-14 protease levels, 8-10 structural and enzymatic properties, 5-8 subcellular distribution of, 18-21 subunits, expression of specific, 10-11 Type I calcium-calmodulin phosphodiesterase, affinity for cGMP and activation of, 64 Type II phosphodiesterase, 64-66 Type III cGI phosphodiesterases affinity for cAMP and cGMP, 66 hormonal regulation, mechanisms for, 80-84, 8 8 - 9 1 insulin-initiated reactions activating, 70 purification and characterization of, 70-74 Type IV phosphodiesterase, 66 Type V phosphodiesterase, 66 Type VI phosphodiesterase, 66 Type VII phosphodiesterase, 66-67 Tyrosine kinase in cell proliferation and metabolism, 85 in type III cGI PDE activation, 70 U Un-»N transition, folding intermediates along, 224 Ub-conjugates degradation of by 26S protease, 7, 14, 2 3 - 2 4 by MCP, 14 phosphorylation and inhibitors of, 11 increase in fasted rat muscle, 22 subcellular concentrations of, 20 Ub-lysozyme conjugates, 14-16 Ub-mediated proteolytic pathway enhancement in fasted rat muscle, 22 subcellular locations of components of, 20

340

INDEX

Ubiquitin, 5-7 Ubiquitinated proteins, 17 Unfolded proteins conformational restrictions of folded versus, 218-219 hydrogen bond breaks, 228 molten globules versus, 214, 220 oligopeptides, 215-216 Unfolding proteins, protein engineering analysis, 235

W Weak interactions protein assembly form determination, 249 protein structure and, 213 Wortmanin, inhibiting insulin action, 88

Valyl-tRNA synthetase complex cDNA sequence, 107 description, 122-124 synthetases associated with, 102 Variable surface glycoproteins, 248-249 Vasodilators, NONOates and, 176-177 Viruses NOx-mediated destruction of zinc-finger motifs and, 171 tRNAs as primers for reverse transcriptase in retroviruses, 128 Vision, photoreceptor type VI PDEs and, 66

Yeast catalytic domains in human RS synthetases versus, 108 differentiating h u m a n tRNA from, 108 high Mr synthetase complexes, 104 HisRS synthetase N-terminal domain, 108

XO. see Hypoxanthine/xanthine oxidase

Zinc-finger motifs, 170-171, 181 Zygotes, see also Embryonic tissue subcellular distribution of MCP in, 18

ISBN D-12-lSBfl3^-D 9001 8

9"780121"528348

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