function of stress proteins, and implications for medicine and disease.

PHYSIOLOGICAL REVIEWS Vol. ‘72, No. 4, October 1992 Printed in U.S.A.

Mammalian Stress Response: Cell Physiology, Structure/Function of Stress Proteins, and Implications for Medicine and Disease WILLIAM Department

I. II. III. IV.

V. VI. VII.

VIII.

I.

of Medicine

and Physiology,

J. WELCH

University

of California,

San Francisco,

California

Introduction ......................................................................................... Induction of Response ............................................................................... Changes in Cell Physiology After Stress ........................................................... Structure and Function of Stress Proteins ......................................................... A. Ubiquitin ......................................................................................... B. Heat shock protein 28 ............................................................................ C. Heat shock protein 60 (groEL) family ........................................................... D. Heat shock protein 90 family .................................................................... E. Heat shock protein 110 .......................................................................... F. Heat shock protein 70 family .................................................................... Role of Heat Shock Proteins as Molecular Chaperones in Facilitating Protein Folding and Assembly ................................................................................... Role of Heat Shock Proteins in Cells Experiencing Stress and Their Possible Mode of Regulation Stress Response in Disease and Medicine ........................................................... A. Stress response as a marker for cell injury ..................................................... B. Stress response and immunology ................................................................ C. Stress response and toxicology .................................................................. Conclusions ..........................................................................................

INTRODUCTION

Cells have developed a number of different strategies to deal with adverse changes in their environment. Examples include the temporary amplification of gene expression (e.g., dihydrofolate reductase) in response to a specific metabolic poison (e.g., methotrexate) or more permanent changes in gene expression that now provide the cell a means by which to survive in an otherwise hostile environment. In addition, cells from all organisms have developed a remarkably similar response to sudden increases in their normal growth temperature. This response, initially referred to as the heat shock response (116), is characterized by the extremely rapid increased expression of a select group of proteins, the so-called heat shock proteins (HSPs). In addition to heat shock, increased expression of these proteins also occurs when cells are exposed to a number of other metabolic insults, including amino acid analogues, various heavy metals, agents that modify protein sulfhydryls, various ionophores, and finally a number of other metabolic poisons. Consequently, owing to the fact that so many deleterious agents/treatments result in similar changes in gene expression, the response is now more often referred to as the stress response and the proteins whose 0031-9333/92 $2.00 Copyright 0 1992 the American Physiological

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expression increases, the stress proteins (for other reviews, see Refs. 5, 26, 83, 93, 98, 107, 139). A significant amount of work has clearly shown that the stress response and in particular the stress proteins are essential for the survival of the cell confronted with a particular environmental insult. In bacteria and yeast, deletion of the genes that encode some of the stress proteins renders the organism unable to survive a heat shock challenge (27,44; for review see Ref. 45). Similarly, mammalian cells injected with antibodies specific to the most highly induced stress protein, HSP 70, quickly die following their exposure to a brief heat shock treatment (114). Finally, when cells are given a mild sublethal heat shock treatment, sufficient to result in the increased expression and accumulation of the stress proteins, the cells now are able to survive a second and what would otherwise be a lethal heat shock challenge (48,57). This phenomenon, referred to as acquired thermotolerance, has been shown to be dependent on the increased expression and accumulation of the stress proteins (58, 78, 82). II.

INDUCTION

OF RESPONSE

Although referred to as heat shock or stress proteins, we now know that most of these proteins are in 1063

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fact expressed constitutively in the normal unstressed biological cell and participate in a number of important pathways. A select few of the stress proteins, however, are expressed only in times of trouble, and therefore, their appearance is often diagnostic that the cell has experienced some type of trauma. The mechanism by which the cell recognizes an adverse change in its environmental circum stance and subseq uent ly increases the expression of the stress proteins is only now beginning to be u nderstood. Owing to the fact that so many of the agents or treatm .ents that lead to the induction of a stress response are known to adversely affect protein conformation, Hightower (60) in 1980 suggested that the intracellular accumulation of abnormally folded proteins likely represented the trigger by which the stress response was initiated. Perhaps the increased expression of the stress proteins, Hightower proferred, somehow was involved in the recognition and removal of such denatured proteins in the traumatized cell. Subsequent work in both bacteria and animal cells began to provide support for this idea. First, Goff and Goldberg (50) demonstrated that feeding amino acid analogues to bacteria resulted in the production of abnormally folded proteins and the induction of a stress response. Moreover, because one of the proteins whose expression increased was shown to be a bacterial protease, these investigators suggested that whenever cellular proteolytic systems became overburdened with the rem .oval of abnormally folded proteins, there occurred an induction of a stress response. Subsequent studies by Kelley and Schlesinger (71) similarly showed that adding various amino acid analogues to animal cells also was sufficient to activate a stress response. Then in 1986, Anathan et al- (1), in w ,hat m any consider a landmark finding, reported that sim Pl.y injecting a collection of denatured proteins into frog oocytes was sufficient to activate the stress response. As is di scussed in sect ion VI, the idea accumu lation of abnormally that the intracellular folded polypeptides represents the triggering mechanism by which the stress response is initiated fits in nicely with our new information regarding the structure and function of the individual stress proteins. Finally, when discussing their regulation, it is important to remember that most of the stress proteins are in fact expressed constitutively in the cell maintained under normal growth conditions. Moreover, their degree of expression appears dependent on the metabolic activities of the cell. For example, the expression of some of the stress proteins appears relatively high in exponentially growing cells compared with their quiescent counterparts or in cells that exhibit high secretory activities (unpublished observations). Finally, increased expression of one or more of the stress proteins often is observed in cells infected with different viruses, in particular many lytic viruses, in which synthesis of the viral proteins is extremely high (23, 96, 108). With the n ew informati on that ind icates that the ltress proteins are essential for protein maturation, th relatively higher expression of the stress proteins under these different conditions is likely due to the increased demand

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on the cell for protein synthesis and secretion events. Thus, whenever the cell encounters 1) an increased demand for protein synthesis, assembly, and/or secretion or Z) a situation in which protein maturation events are sufficiently perturbed, thereby leading to an accumulation of abnormally folded proteins, there occurs an increased synthesis of one or more of the stress proteins. III.

CHANGES

IN CELL

PHYSIOLOGY

AFTER

STRESS

Although most investigators have focused their attention on the molecular biology and biochemistry of the stress proteins, some efforts have been directed at determining the changes in cell physiology that accompany the induction of the stress response. Perhaps not too surprising is the fact that cells from homeothermic organisms exhibit an arrest of most activities associated with proliferation when subjected to metabolic stress (85,112,149). In the case of cells from most poikilothermic organisms, however, growth arrest after heat shock is usually only a temporary event. For example, after a temperature shift from 20 to 37”C, yeast and bacteria will increase the expression of their stress proteins for a short period of time. Even at the higher temperatures, these organisms will eventually return to their normal patterns of protein synthesis and other activities associated with proliferation. In contrast, most mammalian cells do not appear capable of acclimating to higher temperatures (e.g., 42°C). Instead, when maintained at the higher temperature, they will continue to synthesize the stress proteins all the way up to the point of cell death. A number of biochemical pathways appear compromised or redirected in mammalian cells after hyperthermic treatment. One early consequence is a rapid drop in intracellular pH, decreased levels of ATP, and an increase in cytosolic calcium levels (42,128,137). Although accompanying the induction of the stress response, these changes per se do not appear to be sufficient to activate the stress response. For example, simply lowering intracellular pH or increasing cytosolic calcium levels by other means does not usually result in any significant changes in stress protein expression (35). Mitochondrial function, and in particular aerobic metabolism, is adversely affected in cells subjected to elevated temperatures (79). As a consequence, the cell exhibits an increased dependence on glycolytic energy metabolism and an accompanying increased synthesis of at least two glycolytic enzymes, enolase and glyceraldehyde-3-phosphate dehydrogenase (62,92,97). This switch to anaerobic metabolism may be essential for the cell to survive the hyperthermic treatment. This is suggested owing to the fact that yeast cells growing on dextrose, a metabolite used exclusively in glycolysis, generally appear more thermoresistant than those growing on acetate, a metabolite used exclusively in the respiratory pathway (84). A number of interesting morphological changes have been described in mammalian cells subjected to


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heat shock. Within the nucleus one observes an accumulation or aggregation of so-called perichromatin granules, which likely represent unprocessed forms of mRNA (55, 144). Support for this idea is the fact that heteronuclear RNA processing (or “splicing”) appears compromised after heat shock treatment (86, 148). In this regard it is worth pointing out that many of the genes that encode the highly inducible forms of stress proteins themselves do not contain intervening sequences and therefore are not likely to be dependent on the splicing machinery for their proper maturation (for review see Ref. 83). A rather striking abnormality seen after heat shock involves the nucleolus, the organelle responsible for the assembly of ribosomes and some other ribonucleoprotein complexes. Here one observes an aggregation of the maturing preribosomes and/or other ribonucleoprotein complexes, with the extent of such aggregation dependent on the severity of the temperature shock (3, 124, 144). These morphological changes occurring within the nucleolus are correlated with the observed inhibition of proper ribosomal RNA processing and an arrest of ribosome biogenesis following heat shock (38,119). Finally, a rather curious abnormality concerns the appearance of rodlike filaments, comprised in part of actin, within the nucleus of various mammalian cells provided a brief but severe heat shock treatment (105,144). At the present time, we have absolutely no idea regarding the biological significance of these somewhat unusual intranuclear actin filaments. Heat shock treatment results in a rather dramatic change in another of the cytoskeletal networks, the socalled intermediate filaments (IFS). Normally distributed as a fine meshwork of filaments extending throughout the cytoplasm out toward the cell periphery, the IFS quickly become redistributed into a tight “cage” surrounding the nucleus after stress (40, 131). Accompanying the collapse of these filaments is a redistribution of both the mitochondria and polysomes into the perinuclear region (144). At present, the exact biological role of the IF cytoskeleton has escaped elucidation. Therefore the significance of IF collapse observed after stress remains unclear. Via analysis by electron microscopy, those mitochondria that redistribute along with the collapsed IFS after heat shock appear to be significantly altered. Specifically, the mitochondria now appear “swollen, ” with their individual cristae quite prominent and the intracristal spaces abnormally enlarged (144). These morphological changes presumably correlate with the observed inhibition of normal mitochondrial function (and lowered levels of ATP) in cells after stress (42, 79). Finally, certain aspects of the protein secretory machinery, and in particular the Golgi complex, appear significantly altered after heat shock. In contrast to their well-defined and organized collection of membrane stacks within the normal unstressed cell, the Golgi complex after heat shock often appears fragmented (144). Interestingly, however, there are no reports concerning any major impairment of normal secretory activities in the cell after heat shock. In almost all cases, and providing that the stress treatment is not

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too severe, these various morphological alterations appear to be fully reversible. Whether the HSPs themselves are integral to the recovery process, however, remains to be determined.

IV.

STRUCTURE

AND

FUNCTION

OF STRESS

PROTEINS

A substantial amount of work over the past 30 years has led to the identification and characterization of the individual stress proteins from a wide variety of different organisms. For most, the corresponding genes have been isolated and characterized in great detail. This work has revealed that the stress proteins from prokaryotes to eukaryotes represent one of the most conserved group of proteins so far characterized. For example, members of the bacterial stress protein family exhibit as much as 50% sequence identity with that of their human counterparts (for review see Refs. 26, 83). The induction of the stress response, as assayed by the changing patterns in protein synthesis, for five different mammalian cell lines is shown in Figure 1. Here the cells were labeled with [35S]methionine under normal growth conditions (i.e., 37”C), after a 43”C, 90-min heat shock treatment, or after exposure to an amino acid analogue of proline, L-azetidine Z-carboxylic acid. It can be seen that the changes in the patterns of proteins being synthesized after stress appear very similar in each of the five different cell types. Owing to differences in their mode of regulation, the stress proteins are often divided into two major groups: the so-called heat shock proteins (HSPs) and the glucose-regulated proteins (GRPs). Because their exact function was not known at the time they were first identified, the stress proteins are usually referred to according to both their size on sodium dodecyl sulfate (SDS) gels and mode of induction. For example, in mammalian cells, those proteins whose synthesis increases after heat shock are referred to as the HSPs and exhibit apparent masses of -8,28,58,72,73,90, and 110 kDa. The other major family, the GRPs, were first identified as showing increased expression in cells starved of glucose and exhibit apparent masses of 78,94, and 170 kDa (110; J. Subjeck, unpublished observations). Subsequent work has demonstrated that the GRPs also are upregulated in cells exposed to agents that interfere with calcium homeostasis [e.g., ethylene glycol-his@-aminoethyl ether)-N,N,N’,N’-tetraacetic acid or the calcium ionophore A23187], during hypoxia, or after treatment of the cells with a number of agents that interfere with protein secretion (e.g., ,&mercaptoethanol, the glycosylation inhibitor tunicamycin) (for review see Ref. 129). Because some of the stress proteins have very similar molecular masses (e.g., HSP 72 and HSP 73), they are more easily discerned when analyzed by two-dimensional gel electrophoresis. Shown in Figure 2, for example, are the patterns of protein synthesis, as analyzed by two-dimensional gels, in rat embryo fibroblasts maintained at 37OC (A), subjected to a 43”C, 90-min heat shock (B), treated with the calcium ionophore A23187 (C), or ex-


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FIG. 1. Different mammalian cell lines respond similarly to either heat shock treatment or exposure to an amino acid analogue. HeLa cells, baby hamster kidney cells (BHK), gerbil fibroma cells (GFC), Chinese hamster ovary cells (CHO), and rat fibroblasts (RAT) were incubated either at 37°C (lane I), at 43°C for 90 min (lane 2), or at 37°C in presence of 5 mM L-azetidine 2-carboxylic acid (a proline analogue; lane 3) for 5 h. After appropriate incubation period, cells were returned to normal growth conditions and labeled with [%]methionine for 45 min. Cells were then harvested, and labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Shown is autoradiograph of gel with molecular mass markers indicated on left. On right, in descending order, are indicated relative positions of heat shock protein (HSP) 110, glucose-regulated protein (GRP) 94, HSP 90, GRP 78 (BiP), and HSP 72173 stress proteins. (Note that low-molecular-mass HSP 28 is not apparent here owing to its relatively low content of methionine residues.) [From Welch et al. (143).]

posed to the proline analogue L-azetidine 2-carboxylic acid (D). As mentioned in section II, note that most of the stress proteins are in fact expressed constitutively in these cells but exhibit increased expression after stress. Only in one case, HSP 72, is the protein expressed solely as a function of the cells being stressed. Finally, in some cell types and depending on the stress agent used (e.g., amino acid analogues) coordinate increased synthesis of both the HSPs and GRPs is observed (Fig. 20). Members of both families of stress proteins appear to be both structurally and functionally related.

A. Ubiquitin

Increased synthesis of an -8-kDa protein after heat shock has been reported in chick embryo fibroblasts and in yeast. Isolation of a cDNA encoding this smaller polypeptide revealed it to be identical to ubiquitin (12), a highly conserved protein that serves a role in both chromatin structure (being covalently bound to some histones) and in protein degradation events. With regard to the latter, many intracellular proteins that are to be degraded are first covalently modified by the


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FIG. 2. Analysis of mammalian stress proteins by Z-dimensional gel electrophoresis. Rat embryo fibroblasts growing in 35-mm plastic dishes were labeled with [35S]methionine after incubation at 37°C (A), after a 43”C, 90-min heat shock exposure (B), after exposure to 10 PM of calcium ionophore A23187 (C), or after exposure to proline analogue L-azetidine 2-carboxylic acid (D). After labeling, cells were harvested and labeled proteins were analyzed by 2-dimensional electrophoresis. Shown only are those regions of gels analyzing proteins of -40,000 Da or greater (consequently, low-molecular-mass HSP 28 is not shown). Stress proteins are indicated as follows: A, HSP 110; B, GRP 94; C, HSP 90; D, GRP 78 (BiP); E, GRP 75; F, HSP 73; G, HSP 72; and H, HSP 58. ac, Actin. [From Welch et al. (143a).]

addition of ubiquitin. Once ubiquitin conjugated, these proteins are targeted for degradation via a nonlysosoma1 proteolytic pathway. The increases in ubiquitin levels after heat shock (and other forms of stress) presumably facilitate the targeting and removal of proteins denatured as a consequence of the stress event. Although attractive, this idea has not proven quite so straightforward at the experimental level. Specifically, there are reports that protein degradation is actually reduced in cells after heat shock despite an increase in the amount of ubiquitin-conjugated proteins (17). It remains possible that certain proteolytic systems themselves are compromised immediately after heat shock, thereby ac-

counting for the overall decrease in protein degradation events being observed soon after the heat shock treatment. Presumably as the cell recovers from the heat shock treatment, those proteins now covalently linked with ubiquitin will eventually be targeted for degradation and subsequent removal from the cell. B. Heat Shock Protein 28

In both Drosophila and in plants, multiple, but related, forms of the low-molecular-mass HSP have been described (for review see Refs. 5, 98). In contrast, in


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most mammalian or avian cells, only a single low-molecular-mass HSP of -25-30 kDa has been observed. This protein, referred to as HSP 28 in our laboratory (also referred to as HSP 24, HSP 27, or the low-molecularmass HSP), contains relatively few methionine residues and therefore is easily overlooked in studies employing [35S]methionine for metabolic labeling. The protein is expressed at relatively low levels in normal cells but exhibits a lo- to ZO-fold induction in the cell after stress. Four major isoelectric forms of HSP 28 have been reported, with at least three of the isoforms representing different phosphorylation states of the protein (72,138). Although little is known regarding its exact biochemical function, the protein does exhibit a number of interesting properties. First, all of the low-molecular-mass HSPs so far isolated and characterized from different organisms exhibit sequence homology with the cu-crystallin proteins, abundant components of the lens (63). Like the cu-crystallins, the low-molecular-mass HSPs exist in the cell as very large structures of -400,000 Da (4). colnsequently, one suspects that those domains shared in common by these two families of pro teins are responsible for their similar oligomeric structures. After heat shock treatment, mammalian HSP 28 forms even larger structures, approaching 2 X IO6 Da in size (4). With the use of indirect immunofluorescence analysis, along with biochemical methods, most of HSP 28 has been shown to be present within the cytoplasm in proximity to the Golgi complex. After heat shock, however, much of the protein relocates into the nucleus (4). In addition, whereas HSP 28 is easily extracted from the n ormal unstressed cell by nonioni c detergents, after h eat sh.ock treatme nt th e majority of the protein now appears largely resistant to detergent extraction. As the cells recover from the thermal stress, HSP 28 slowly returns to the cytoplasm and now again is detergent extractable (4). The increased expression of HSP 28, at least in mammalian cells, appears to be important for the cell to survive the hyperthermic treatment. For example, cells constitutively expressing high levels of HSP 28, via transfection of its corresponding cDNA, exhibit significantly higher thermotolerance than do their untransfected counterparts (77). One very interesting aspect of the low-molecularmass HSPs concerns their mode of regulation and/or posttranslational modification in response to a number of biological stimuli. For example, in Drosophila, where there are four or five related but neverthel ess distinct low-molecular- -mass HSPs, these protei ns exhibit changes in both their expression and tissue localization during development and differentiation (19, 104, 125, 150). For at least some of these Drosophila low-molecular-mass HSPs, their regulation appears to be under the control of steroid hormones (64). In mammalian cells, the constitutive expression of HSP 28 similarly appears to be growth regulated with the relative amounts of the protein increasing as cells reach the stationary phase (unpublished observation). In various tumor cell lines derived from the breast, HSP 28 synthesis increases in response to the steroid hormone estrogen (37). Perhaps

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most intriguing are the observations that HSP 28 exhibits rapid increased phosphorylation in response to various mitogens, as well as after treatment of the cells with calcium ionophores and certain tumor promoters (138). Moreover, a HSP 28 appears to undergo rapid phosphorylation following the differentiation of HL-60 cells in culture (G. Minowada and W. Welch, unpublished observations). Taken together, these studies indicate that both the levels and/or phosphorylation state of HSP 28 appear to correlate with the growth status of the cell. Further studies are needed to delineate the exact biochemical function of HSP 28 and how phosphorylation may be important in the regulation of its function. C. Heat Shock Protein 60 (groEL) Family First described in tetrahymena (88), HSP 60 now has been identified, cloned, and sequenced from a number of eukaryotic organisms (for review see Ref. 39). Such analysis has revealed its homology to the previously characterized groEL HSP of bacteria. Earlier studies had shown that mutations in the groEL gene resulted in an inability of the bacteria to support the growth of various bacteriophages (25,127). Subsequent biochemical work demonstrated a requirement for a functional groEL protein in the correct morphogenesis of the bacteriophage prohead. Specifically, groEL was shown to exist as a large homooligomeric structure that functioned in facilitating the correct assembly of the bacteriophage head and tail proteins into their final prohead structure. Such assembly events were also shown to require a second component, groES, a IO-kDa polypeptide related to groEL and that also appears to exist as a oligomeric complex in bacteria (46, 47). In plants, a groEL homologue has been identified and shown to be equivalent to the so-called Rubisco binding protein, a chloroplast component involved in the posttranslational assembly of Rubisco (ribulose bisphosphate carboxylase-oxygenase), the enzyme involved in CO, fixation (56). Finally, more recent studies have described groEL-related proteins in both mammalian cells and yeast (X,65,89). These proteins, also referred to as HSP 58 or HSP 60, are encoded by nuclear genes, are synthesized within the cytoplasm, and are then translocated into the mitochondria. Through both genetic and biochemical means, all of these related groEL proteins have been shown to facilitate the folding of monomeric proteins and/or to catalyze the higher ordered assembly of oligomeric complexes (10, 20, 46, 47, 51, 56, 101). In particular, the groEL complex has been suggested to function as a “scaffold” or “workbench” by which newly synthesized proteins are correctly folded and/or assembled into their final and mature structure. These assembly events mediated by groEL require ATP, ATP hydrolysis, and most likely other cofactors, such as the smaller bacterial groES proteins (and its corresponding eukaryotic equivalent) (for review see Ref. 39). Another possible cofactor, at least in mammalian cells, may be the mitochondrial form of HSP 70 (90; see sect. IvF).


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Owing to their apparent role in facilitating monomeric protein folding and/or the proper assembly of oligomeric protein complexes, members of the HSP 60 or groEL family now are often being referred to as “molecular chaperones” (39). As discussed in section V, the term molecular chaperone is being used to classify a number of unrelated proteins that appear to function collectively in facilitating the proper maturation of other proteins within the cell. If members of the HSP GO/groEL family do indeed serve such a role, workers within the field suspect that there must exist additional members of the family, distinct from those so far characterized only within the mitochondria and chloroplast. Recently, a cytosolic protein of ~60,000 Da has been identified in mice and shown to exhibit some sequence homology with the groEL family of stress proteins (53, 132, 146). D. Heut Shock Protein 90 Family 1. Heat sh,ockprotein

90

Heat shock protein 90 represents one of the most abundant proteins in mammalian cells, yet its synthesis still increases after stress. By two-dimensional gel electrophoresis, the protein appears extremely heterogeneous, consisting of multiple isoforms, many of which contain phosphate (143). The protein is encoded by at least two genes, designated cyand ,8, that appear -70% related at the protein sequence level (59,113). The purified protein exists as a dimer with an unusually large Stokes radius, indicative of the protein having an extended rodlike structure (140). Whether the dimer exists as a homo- or heterodimer of the cy- and ,&subunits is not known. Although the exact function of HSP 90 is not known, a considerable amount of interesting phenomenology has been described for this protein in mammalian cells. Although the purified protein exists as a dimer, analysis of whole cell lysates by gel filtration has revealed an extremely heterogeneous profile of HSP 90, a result likely indicative of HSP 90 interacting with a large number of other cellular proteins (unpublished observations). Indeed, most of what we know regarding HSP 90 concerns its interaction with other, rather interesting, proteins. For example, our first clue regarding the function of HSP 90 followed from observations concerning its interaction with the tyrosine specific protein kinase, pp60”‘“, the transforming gene product of Rous sarcoma virus (15, 100, 123). Here, newly synthesized ~~60”‘” was observed to coprecipitate with HSP 90 and another as yet characterized EiO-kDa cellular protein. When present in this complex, newly synthesized pp60”‘” appeared inactive, as evidenced by its lack of both tyrosine kinase activity and autophosphorylated tyrosine residues. As this complex containing pp60”‘” reached its final destination at the inner side of the plasma membrane, both HSP 90 and the 50-kDa protein were re-

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leased, with pp60”‘” now exhibiting its full tyrosine kinase activities. Subsequent studies have shown that many retrovirus-encoded oncogene products, all of which exhibit tyrosine specific protein kinase activity, similarly interact transiently with HSP 90 and the 50kDa protein. These include the fps gene product from Fuginami and PRCC II sarcoma viruses, the yes gene product from Yamaguchi 73 sarcoma virus, the ros gene product of the UR-2 sarcoma virus, and the fes gene product from Synder-Theilen feline sarcoma virus (for review see Ref. 14). To date, however, the protooncogene equivalents have not been reported to be associated with HSP 90. In a somewhat similar scenario, HSP 90 has been implicated in the regulation of another important class of proteins, the steroid receptor family. Different members of the steroid hormone receptor family (e.g., progesterone, testosterone, glucocorticoid) are present in an inactive form within the cell. When steroid hormone diffuses into the cell, it binds to its appropriate steroid receptor and thereby activates the receptor to bind to its target genes and activate transcription (for review see Ref. 8). Biochemical studies have shown that the inactive form of most steroid hormone receptors exists in a complex with a number of other cellular proteins. On binding to its appropriate steroid hormone, the receptor releases at least some of these associated proteins, undergoes a dimerization event, and now appears “activated” as evidenced by its ability to bind DNA and activate transcription. Isolation and characterization of the inactive form of a number of different steroid hormone receptors has shown that at least two stress proteins, HSP 90 and HSP 70, are in close contact with the receptor (l&74,121). The current idea is that in the absence of steroid hormone, HSP 90 binds to the steroid receptor and thereby prevents its inappropriate interaction with DNA. Although some investigators have suggested that HSP 90 might interact directly with the DNA binding domain present within the receptor, others have suggested that HSP 90 interacts with those domains of the receptor that are critical for receptor dimerization, a step necessary for its subsequent binding to DNA (discussed in Refs. 29 and 111). At present, the role of HSP 70 within the steroid receptor complex is unknown. One possibility is that HSP 70 somehow serves to facilitate the dimerization of the receptor following its binding to the steroid hormone. Further biochemical studies are needed to delineate the exact mechanism by which HSP 90 and HSP 70 participate in the regulation of this important class of transcription factors. Finally, other data have implicated a role for HSP 90 in the regulation of yet another protein kinase, the so-called heme-regulated eukaryotic initiation factor (EIF) 2cukinase. In heme-deficient reticulocyte lysates, this kinase is activated and phosphorylates the cu-subunit of EIF-2, thereby resulting in an inhibition of overall protein synthesis. When complexed with HSP 90, EIF-2cu kinase activities appear enhanced (117). When cells are provided a brief but severe heat shock treat-


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ment, EIF-2cu kinase exhibits increased phosphorylation, and a temporary inhibition of protein synthesis occurs (36). Consequently, a number of laboratories are examining in detail the interaction of HSP 90 with the EIF-2cu kinase and whether such interactions are important in regulating the changes in protein synthesis that occur in the cell after stress. In summary, HSP 90 has been shown to interact with a number of rather interesting proteins in the cell. The current idea is that HSP 90 serves a regulatory role by binding to and either inhibiting or stimulating the aitivities of its target proteins. Considering the relatively high levels of HSP 90 in the normal unstressed cell, one suspects that there are additional protein targets to be discovered that interact with and are regulated by HSP 90.

The GRP 94 exhibits significant sequence homology with HSP 90 (76, 87). Whereas HSP 90 is primarily a cytosolic protein, GRP 94 is a compartmentalized protein found within the endoplasmic reticulum (ER) and perhaps on the plasma membrane (80, 99). The protein contains simple N-linked oligosaccharides and is phosphorylated (99, 143). Analysis of its predicted amino acid sequence has revealed a ZO-amino acid hydrophobic domain within the first one-third of the molecule, and therefore, it has been suggested that the protein spans the ER membrane with its NH, terminus inside the ER and a considerable portion of its COOH terminus present on the cytosolic side of the membrane (87). At its extreme COOH terminus, however, there is a putative ER retention signal sequence (KDEL), thereby prompting others to suggest that the protein is present entirely within the ER lumen (13). Biochemical studies have not helped in resolving this issue of GRP 94 topology. For example, some investigators have reported that GRP 94, when present in isolated microsomes, is completely protected from added proteases, a result consistent with it being present entirely within the ER lumen (73). Others, however, have observed its proteolytic digestion in isolated microsomes, a result indicative of the protein having a transmembrane orientation (66, 81). Unfortunately, other biochemical approaches to address the question of GRP 94 topology also have provided mixed results. For example, when analyzing the extractability of the proteins from isolated microsomes, GRP 94 exhibited properties of both a soluble and integral membrane protein (66). My own hypothesis is that GRP 94 is in fact a transmembrane protein that crosses the membrane twice, with its NH,- and COOH-terminal domains both present within the lumen of the ER. Despite a knowledge of its amino acid sequence, intracellular locale, and various posttranslational modifications, very little is known regarding the biochemical function of GRP 94. Like a number of other proteins present within the ER, GRP 94 exhibits calcium binding properties, similar to that observed for its related cyto-

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solic counterpart, HSP 90 (13,66). Owing to its locale, its relatively high abundance in cells with high secretory activities, and finally, the fact that its synthesis increases in response to agents that perturb protein secretion, one suspects a role for the protein in some aspect of the secretory pathway. Because its related counterpart, HSP 90, regulates the activities of a number of proteins through which it interacts, we are currently examining whether GRP 94 might similarly interact with and regulate the activities of other resident ER proteins and/or proteins that traffic through the ER. E. Heat Shock Protein 110 Although having been identified as major stress proteins a number of years ago, studies are just now beginning to address the structure and function of HSP 110. Initially described in mammalian cells by Subjeck et al. (130), HSP 110 is constitutively expressed at low levels and appears present within the cytoplasm, nucleus, and nucleolus. After heat shock, HSP 110 levels increase, with much of the protein now appearing heavily concentrated within the nucleolus in proximity to that region of the organelle involved in ribosomal RNA transcription (130). Recently, the gene for what appears to be the yeast homologue of mammalian HSP 110 has been cloned and sequenced (103). This protein, referred to as yeast HSP 104, appears to contain a putative nucleotide binding site. In addition, the protein exhibits homology to the so-called bacterial ClpA/ClpB proteins, which appear to play a role in ATP-dependent proteolysis events (103). In yeast, deletion of the HSP 104 gene has little effect on the ability of the cells to grow either at normal temperatures or following heat shock. Interestingly, however, such HSP 104 deletants appear compromised in their ability to acquire thermotolerance (122). Specifically, whereas wild-type cells subjected to a prior mild heat stress are able to develop tolerance to a second and more severe heat stress, the HSP 104 mutants appear unable to develop the normal thermotolerant phenotype. Subsequent work is required to define the exact biochemical function of HSP 104 and its intimate role in the development of thermotolerance. F. Heat Shock Protein 70 Family Both biochemical and genetic studies have revealed there to be a family of HSP 70-related proteins, some of which are constitutively expressed, whereas others appear strictly stress inducible (for review see Refs. 26, 139). All of the related HSP 70 proteins share the common property of binding nucleotides, especially ADP and ATP, but are distributed within different intracellular compartments (142, 151). In mammalian cells, the various HSP 70-related proteins are usually referred to on the basis of their apparent size on SDS-polyacrylamide gel electrophoresis. Unfortunately, due to different gel systems employed by different investigators, the


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nomenclature used to refer to the family is somewhat complicated and confusing. In our laboratory we refer to those forms of HSP 70 that are present within both the cytoplasm and nucleus as HSP 73 and HSP 72. Heat shock protein 73 is synthesized constitutively in all mammalian cells and therefore is often referred to as the constitutive HSP 70, or the HSP 70 cognate. The synthesis of HSP 72 is usually restricted to the cell experiencing stress and therefore is often referred to as the inducible form of HSP 70. The two proteins exhibit extremely high sequence homology (-95%) and similar biochemical properties. The third major form of HSP 70 is referred to as the glucose-regulated 7%kDa protein and is identical to a previously described protein referred to as BiP. Glucose-regulated protein 78 or BiP resides within the lumen of the ER. A fourth form of HSP 70 is found within mitochondria and chloroplasts and is referred to in our laboratory as the GRP 75. Although the corresponding mammalian gene for GRP 75 has not yet been isolated, in yeast the SSCl gene has been shown to encode this mitochondrial form of HSP 70 (27). Of all of the members of the yeast HSP 70 family, this mitochondrial form exhibits the highest sequence homology with the bacterial homologue of HSP 70, the so-called dnaK protein (26). Because HSP 70 represents the most highly induced member of the stress protein family, it has attracted considerable attention from workers within the heat shock field. Our first clue regarding the role of HSP 70 followed from the work of Bardwell and Craig (7), who were the first to isolate the gene encoding HSP 70 from Drosophila. Sequence analysis revealed that the Drosophila HSP 70 gene was -50% homologous to the Escherich,ia coli 70-kDa HSP referred to as dnaK (7). Earlier work had shown that mutations in dnaK resulted in a number of interesting phenotypes, including an inability of the mutant bacteria to support the growth of various bacteriophages (for review see Ref. 45). Subsequent work demo nstrated that such mutants also exhibimpaired DNA ited alterat #ions i n cellular metabolism, and RNA synthesis, compromised cell division, and finally an inability to grow at high temperatures. The fact that the Drosophila HSP 70 gene was homologous to a protein in E. coli that appeared essential for so many different biological phenomenon was indicative that HSP 70 also served an important role in eukaryotic cells. Our first insights regarding the biochemical role of the HSP 70 family of stress proteins followed from the observation that the form of HSP 70 present within the ER, GRP 78, was identical to a previously described and somewhat characterized protein referred to as BiP (54, 95). The BiP, short for binding protein, had been shown to interact transiently with newly synthesized immunoglobulin (Ig) heavy (H) and light (L) chains within the ER lumen (11, 54). For example, using metabolic pulsechase labeling techniques, it was shown that BiP interacted with the individual IgG H and L chains before their assembly into the final H,L, immunoglobulin structure. Within a few years, a number of other secretory proteins were shown to similarly interact with BiP

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RESPONSE

before their assembly into higher ordered structures (24, 49). In each case, the interaction of BiP with the particular secretory protein appeared transient, with the release of BiP from its target requiring ATP hydrolysis. Moreover, those secretory proteins that failed to properly assemble within the ER, either due to specific mutations or when not properly glycosylated, were observed to remain within the ER in a relatively stable complex with BiP (69, 75). Consequently, it was suggested that BiP likely served as some part of a quality control system within the ER, involved in monitoring the proper assembly of proteins that were being readied for their secretion out of the cell. Subsequent work began to show that the cytosolic forms of HSP 70 (HSP 72 and 73) also were involved in various aspects of protein maturation. For example, the cytosolic HSP 70 proteins were reported to be essential for the proper posttranslational translocation of newly synthesized proteins from the cytosol into either the ER or mitochondria of yeast (22, 30). It was suggested that the cytosolic HSP 70 proteins directly interacted with those proteins to be translocated and that such interactions likely served to maintain the targets in an unfolded or “translocation competent” state within the cytoplasm until their subsequent import into the appropriate organelle. Again, these processes appeared to require ATP and in particular ATP hydrolysis. On the basis of more recent studies, there is now evidence that implicates a rather widespread role for the cytosolic forms of HSP 70 (HSP 72/73) in facilitating the early steps of protein maturation (9). With the use of metabolic pulse-chase labeling techniques, it was observed that a large number of newly synthesized proteins could be isolated in a complex with HSP 72/73. Such interactions appeared transient such that 15-30 min after their synthesis, the newly synthesized proteins no longer were found complexed with HSP 72/73. These interactions of HSP 72/73 with newly synthesized proteins appeared to take place as the target proteins were undergoing their synthesis on the ribosome. Indirect support for this notion was the fact that newly synthesized proteins, released prematurely from the ribosome via the addition of puromycin, could be isolated in a complex with HSP 72/73. V.

ROLE

OF HEAT

CHAPERONES AND

SHOCK

PROTEINS

IN FACILITATING

AS MOLECULAR PROTEIN

FOLDING

ASSEMBLY

These studies, in sum, have led to the idea that the family of related HSP 70 proteins is intimately involved in various aspects of protein maturation. Although it has been generally accepted that protein folding is dictated only by the primary amino acid sequence of the polypeptide chain (and suitable posttranslational modifications), these new results regarding the properties of both the HSP 70 and HSP 60 families of stress proteins have forced us to reconsider this notion (2). Specifically, in the cell protein folding and assembly now is believed


WILLIAM

HSP 70

FIG. 3. A model by which HSP 70 interacts with and facilitates maturation of newly synthesized proteins. During protein synthesis, nascent polypeptide chains emerge from ribosome and interact with HSP 70. Multiple copies of HSP 70 may be necessary to interact with nascent polypeptide chain, the number likely depending on size of target protein. Binding to HSP 70 prevents premature folding of nascent chain. Once synthesis is complete, target proteins may undergo different routes. For monomeric proteins (I), folding occurs through orderly release of HSP 70 proteins from peptide domains as they fold together into their final conformation. Such release is facilitated by ATP hydrolysis. For proteins that are to be assembled into an oligomerit structure (2), partial folding of monomers with concomitant release of some HSP 70 molecules may occur. These “assembly competent” monomers now proceed to assemble into their proper oligomeric structure, accompanied by release of remaining HSP 70 chaperone. For those proteins that are translocated from cytosol into organelles (endoplasmic reticulum or mitochondria) (3), target protein is maintained in a semifolded, or translocation competent, form by virtue of bound HSP 70 protein. As translocation proceeds, HSP 70 proteins are released. [From Beckmann et al. (9), copyright 1990 by the AAAS.]

to require the participation of additional components, some of which represent members of the HSP 70 and HSP 60 families of stress proteins (for review see Refs. 39,118). A possible role for some of the stress proteins in mediating such events is outlined in Figures 3 and 4. As proteins are being synthesized, the nascent chains emerge from the ribosome and become complexed with the cytosolic forms of HSP 70 (Fig. 3). Although not yet proven, we suspect that the interaction of HSP 70 with the nascent polypeptide chain prevents its premature folding until its translation has been completed. As is indicated in Figure 3, multiple copies of HSP 70 might be required to bind to the various peptide domains of the maturing polypeptide. Perhaps, as each of the individual domains of the polypeptide is synthesized, its folding is transiently delayed via binding to HSP 70. Whether the individual domains of the polypeptide begin to fold cotranslationally or whether folding only commences once the entire polypeptide has been synthesized remains to be established. Whatever the case, the release of HSP 70 from the newly synthesized protein appears to require ATP hydrolysis. For proper folding to ever take place, the affinity of HSP 70 for its target (e.g., a peptide domain) would have to be less than the

J. WELCH

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72

affinity of the neighboring peptide domains for one another as they fold together. That HSP 70 does indeed bind to short synthetic peptides has been observed in vitro. In addition, the binding constants so determined (in the micromolar range) indicate that although such interactions do occur, they are relatively weak (43). Although this proposed model is supported by some experimental data, albeit limited, it has not been shown that all newly synthesized proteins do in fact interact with HSP 70 during the course of their synthesis. An alternative possibility is that HSP 70 interacts with only a subset of newly synthesized proteins that for some reason are experiencing difficulties with respect to their normal folding pathway. Through such an interaction, this subset of newly synthesized protein might be stabilized in their “unfolded” form and, with time, recommence along their productive folding pathway. Thus whether HSP 70 is an essential component in all protein folding events or whether it serves a more specialized “quality control”-like role will require additional experimentation. In the case of those proteins destined for posttranslational translocation into an intracellular organelle (e.g., ER, mitochondria) the completed polypeptide might remain in an unfolded or translocation competent state by virtue of its continued interaction with HSP 70 (Fig. 4). The pertinent question here is why, unlike a normal cytosolic protein, would the protein destined for translocation not spontaneously begin to fold? A likely answer is the fact that most proteins that are to be transferred from the cytosol into an organelle are synthesized with a leader or “signal” sequence, usually present at the extreme NH, terminus. In addition to dictating where the protein will be transferred, recent studies have shown that the presence of a signal sequence also can act to retard or slow down the final folding of the polypeptide (102). As the preprotein arrives at its appropriate organelle, the partially unfolded or translocation competent preprotein (either already bound to HSP 70 or now requiring HSP 70 to facilitate further unfolding) begins its entry into the organelle accompanied by the ATP-dependent release of its cytosolic chaperone, HSP 70. Once the translocating polypeptide has entered into the appropriate organelle, the signal peptide is removed, and the compartmentalized forms of HSP 70, GRP 78 (BiP) within the ER or GRP 75 within the mitochondria, now are called into play. (These events are outlined in Figure 4 for GRP 75 within the mitochondria. Similar events likely occur with respect to BiP within the ER.) Binding of BiP or GRP 75 to the transloeating and unfolded polypeptide would again serve to stabilize or prevent the premature folding of the incoming protein until the translocation event has been completed. Some indirect support for this proposed scenario has recently been provided via the analysis of temperature-sensitive mutants of either BiP of GRP 75. Specifically, in the absence of nonfunctional BiP or GRP 75 (e.g., using temperature-sensitive yeast mutants), those proteins entering into either the ER or mitochondria, respectively, become arrested within the translocation


HEAT

SHOCK

1073

RESPONSE ___

FIG. 4. Model describing possible role of GRP 75 and HSP 58 in mitochondrial protein import, folding, and assembly is shown. Newly synthesized proteins destined for mitochondria are maintained in unfolded or translocation competent state within cytoplasm by virtue of their interaction with cytosolic HSP 72/73. Translocation of polypeptide into mitochondria is accompanied by ATP-dependent release of HSP 72/73. As transloeating and unfolded polypeptide enters into mitochondria, it becomes complexed with GRP 75, and mitochondrial signal sequence is removed by signal peptidase. Once entirely inside mitochondria, folding of polypeptide commences, accompanied by ATP-dependent release of GRP 75 (la). For some monomeric mitochondrial proteins, it remains possible that folding is also dependent on an interaction with HSP 58 (lb). For assembly of oligomeric proteins, GRP 75 is released from monomer (k), and monomer then moves to HSP 58 ($b) and is assembled into its oligomeric form (9). Alternatively, monomer, still bound to GRP 75, moves to HSP 58 (k) and is assembled into its oligomeric form (3). [From Mizzen et al. (go), Cell R~!!!ulution(c>1991. The American Society for Cell Biology.]

Polypeptlde

CYTOPLASM

INTERMEMBRANE

SPACE

channel and consequently do not complete their translocation (67, 136). One interpretation of these results is that in the absence of binding to its GRP 75 or BiP chaperone, the translocating polypeptide, with a large part of its chain still present in the cytoplasm, begins to fold prematurely and therefore incorrectly, and as a result may “precipitate” within the translocation channel. Under normal conditions, once translocation into the organelle has been completed (and now with the signal sequence having been removed), the translocated protein now commences along its proper folding pathway, again accompanied by release of its particular HSP 70 chaperone. In the case of those proteins that are to be assembled into higher ordered oligomeric structures, the translocated polypeptide may likely remain bound to its chaperone until its oligomeric assembly has been accomplished. This would be consistent with previous observations showing that IgG H and L chains, even after their translocation into the ER lumen has been completed, remain bound to BiP until their subsequent assembly into the final H2L, oligomeric structure. In the case of mitochondrial (or chloroplast) proteins that are to be folded and/or assembled into oligomeric structures, the groEL homologue, HSP 58 or 60, is called into action. For example, HSP 60, perhaps in conjunction with the mitochondrial form of HSP 70, acts in concert to ensure that folding and/or oligomerization events proceed in an orderly fashion. Considering that similar posttranslational folding and assembly events also occur within other organelles (e.g., cytoplasm, ER, and nucleus), I suspect that other forms of groEL will eventually be discovered. Indeed, very recent studies have

iLlonomer

3

described a HSP 58/groEL-related protein within the cytoplasm that similarly may play a role in the folding and assembly of various cytosolic proteins (132,146). VI.

ROLE

OF

HEAT

EXPERIENCING

SHOCK STRESS

PROTEINS AND

THEIR

IN CELLS POSSIBLE

MODE

OF REGULATION

These new results implicating a number of the stress proteins as being essential in protein maturation events may explain why their expression is increased in the cell experiencing stress. Presumably their increased expression allows the cell under stress to better cope with the problems it faces with respect to protein maturation events. As discussed in section I, most of the agents/treatments known to induce the stress response fall under the category of being protein denaturants. Examples include elevated temperatures, amino acid analogues, and various heavy metals or other agents (e.g., arsenite) that attack sulfhydryl groups. The protein targets to be affected would either be mature proteins or proteins in the process of maturation. Because some of the stress proteins appear integral in facilitating protein maturation events, one suspects that it is probably nascent (i.e., maturing) proteins that represent the major labile target in the cell experiencing stress. This proposal is supported by a number of relevant observations. First, the simple fact that amino acid analogues induce high-level expression of all of the stress proteins indicates that it is the newly synthesized and analogue-containing proteins that somehow are recognized by the cell


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WILLIAM

as being abnormal and thereby trigger the stress response. Indeed, it has been shown that the interaction of cytosolic HSP 70 with newly synthesized proteins is transient in the normal cell, but the interaction of HSP 70 with newly synthesized proteins containing an amino acid analogue is relatively long lived (9). Presumably, if the newly synthesized and completed polypeptide is unable to commence proper folding, due to the presence of the incorporated amino acid analogue, the polypeptide continues to appear unfolded and consequently remains bound to its HSP 70 chaperone. Similar observations have been made for BiP in the ER. Specifically, various secretory proteins that are unable to properly fold or assemble into their final oligomeric structures, for example, due to underglycosylation or specific mutations, remain bound to their BiP chaperone and are not efficiently secreted (11, 34, 69, 75). The idea that proteins that are in the process of maturation represent a particularly labile target in the cell under stress is also supported by previous observations examining the intracellular locale of HSP 72/73. For example, soon after heat shock treatment, a significant portion of HSP 72/73 was found to accumulate within the nucleus and, in particular, the nucleolus (106, 135, 141; Fig. 5). As discussed in section III, previous studies had demonstrated significant alterations in nucleolar integrity after heat shock and an accompanying inhibition of ribosome biogenesis events. Analysis of the nucleolus after heat shock by high-resolution electron microscopy has revealed significant amounts of denatured and/or aggregated preribosomal components (3, 124, 145). Most likely after heat shock treatment, maturing preribosomes experience difficulty in their folding and assembly, perhaps begin to denature or unfold, and now become targets for HSP 70. Whether binding to HSP 72/73 facilitates their refolding (i.e., renaturation) or, alternatively, simply allows for their solubilization and subsequent turnover remains to be determined. With time of recovery from the stress event and as the nucleoli regain both their normal morphology and function, HSP 72/73 appears to exit the organelle and now accumulates to relatively higher levels within the cytoplasm (Fig. 5). Thus, in the normal unstressed cell, members of the HSP 70 family appear to interact with the unfolded forms of proteins that are in the course of maturation. Presumably via their interaction with the particular HSP 70 family member, the maturing polypeptide is stabilized and/or prevented from premature folding. Once synthesis or translocation of the polypeptide has been completed and as it now begins to assume its final three-dimensional structure, the HSP 70 chaperone is released presumably through its ability to bind and hydrolyze ATP. The released HSP 70 chaperone is now ready to participate in a subsequent folding pathway. In cells experiencing stress, usually due to their exposure to agents/treatments that lead to the accumulation of abnormally folded proteins, many of the abnormally folded proteins appear to interact stably with the different forms of HSP 70. Over time and as more proteins become stablv complexed with their HSP 70 chaperones,

Volume

J. WELCH

72

the available levels of the particular HSP 70 chaperone are eventually exhausted. The cell somehow senses this reduction and responds by the increased expression of new HSP 70 chaperones. This proposed model by which synthesis of the various HSP 70 family members is rec,ulated as a function of substrate levels appears consistent with a number of previous observations. First, injection into cells of a collection of denatured proteins results in an induction of a stress response and, in particular, increased expression of cytosolic HSP 70 (1). Presumably, the denatured proteins introduced into the cell are recognized as being unfolded, become complexed with cytosolic HSP 70, and thereby reduce the available levels of free HSP 70. The cell now responds by increased expression of cytosolic HSP 70. Second, a number of studies have demonstrated that the relative increases in HSP 70 synthesis appear to depend on both the preexisting l!:vels of HSP 70 and the severity of the stress treatment (31, 91). Third, in cells expressing secretory proteins that are not efficiently secreted due, for example, to specific mutations and/or improper glycosylation, the secretory protein adversely affected appears to remain stably complexed with its HSP 70 chaperone, BiP (69, 75). Again, as a consequence, the cell responds by the upregulation of new BiP synthesis. How the cell monitors the reduction in the available pool of the particular HSP 70 family member, and subsequently triggers its increased expression, remains unclear and represents an interesting aspect of signal transduction. One attractive idea, at least for the cytosolic HSP 70 proteins, is that the transcription factor that regulates HSP 70 expression may itself exist in a complex with HSP 70. Under normal growth conditions, when HSP 70 levels are in excess, the transcription factor would be maintained in an inactive form, perhaps by virtue of its being complexed with HSP 70. In response to conditions of stress and as HSP 70 is recruited into longer lived complexes with abnormally folded proteins, the equilibrium would be shifted such that the transcription factor is released from HSP 70 and is now able to bind to its target gene and activate HSP 70 transcription. As the levels of free HSP 70 are restored to some critical level, the equilibrium is shifted backward such that the transcription factor again becomes comple;,ed with HSP 70 and there occurs a corresponding cessation of HSP 70 transcription. Alternatively, the transcription factor might exist in an inactive complex with some other protein cofactor. On stress, this cofactor itself may begin to denature, become a target for HSP 70, and thereby liberate the transcription factor to now bind to its target genes. Now with the heat shock transcription factor having been purified and its corresponding gene isolated, the exact molecular details by which this transcription factor is regulated should be shortly forthcoming. VII.

STRESS

RESPONSE

IN DISEASE

AND

MEDICINE

A. Stress Response as a Marker for Cell Injury

Considering that the stress response represents a basic cellular defense mechanism employed bv all cells


FIG. 5. Distribution of HSP 72 in rat fibroblasts after heat shock and during recovery. Rat fibroblasts were subjected to a 42.5”C, 3-h heat shock treatment. After treatment, 1 cover slip was removed, cells were fixed, and distribution of highly stress-inducible HSP 72 was determined by indirect immunofluorescence using antibodies specific for HSP 72. To remaining cells, cover slips were removed from heat shock incubator and further incubated at 37°C for either 4, 8, or 24 h. At each time point of recovery, a cover slip was removed and again HSP 72 locale was determined by indirect immunofluorescence. A, C, E, and G: phase micrographs. B, D, F, and H: corresponding fluorescent micrographs of cells. Note intense nuclear and, in particular, nucleolar staining in cells immediately after heat shock treatment (A and B) and persisting for as many as 4 h after recovery (C and D). With time of further recovery, most of protein is no longer found within nucleolus but instead accumulates within cytosol. A and B: cells heat shock treated for 3 h. C and D: cells heat shock treated and recovered for 4 h. E and l? cells heat shock treated and recovered for 8 h. G and H: cells heat shock treated and recovered for 24 h. [From Welch and Suhan (145), by copyright permission of the Rockefeller University Press.] 1075 Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (130.064.011.153) on July 26, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

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in times of trouble, it is not too surprising that investigators are finding the response to be important in various aspects of medicine and disease. One area that is beginning to attract considerable attention concerns the potential use of the stress response as a diagnostic tool for determining the extent of tissue/organ traumas. Particularly relevant here are studies examining the stress response in various organs and tissues following ischemia and reperfusion. In the case of the central nervous system, increased levels of the stress proteins are observed within certain regions of the brain following transient ischemia (3.2). Perhaps more interesting are the observations showing that the ability of certain neuronal populations to survive an ischemic trauma appears to be correlated with their increased expression of the stress proteins. For example, induction of the highly stress-inducible HSP 72 protein following transient ischemia was most pronounced within both the dentate granule cells and the hippocampal CA3 cells, regions in the brain that are known to exhibit the highest survivability following an ischemic trauma. Conversely, HSP 72 accumulation appeared minimal in those regions of the brain, like the CA1 region, that appeared to be the most sensitive to the ischemic episode (134). In yet another study, increased levels of the stress proteins within rat photoreceptor cells appeared to confer an additional degree of protection to the cells following their exposure to otherwise damaging bright light. Here a positive correlation was observed between maximal photoreceptor protection and the levels of HSP 72 (6). In the heart, increased expression of stress proteins is observed during either transient ischemia-reperfusion or after hemodynamic overload (28, 33). Moreover, increased expression of the stress proteins in the heart, by prior hyperthermic exposure of the whole animal, again appeared to confer a protective effect to the organ upon a subsequent ischemic episode (68). Thus clinicians are beginning to use stress protein expression as a sensitive indicator by which to diagnose injury in vivo. Perhaps more exciting, however, is the therapeutic potential of the stress response. Being able to render tissues or organs thermotolerant, perhaps via site-directed hyperthermia or by pharmacological means, could be exploited in the whole animal and help in reducing injury from relevant traumas such as ischemia-reperfusion that often accompany different types of elective surgery and/or during organ transplantation. B. Stress Response and Immunology Yet another area receiving considerable attention regards the role of the stress response as it relates to various aspects of the immune response (extensively reviewed in Ref. 70). Fever has long been argued to enhance overall white blood cell function and enhance the activities of various components associated with the febrile response. For example, so-called endogenous pyrogens such as the interleukins, tumor necrosis factor, and interferons appear to exhibit enhanced activities at ele-

J. WELCH

Voluwle

22

vated temperatures such as those encountered during fever (for review see Ref. 109). It is tempting to speculate that one role of the febrile response is to augment white blood cell function via the increased expression of the stress proteins. Whether the stress proteins, distinct from their role in facilitating protein maturation events, directly play a role in other aspects of the immune response is not clear. It has been shown that various lymphokines affect either the expression and/or phosphorylation of a number of the stress proteins in different white blood cell types (41, 108). For example, B-cells, when activated via their antigen receptor, exhibit a rapid increased expression of the stress proteins. These activated B-cells now exhibit increased thermotolerance in vitro compared with their resting counterparts (126). Recent reports have described what appears to be yet another member of the HSP 70 family that may be involved in some aspect of class II histocompatability-mediated antigen presentation (133). This observation takes on added interest considering the fact that I) the HSP 70 proteins, like the class I and class II histocompatability proteins, appear to bind somewhat promiscuously to short synthetic peptides (43); 2) the putative peptide binding site of HSP 70 has been suggested to be remarkably similar, in its overall three-dimensional structure, to that already determined for the peptide binding domains of the histocompatability proteins (115,120); and 3) one or more of the genes encoding HSP 70-related proteins are found within the major histocompatability locus (for review see Ref. 52). Also intriguing are recent results examining the role of stress proteins as targets for the immune response following microbial and parasitic infections. After invasion into the warm-blooded host, there occurs a vigorous production of the microbial stress proteins, some of which appear to represent immunodominant targets for both T- and B-cell responses. Attention in this regard also has begun to focus on a somewhat novel subset of T-cells referred to as y/6 T-cells. This class of T-cells, found primarily in the epithelial of the skin, small intestine, and reproductive organs of mice, appears less mobile than their a&counterparts, exhibit cytolytic activity, and so far have been shown to express a rather limited repertoire of T-cell receptors (for review see Ref. 70). Many of the y/&receptors so far characterized appear specific for the microbial forms of the stress proteins and, in particular, an epitope present within the groEL family of stress proteins. Hence, one idea is that these y/6 T-cells may represent an immunologically predetermined “front line” defensive mechanism by which the immune system confronts various types of microbial infections. In addition, there also are reports that some autoimmune diseases may arise as a result of the activation of T- and B-cells that appear specific for foreign stress proteins but that may also recognize the closely related stress proteins of the host. Indeed, antibodies to self stress proteins have been reported in a variety of autoimmune disorders including rheumatoid arthritis, systemic lupus, ankylosing spondylitis, Reiter’s syndrome, and Crohn’s disease (for re-


October

1992

view see Ref. 147). Whether such autoantibodies rectly responsible for each of these particular mune disorders still remains to be established.

HEAT

SHOCK

are diautoim-

C. Stress Response and Toxicology Finally, the stress response is being considered as a new tool for toxicology, either using in vitro cell lines or in the whole organism (61,94). The rationale here is that so many environmentally relevant insults (e.g., pollutants) can elicit the increased expression of one or more of the stress proteins. Thus, being able to monitor the induction of those stress proteins that are only expressed in the traumatized cell (e.g., HSP 72) could be exploited for the development of new and rapid toxicological assays. For example, cell lines have been constructed with stress protein promoter elements driving the expression of reporter genes (R. Voellmy, unpublished observations). Using such reporter cell lines, one could potentially screen, in large numbers and relatively quickly, whether various drugs or cosmetics, result in an induction of a stress response. Indeed, some success in this regard has already been realized for the screening of teratogens, agents that result in developmental abnormalities (16; Voellmy, unpublished observations). With the use of a similar approach, such stress protein promoter/reporter systems could be introduced into animals (e.g., transgenic fish) and the animals then used as “scouts” to determine whether a particular environment (lake or stream) contains levels of pollutants that might be sufficient to activate a stress response. If so, this might be indicative that the pollutants are reaching levels that may be deleterious to the resident organisms and thereby alert us to subsequent clean up activities.

VIII.

CONCLUSIONS

The stress response represents an evolutionary conserved mechanism by which cells respond and defend against abrupt and adverse changes in their environment. Although expressed at relatively higher levels in the traumatized cell, most of the stress proteins are in fact synthesized under normal growth conditions and appear integral in a number of important cellular processes. Perhaps most exciting is the story developing that implicates a central role for many of the stress proteins in facilitating protein maturation. While it is still believed that the primary amino acid sequence of a protein (as well as its various posttranslational modifications) dictates its final three-dimensional structure, these new studies indicate that inside the cell protein folding and assembly may also require the participation of additional components, so-called molecular chaperones, which include members of the stress protein family. When chaos is introduced into the folding process, as appears to occur in cells placed under stress by exposure to agents/treatments that interfere with protein folding, the cell responds by increasing the expression of its

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1077

molecular chaperones. In addition to these new developments at the biochemical level, investigators are beginning to explore the role of the stress response and stress proteins in various aspects of disease. In this regard, the potential role of the stress proteins as sensitive markers of cell injury, and their possible connection with the immune response and autoimmune diseases are under investigation. Finally, the idea that their expression may be expolited as a new way by which to monitor the status of our environment is also being explored. I gratefully acknowledge my fellow co-workers, both past and present: J. R. Feramisco, P. T. Thomas,A. P. Arrigo, L. A. Mizzen, R. P. Beckmann, H. S. Kang, R. Brown, R. Martin, G. Minowada, and W. J. Hansen. The assistance of M. Lovett, A. N. Kabiling, and T. M. Kleven is also acknowledged. In addition, thanks to all of my many collaborat.ors. Our work has been supported over the past 9 years by National Institute of General Medical Sciences Grant GM33551, American Chemical Society Grant CD-502, and National Science Foundation Grant DCB-9018320. REFERENCES 1. ANATHAN, J., A. L. GOLDBERG, AND R. VOELLMY. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. S&rice Walsh. DC 232: 252-254, 1986. 2. ANFINSEN, C. B. Principles that govern the folding of protein chains. Scieqlce Wash. OC 8: 223-230, 1973. 3. AMALRIC, F., R. SIMARD, AND J. P. ZALTA. Effect de la temperature supraoptimale sur les ribonucleoprotines. II. Etude biochmique. Exp. Cell Rex 55: 370-377, 1969. 4. ARRIGO, A. P., J. P. SUHAN, AND W. J. WELCH. Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat shock protein. Mol. Cell. Biol. 8: 5059-5071, 1988. 5. ASHBURNER, M., AND J. J. BONNER. The induction of gene activity in Drosophila by heat shock. Cell 17: 241-254, 1979. 6. BARBE, M. F., M. TYTELL, D. J. GOWER, AND W. J. WELCH. Hyperthermic protects against light damage in the rat retina. Science Wash. DC 241: 1817-1820, 1988. 7. BARDWELL, J. C., AND E. A. CRAIG. Major heat shock gene of fiosophila and the heat inducible dnaK gene are homologous. Proc. Nutb Acad. Sci. USA 81: 848-849, 1984. M. Gene regulation by steroid hormones. Cell 56: 3358. BEATO, 344,1989. 9. BECKMANN, R. P., L. A. MIZZEN, AND W. J. WELCH. Interaction of hsp 70 with newly synthesized proteins: implications for protein folding and assembly events. Science Wush. DC248: 850854,199O. 10. BOCKKANEVA, E. S., N. M. LISSIN, AND A.-S. GIRSHOVICH. Transient association of newly synthesized unfolded proteins with the heat shock groEL protein. Nature Land. 336: 254-257, 1988. 11. BOLE, D. G., L. M. HENDERSHOT, AND J. F. KEARNEY. Posttranslational associations of immunoglobulin heavy chain binding protein with nascent heavy chains in non-secreting and secreting hybridomas. J. CeZr! Biol. 102: 1558-1566, 1986. 12. BOND, U., AND M. J. SCHLESINGER. Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Mol. CeZI. Biol. 5: 949-956, 1986. C., AND G. L. E. KOCH. Perturbation of cellular calcium 13. BOOTH, induces secretion of luminal ER proteins. Cell 59: 729-737, 1989. 14. BRUGGE, J. S. Interaction of the Rous Sarcoma virus protein, proteins pp50 and pp90. Curr. Top. Mipp60”‘“, with the cellular crobiol. Immunol. 123: l-23, 1986. 15. BRUGGE, J. S., E. ERIKSON, AND R. L. ERIKSON. The specific interaction of the Rous sarcoma virus transforming protein, with cellular proteins. Cell 25: 363-372, 1981. PP6C


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