Role of the Heat-Shock Response in the Life and Death of...

Role of the Heat-Shock Response in theLife and Death of Proteins

ANU MATHEW AND RICHARD I. MORIMOTOa

Department of Biochemistry, Molecular Biology and Cell Biology, RiceInstitute for Biomedical Research, Northwestern University, Evanston,Illinois 60208 USA

Changes in protein levels and the folded states of proteins are sensed by molecularchaperones and the degradative machinery that function to maintain cellular pro-tein homeostasis. The importance of these systems is highlighted by their highdegree of functional and structural conservation across evolution, from prokaryotesto eukaryotes, and their essential roles in a variety of cellular functions.1–3 The chap-erone network, proteases, and components of the degradative machinery are alsolinked by common genetic regulatory pathways. For example, many of the proteinsinvolved in protein folding and degradation are also heat-shock proteins whoseexpression is induced when cells are stressed and accumulate denatured or mal-folded proteins.1,3–6 The family of heat-shock proteins (HSPs) encompasses a num-ber of functionally related proteins that are expressed constitutively and/or atelevated levels upon exposure of cells to a variety of stress conditions including ele-vated temperature, arsenite, heavy metals, amino acid analogues, and oxidants.1,4,6

The chaperones and proteolytic systems often function together to determinethe ultimate fates of proteins and are often coordinately regulated, as outlined inFIGURE 1. The heat-shock response regulates proteolysis by enhancing the expres-sion of proteolytic factors and proteases, as well as of chaperones which may func-tion as cofactors for proteolysis.2,3,5 The stress response in both prokaryotes andeukaryotes is, in turn, regulated by the proteolytic machinery7–10 (also A. Mathew,S. K. Mathur, and R. I. Morimoto, unpublished observations). This review willaddress the chaperone requirements in eukaryotic proteolysis and the regulationof eukaryotic chaperone expression by the ubiquitin–proteasome proteolytic system.A number of recent reviews have also addressed chaperone activities associated withproteolysis in prokaryotes and eukaryotes.3,11,12

BIOCHEMICAL PROPERTIES OF MOLECULAR CHAPERONES

Chaperones are involved at various stages in protein biogenesis, regulatingtheir structure and function under normal physiological conditions, as well as dur-ing and following stress which results in protein unfolding and misfolding. Thefamily of heat-shock proteins and molecular chaperones includes Hsp100, Hsp90,Hsp70 (dnaK), Hsp60 (GroEL), Hsp40 (dnaJ), and the small HSPs. These chaper-ones are abundant and ubiquitous and function constitutively in protein synthesisand folding, protein translocation into membrane compartments, and the assemblyand disassembly of oligomers.2,13 Chaperones often function in concert with other

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aAddress for correspondence: Dr. Richard I. Morimoto, Department of Biochemistry,Molecular and Cell Biology, Northwestern University, 2153 North Campus Drive, Evanston,IL 60208. Telephone: 847-491-3340; Fax: 847-491-4461; e-mail: [email protected]

protein-folding activities such as protein disulfide isomerases and peptidylprolylisomerases (immunophilins).13 Molecular chaperones including Hsp90, Hsp70,Hip, p23, and immunophilins regulate steroid hormone aporeceptors and kinaseactivities.14–18 The activities of the Hsp70 and Hsp60 chaperones are also modu-lated by their associations with cochaperones.13 The prokaryotic cochaperonesDnaJ and GrpE stimulate the ATPase activity of Hsp70 (DnaK) and promote effi-cient nucleotide release, respectively. In eukaryotes multiple DnaJ homologues areexpressed in addition to other Hsp70 regulatory proteins such as Hip andBag1.19–20a Similarly, the ATPase activity of GroEL (bacterial homologue of hsp60)is modulated by GroES.13

Under conditions of stress, heat-shock proteins prevent inappropriate inter-and intramolecular interactions, which may lead to protein misfolding or aggre-gation.1,2,13,21,22 Prolonged association with chaperones may occur in the event thatproteins are irreparably defective in conformation or oligomer assembly.3 Variousactivities of the HSP family members are outlined in FIGURE 2. In vitro studies toelucidate chaperone activities have shown that Hsp90, Hsp70, Hsp60, and thesmall HSPs are effective in maintaining unfolded proteins in soluble nonnativestates conducive to refolding to native state.13,23,24 The Hsp70 and Hsp60 chaper-ones, together with their respective cochaperones and ATP, have the ability torefold nonnative intermediates to the final native state13 whether the substrate is anascent polypeptide25 or thermally unfolded.26,27 Malfolded proteins not rescuedby the action of these chaperones are likely to form aggregates. The Hsp100 chap-erones have the unique ability to disaggregate protein aggregates in an ATPdependent manner.28

100 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Coordinate regulation of expression and activity of chaperones and proteolyticfactors.

THE UBIQUITIN–PROTEASOME SYSTEM OF PROTEIN DEGRADATION

Denatured and misfolded proteins that appear during heat shock and otherstresses that cannot be refolded by the chaperone machinery must be rapidlyremoved by proteolysis. The ubiquitin–proteasome machinery is the major routeof removal of soluble cytosolic and nuclear proteins in higher eukaryotes and con-sequently has a central role in a variety of regulatory events of the cell.29–34 Thesteps involved in the ubiquitin–proteasome-dependent degradation of proteinsare summarized in FIGURE 3. Many of the proteins targeted to this system fordegradation by the proteasome (a multicatalytic protease complex) are modifiedby polyubiquitination. However, it must be noted that not all proteasome sub-strates are ubiquitinated35–37 and not all ubiquitinated proteins are metabolicallyunstable5 or targeted to the proteasome (some are targeted to the lysosome/vac-uole).38– 40 The 26S proteasome32,34,41,42 comprises a cylindrical 20S catalytic coreassembled from four heptameric rings. Deletion of even one of its subunits islethal to the cell, and, furthermore, mutations in these subunits often result in sen-sitivity to stress.43 A 19S particle that associates with each end of the 20S cylinderhas been suggested to participate in the unfolding of the substrate and facilitatingthe translocation of the substrate into the core.42 The composition of the 19S“mouth” particle is complex and contains a ubiquitin-binding site, severalATPases, and isopeptidases (to cleave and release ubiquitin chains and moi-eties),32,41 which all probably contribute to the selective nature of the proteasome.

Ubiquitination is mediated by a cascade of enzyme activities, namely, the ubiquitin-activating enzyme, E1, ubiquitin-conjugating enzymes (E2), and theubiquitin protein ligases (E3), as outlined in FIGURE 3.5 Ubiquitin is activated to anactive thiol ester intermediate by the action of E1. The activities of the E2 proteinsfacilitate covalent attachment of the activated ubiquitin to the lysine residues of

MATHEW & MORIMOTO: HEAT-SHOCK RESPONSE 101

FIGURE 2. Functions of the chaperones. Proteins in their native state (1) undergo unfold-ing as a result of stress. This transient unfolded state (2), can be prevented from aggregat-ing by association with the Hsp70, Hsp60, Hsp90, and small HSP proteins (3). Onceaggregate formation occurs, the activity of the Hsp100 proteins may facilitate disaggrega-tion (4). The transient unfolded intermediate of the protein can also be refolded by theactions of Hsp60, Hsp70, and their cochaperones, in the presence of ATP (5). The latter fac-tors mediate the folding of nascent polypeptides as well (6).

target protein substrates by formation of an isopeptide bond, which may or maynot be associated with an E3 activity. Polyubiquitin chains are normally added tothose substrates destined for degradation. The abundant protein ubiquitin isexpressed constitutively in all cells and expression is upregulated upon exposureto stress. At least two yeast E2 proteins, UBC4 and UBC5, are also heat-shockinduced and are responsible for most of the ubiquitin-conjugating activity duringstress.44 Exposure of yeast cells to cadmium results in significantly elevatedexpression of an alternate pair of E2 proteins, namely, UBC5 and UBC7.45 The exis-tence of multiple E2 and E3 enzymes that respond to different stresses, togetherwith the ability to form alternative ubiquitin chain conformations, may provide ahigh degree of substrate specificity.5,46 The fate of proteins that accumulate duringexposure of cells to stress is dependent on the interplay of chaperones and theubiquitin–proteasome system.3,12 The stress signal, in turn, leads to the transcrip-tional activation of genes encoding components for proteolysis and chaperoneactivity1–7,8a,10 (also, A. Mathew, S. K. Mathur, and R. I. Morimoto, unpublishedobservations).


FIGURE 3. The ubiquitin–proteasome pathway of protein degradation.

ROLE OF MOLECULAR CHAPERONES IN PROTEIN DEGRADATION

Molecular chaperones are required for normal energy-dependent proteindegradation in various prokaryotic systems, and furthermore, the levels of certainchaperones are often rate limiting to proteolysis.3,47– 49 Mutations in genes encodingchaperones result in both a general decrease in protein degradation and the con-stitutive activation of the heat-shock response, which leads to the elevated expres-sion of chaperones and proteases.50 Chaperones may function both in the refoldingof misfolded proteins and in the presentation of substrates to proteolytic activitiesif the substrate cannot be refolded. The fate of chaperone-bound substrates can bealtered by other chaperones or modulators of chaperone activities; for example,the affinity of GroEL for substrates may be increased by either its association withtrigger factor or by heat-shock-induced phosphorylation.51,52

Several lines of genetic evidence point to a link between the chaperone activitiesand ubiquitin–proteasome-mediated protein degradation. A ubiquitin-processingenzyme was identified as a suppressor of an Hsp70 mutation,21 suggesting an inter-action between Hsp70 and the ubiquitination machinery. A more direct role forchaperones in the degradative process has been suggested from yeast mutations inHsp70, which result in a general reduction in ubiquitin–proteasome-mediateddegradation of proteins. The phenotype observed for these Hsp70 mutations is sim-ilar to that described for mutations in ubc4/ubc5.1,44 Likewise, mutation of the yeastDnaJ homologue, Ydj1, affects ubiquitination of abnormal and short-lived proteins,whereas mutation of the other yeast DnaJ homologue, Sis, primarily affects protea-somal digestion of ubiquitinated proteins.3,53

Exposure of cultured mammalian cells to proteasome inhibitors leads to theaccumulation of polyubiquitinated proteins that are found to be associated withHsp90 and Hsp70. Coassociated polyubiquitinated proteins are released fromboth Hsp90 and Hsp70 immunoprecipitates upon ATP hydrolysis, which is anindication of the specificity of this interaction (A. Mathew, S. K. Mathur, and R. I.Morimoto, unpublished observations). The association of polyubiquitinated pro-teins with Hsp70 and Hsp90 is lost upon removal of the proteasome inhibitors,which reveals that the interaction is transient and reversible and that the chaper-one-associated substrates are either degraded or refolded. Direct chaperone asso-ciation may be required for presentation of specific substrates to theubiquitination machinery; however, the evidence for such a requirement isderived from in vitro experiments using lysates. Immunodepletion of Hsc70 froman in vitro degradation assay revealed a requirement for this chaperone in ubiqui-tination of some, not all, protein substrates.54 Chaperone association with specificubiquitin–proteasome substrates has been detected and proposed to be requiredto expose sites for ubiquitin modification.54 Association of Hsp90, Hsp70, andother components of the Hsp90 heterocomplex with a denatured substrate resultsin either refolding or degradation; prolonged association with Hsp90 favored thelatter.55 Specific inhibition of Hsp90 release (and consequently of the other mem-bers of the chaperone complex) with the drug herbimycin A also results inincreased degradation of other proteins known to normally associate with Hsp90(Raf-1 and transmembrane receptor tyrosine kinases).55,56 Specific Hsp70-associatedproteins shown to be degraded by the proteasome include CFTR56a,56b and theapolipoprotein B100.56c Experiments in yeast provide additional support for theroles of the chaperones Ydj1 and Hsp70 homologues in proteolysis. Both chaper-ones associate with the G1 cyclin, Cln3, an important yeast cell cycle regulator and


a ubiquitin–proteasome substrate. The association with Ydj1 is required for effi-cient Cln3 phosphorylation, a step that necessarily precedes its ubiquitination.57

CHAPERONES AND PROTEOLYSIS IN OTHER CELLULAR COMPARTMENTS

In conjunction with the proteolytic systems located in each subcellular com-partment are corresponding chaperone activities. For example, mitochondrial pro-teases require chaperones for efficient degradation of abnormal proteins.11,58,59 Forinstance, in the absence of functional mitochondrial Hsp70 or Mdj1 (DnaJ homo-logue), Pim1 proteolysis of misfolded polypeptides is severely diminished and theefficiency of protein folding declines.59 The lysosome has been shown to requireconstitutive Hsp70 for selective degradation of substrates. The purported Hsp70recognition motif is an amino-terminal localized sequence, KFERQ (or relatedsequences), which presumably targets substrates to lysosomes for degradation.60

Only lysosomal subpopulations that contain Hsp70 are capable of degradingthese specific substrates,61 supporting a role for the chaperone in translocation ofthe proteins across the organellar membrane. In addition, a specific lysosomalmembrane protein, LPG96, may be involved in this targeting mechanism, since itsassociation with the Hsp70 protein causes increased affinity for model substrateproteins. The overexpression of LPG96 was furthermore shown to result inincreased lysosomal proteolytic activity.62

The removal of mutant or misfolded proteins from the endoplasmic reticulumrequires selective elimination via the cytosolic ubiquitin–proteasome pathway.37,63

This suggests a role for chaperones in the retrograde transport of proteins to thecytosolic side of the membrane for accessibility to the proteasome. The lumen-localized homologue of Hsp70, BiP, is required for the degradation of some, butnot all, lumen proteins; however, its role in this process is unclear.3,13

In summary, chaperone activity, whether corresponding to the known bio-chemical properties of the classical chaperones or the potential role of proteasesubunits in unfolding substrates, is likely to be required for efficient proteolysis ofpolypeptides that cannot be productively renatured. Chaperones may also facili-tate the modification of obsolescent substrates (e.g., phosphorylation, ubiquitina-tion) and participate in the presentation of substrates directly to the proteolyticmachinery. Hence, chaperones constitute a major factor in the protein quality con-trol mechanism, which has recently been described as a triage system.11

THE REGULATION OF THE HEAT-SHOCK TRANSCRIPTIONALRESPONSE BY MISFOLDED PROTEINS AND PROTEOLYSIS

Heat-shock proteins that function as chaperones or proteases regulate theirexpression by their effects on the stress-induced activities of heat-shock transcrip-tion factors.8,9,64– 66 In eukaryotes, a family of heat-shock transcription factors(HSFs) mediates the transcription of genes encoding heat-shock proteins by acti-vation of preexisting forms of these proteins to transcriptionally active states. Inyeast, HSF is present as a constitutively active DNA-binding protein that acquirestranscriptional activity upon exposure to stress. In higher eukaryotes, HSFs exist


in a non-DNA-bound state, which upon heat-shock or other forms of stress under-goes oligomerization and binds DNA. HSF is trimeric in its active state and bindsto a consensus target sequence, the heat-shock element (HSE), present in promoterregions of heat-shock genes.67 The attenuation of HSF activity is likely to involvenegative regulation by Hsp70 without affecting the level of HSF.65,66 This is likelyto be at least partly due to repression of transcriptional activity by direct chaper-one association with the HSF transactivation domain, as has been shown forHSF1.67a Because the level of free Hsp70 seems to be critical and Hsp70 interactswith stress-damaged proteins, the processes by which such damaged proteins aredisposed of may be important to this homeostatic control mechanism. Of the fourHSFs in vertebrates, HSF1 corresponds to the ubiquitous heat-shock-induced fac-tor; HSF2 activity has been detected in conditions of differentiation; HSF3 exhibitsthe properties of an extreme stress-induced factor activated under non-stress con-ditions by association with c-Myb, and the expression of which is required forHSF1 activation in avian systems; and HSF4 lacks the activity of a positive activa-tor and may be a negative regulator of heat-shock gene expression.65,66,67b,67c


FIGURE 4. Regulation of chaperone expression by proteasome activity. Malfolded,mutant, and short-lived proteins are polyubiquitinated (1) and normally degraded by theproteasome (2). Inhibition of proteasome activity by the addition of MG132, lactacystin, orhemin results in accumulation of polyubiquitinated substrates (3). HSF2 is activated fromthe inert dimer to DNA-binding trimer in response to this accumulation (4). HSF2 inducesexpression of molecular chaperones such as Hsp70, Hsp90, and Hdj-1 (5). These molecularchaperones associate with the nonnative polyubiquitinated substrates (6) and are suggestedto prevent their aggregation (7), maintaining the polyubiquitinated proteins in an interme-diate folded state primed for degradation upon resumption of proteasome activity.

In addition to the classical stress induced activation of HSF1, recent work froma number of groups have shown that inhibition of the ubiquitin–proteasome path-way by specific inhibitors (reversible peptide aldehydes such as MG132 andALLN68,69 or the irreversible inhibitor lactacystin70) results in HSF activation andheat-shock protein expression in yeast and several mammalian cells7,10 (also, A.Mathew, S. K. Mathur, and R. I. Morimoto, unpublished observations) (FIG. 4). Inmammalian cells, the HSF that is primarily activated is not the stress-activatedfactor HSF1, but HSF2 as determined by gel shift and antibody supershift assays.These results are further supported by analysis of the ts85 cell line, which carriesa temperature-sensitive mutation in its ubiquitin-activating enzyme (E1)71 andactivates HSF2 when the degradative machinery is arrested. At the nonpermissivetemperature, there is a reduced ubiquitination of protein substrates11,71 that is suf-ficient to activate HSF2 and heat-shock protein expression. These results revealthat inhibition of the ubiquitin–proteasome pathway at two distinct steps resultsin activation of HSF2 and the expression of heat-shock proteins. This activationmay be associated with the observed accumulation of HSF2 under conditions ofubiquitin–proteasome inhibition, due to the combined effects of reduced HSF2degradation as well as increased HSF2 synthesis (A. Mathew, S. K. Mathur, and R.I. Morimoto, unpublished observations). This suggests that the accumulation of alabile factor, which may be HSF2 itself, that is normally ubiquitinated anddegraded by the proteasome is required for HSF2 activation.

HSF2 activity has been previously observed in a number of developmentalmodel systems such as mouse embryogenesis, spermatogenesis, hemin-inducedK562 cell differentiation, and in embryocarcinoma cell lines.65,66 In vitro terminaldifferentiation of various cell lines, including K562 cells, results in a markeddownregulation of proteasome and ubiquitin gene expression,72,73 although thelevels of certain components of the proteasome do not change dramatically.Downregulation of proteasome subunit expression during hemin-induced differ-entiation of K562 cells may contribute to the activation of HSF2; however, the sig-nal for activation of HSF2 is unlikely to be differentiation alone, as treatment ofK562 cells and other erythroleukemic cell lines with alternative inducers of differ-entiation does not lead to HSF2 activation (L. Sistonen and R. I. Morimoto, unpub-lished observations). Why then does hemin treatment result in activation of HSF2?The answer may lie in the properties of hemin, which is a potent inhibitor of theproteasome.74–76 Conversely, hemin treatment results in HSF2 activation only in ahematopoietic cell line, suggesting that perhaps both the proteasome inhibitoryproperty of hemin and the induction of erythroid differentiation are involved.

During mouse spermatogenesis the increase in HSF2 activity peaks in sperma-tocytes and round spermatids77 and coincides with the expression of the rat homo-logue of UBC4 and UBC5.78 It remains to be established, however, whether HSF2regulates the expression of these E2 proteins upon proteasome inhibition, orwhether the increase in HSF2 activity corresponds to an increase in the expressionof heat-shock genes. HSFs have not been shown to be required for heat-shock-induced expression of ubiquitin, UBC4, and UBC5, though their gene promoterregions possess putative HSEs.1,44,79 Changing patterns of expression of E280 andother ubiquitination-associated proteins, such as deubiquitinating enzymes, havebeen proposed to influence the differentiation process in various systems and areat least partly responsible for the selective turnover of proteins characteristic ofthe changes inherent with differentiation.33,81 The resulting alterations in proteinflux may contribute to changes in HSF2 activity observed in differentiation sys-tems, giving it the properties of a differentiation-associated HSF.


Changes in the flux of proteins in the cell, whether induced by normal physio-logical signals such as differentiation, or by stress conditions that lead to the accu-mulation of oxidatively modified proteins (which affect proteasome function82) canbe immediately transmitted to the chaperone network by activation of HSF2/HSF,resulting in increased expression of heat-shock proteins. During inhibition of pro-teasome activity, heat-shock protein synthesis remains elevated, but is rapidlyreversed upon relief of the inhibition (A. Mathew, S. K. Mathur, and R. I. Morimoto,unpublished observations). As illustrated in FIGURE 4, a consequence of diminishedproteasome activity is the accumulation of polyubiquitinated proteins that may beprone to aggregation. The HSPs would serve to prevent aggregation, facilitate pro-tein repair/refolding, participate in ubiquitination/deubiquitination, and preparefor presentation to the proteasome for degradation. In support of such a scenario,HSP association with polyubiquitinated proteins is observed when proteasomeactivity is inhibited, and once proteasome activity is restored this association is lost.

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