Molecular chaperones and the heat shock response

Biochimica et Biophysica Acta 1423 (1998) R13^R27

Meeting report

Molecular chaperones and the heat shock response Sponsored by Cold Spring Harbor Laboratory, 6^10 May 1998 Randal J. Kaufman * Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA Received 30 July 1998; accepted 8 October 1998 Keywords: Stress response; Hsp70; Hsp90; BiP; DnaJ; DnaK; GroEL; GroES; Protein folding; Protein aggregation; Heat shock factor; Unfolded protein response

1. Introduction

2. Protein aggregation in human disease

The sixth Cold Spring Harbor heat shock meeting, organized by Carol Gross, Arthur Horwich, and Susan Lindquist, was held 6^10 May 1998. The meeting was ¢lled to capacity (400 participants). In contrast to previous meetings where heavy emphasis was on structural and functional aspects of molecular chaperones, this meeting had a signi¢cant larger percentage of abstracts dealing with the physiological responses to stress in eukaryotic cells and the role of protein folding and chaperones in human disease. There were over 70 oral presentations and over 300 abstracts presented. Unfortunately, for lack of space, I have particularly limited this summary to those few reports that provide signi¢cant new insight into the role of protein folding and chaperones in disease and in the physiological response to stress. I hope that colleagues with di¡erent interests and perspectives will contribute additional reviews on this exciting conference.

Recently it has become clear that many human diseases result from improper protein folding. Despite the presence of abundant molecular chaperones in vivo, proteins still fail to attain their native stable structure. The diseases of protein misfolding are poorly understood at the molecular level but involve the deposition of soluble proteins as amyloid ¢brils. This process is a consequence of conversion of one of about 20 normally functional proteins into a L-sheet quaternary structure that is often ¢brillar. More than any heat shock or chaperone meeting in the past, the characterization of aggregate formation was a significant focus. S. Radford described the characteristics of instability, unfolding and aggregation of human lysozyme variants that result in amyloid ¢brillogenesis. Two naturally occurring mutants, Ile56 The and Asp67 His, of human lysozyme are the cause of autosomal dominant hereditary amyloidosis. Patients heterozygous for these mutations have ¢brils composed exclusively of the mutant form of the protein. Their aggregation into amyloid ¢brils involves transformation of primarily K-helical structures known from the crystal structure, into stacked insoluble L-sheets with strands perpendicular to the long axis of the ¢ber [1].

* Fax: (734) 7639323; E-mail: [email protected]

0304-419X / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 9 X ( 9 8 ) 0 0 0 2 9 - 8

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The process involves partially folded intermediates and a molten globule-like state. The mutations that cause disease are thought to disrupt the maintenance of the hydrophobic core. In£uences that destabilize native conformations in proteins, such as high temperature or high salt, result in a highly more cooperative process that generates ¢brils. This process can be reproduced from oligopeptides that aggregate into long semi-£exible L-sheets by side chain-side chain interactions [2]. The notion that these peptide gels are potentially biocompatible and biodegradable suggests that they may be used for a number of therapeutic applications. In addition, it may also be possible to design molecules to alter protein folding conformations to prevent protein misfolding in vivo. This notion was demonstrated by J. Kelly using transthyretin which is a tetrameric protein that binds thyroxin and retinol binding protein in plasma and cerebral spinal £uid in humans. In certain individuals transthyretin is converted into insoluble ¢brils that cause senile systemic amyloidosis. J. Kelly demonstrated that certain phenols, biphenyl ethers and biphenyl amines exhibit high a¤nity binding to transthyretin and prevent the conformational changes required for amyloid ¢bril formation. The ability to engineer mice provides an avenue to study the process of ¢bril formation in an animal model in order to study factors that promote or suppress the process in vivo. J. Buxbaum demonstrated that mice expressing a human transthyretin transgene exhibit late onset cardiac amyloidosis due to transthyretin deposits. A majority of the males had the disease, where only 13% of the females displayed symptoms, similar to the disease in Caucasians. These animals provide a model to study late onset and gender-dependent amyloid formation. The slow-onset neurodegenerative diseases that are characterized by the presence of ordered protein aggregates in the brain include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and the prion diseases such as bovine spongiform encephalopathy. Each involves the deposition of a speci¢c protein: APP/AL in AD, K-synuclein in PD, Htn in HD, and PrP in bovine spongiform encephalopathy. However, it is not known whether these deposits are a cause or an e¡ect of the neurodegeneration. Age is the most important factor in ¢bril formation. AD is the most common

dementia with 3^4 million a¡ected in the USA. There is no treatment for these diseases, thus understanding their etiology is of primary importance. P. Lansbury described the requirement for a seeding reaction in the formation of APP/AL ¢brils. The amyloid precursor protein APP is proteolyzed to yield either a 40 amino acid peptide or a 42 amino acid peptide. To release these peptides, two secretases act sequentially: ¢rst, L-secretase cleaves close to the membrane within the extracellular domain and then Q-secretase cleaves within the transmembrane domain. The sites of Q-secretase cleavage are after residues 40 or 42 of AL. AL is also present in intracellular compartments. The two Q-secretase cleavage products, AL42 and AL40, were found in di¡erent compartments: AL42 in the endoplasmic reticulum (ER)/intermediate compartment, and AL40 in the trans-Golgi network (TGN). The cellular compartments that harbor AL are target sites for therapeutic intervention. In the brain, the principal compartment in which AL resides, AL is in a detergent-insoluble glycolipid-enriched membrane domain (DIG). Also present in the DIG fractions are the endoproteolytic fragments of presenilin-1 (PS1) and APP. The presence of these proteins, which all contribute to the generation of AL, indicates that the DIG fraction is probably where the intramembranous cleavage of APP occurs [3]. P. Lansbury also described the use of solid-state NMR and atomic force microscopy to study the assembly state of AL into ¢brils. The results show that the proto¢bril-to-¢bril conversion is nucleation-dependent and that proto¢bril unwinding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The proto¢brillar assembly intermediate may be a useful target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease. NAC is a 35 amino acid peptide isolated from the insoluble core of AD and PD amyloid plaques. It is a fragment of K-synuclein (or NACP), a neuronal protein of unknown function. Two point mutations within the NACP gene cause early onset PD. P. Lansbury demonstrated these plaques form Lewy bodies found in the cytoplasm in PD and in the extracellular space in AD. Both conditions are related and PD is very frequently observed in AD, and vice versa. There is a striking sequence similarity between NAC, the car-

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boxyl terminus of the AL-amyloid protein, and a region of the scrapie prion protein (PrP) which has been implicated in amyloid formation. NAC was prepared by chemical synthesis and found to form amyloid ¢brils via a nucleation-dependent polymerization mechanism. NAC amyloid ¢brils e¡ectively seed AL40 amyloid formation. Amyloid ¢brils comprising peptide models of the homologous AL and PrP sequences were also found to seed amyloid formation by NAC. The in vitro model studies suggest that seeding of NAC amyloid formation by the AL-amyloid protein, or seeding of amyloid ¢brils of the ALamyloid protein by NAC, may occur in vivo. S. Sisodia described the biology of mutant proteins that result in AD. Mutations in APP or the presenilins (PS1 or PS2) cosegregate with autosomal dominant, familial AD. PS1 and PS2 are polytopic membrane proteins resident in the ER and are subject to endoproteolysis to generate derivatives that stably associate and accumulate [4]. The Sel12 gene in Caenorhabditis elegans is homologous to PS1 and PS2. Surprisingly, defects due to deletion of Sel12 can be rescued by human PS1 or PS2. Inactivation of the PS1 gene in the mouse results in death late in embryogenesis, associated with axial skeletal defects and cerebral hemorrhage. The developmental defects can be rescued by a mutant PS1 Ala246 Glu, a mutant that results in familial AD, indicating the mutations in PS1 cause AD by gain of deleterious function. In addition, expression of PS1 mutants associated with familial AD lead to increased production of AL42 peptides and acceleration of AL deposition in brains of transgenic mice. Analysis of primary neuronal cultures from PS1 deleted mice indicated that PS1 has multiple e¡ects on tra¤cking and metabolism, including failed secretion of AL peptides, and accumulation of APP carboxy-terminal fragments. Huntington's disease (HD) is caused by expansion of a CAG repeat, encoding polyglutamine, in the huntingtin protein. The result is a neurodegenerative disorder. Longer expansions are associated with early age onset. C. Ross described that the protein is normally soluble or loosely associated with the cytoskeleton or vesicles in the cytoplasm. In patients with the disease, an N-terminal fragment of the protein is generated and forms insoluble aggregates that result in dystrophic neurites and intranuclear neuronal inclusion bodies. The Gln repeat acts as a polar zipper

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composed of an abnormal L-sheet structure. The cleavage, which can be mediated by caspase, results in nuclear translocation and aggregation. It is possible that the Gln repeat acts as a nuclear translocation signal. The aggregated protein is also ubiquitinated, but not e¡ectively degraded. The genetics and requirements for protein aggregation can be studied in the yeast Saccharomyces cerevisiae. The laboratories of R. Wickner and S. Lindquist demonstrated that a phenotypic trait, PSI, is dependent upon inheritance of an alternate conformation of the Sup35 protein rather than a novel nucleic acid. The highly conserved carboxy terminus of Sup35p is an essential component of the translation termination apparatus, required for the faithful termination of translation at all stop codons. However, the function of Sup35p is compromised if the protein adopts an alternate conformation that is promoted by the divergent amino terminus. The altered form of Sup35p does not kill cells but reduces the ¢delity of translation termination. The altered form of Sup35p in PSI+ cells is aggregated and protease resistant whereas in psi cells, it is mostly soluble and protease sensitive. The laboratory of S. Lindquist demonstrated that the amino terminus of Sup35p is responsible for the assembly into amyloid ¢brils. This reaction has the characteristics of a seeded reaction where the peptide is ordered ¢rst and then assembles into larger ¢brils. In addition, the heat shock protein Hsp104 plays a role in the propagation of the PSI+ phenotype. Hsp104 can promote refolding of proteins denatured upon heat shock. Hsp104 and Sup35p directly interact and this interaction inhibits the ATPase activity of Hsp104 [5]. Over-expression of Hsp104 cures cells of PSI+ and the condition is heritable, even when over-expression of Hsp104 ceases. However, deletion of HSP104 also cures cells of PSI+. Under both conditions, Sup35p becomes soluble, indicating a complex relationship between Hsp104 and the PSI+ phenotype where a minimal amount of Hsp104 is required to perpetuate the PSI+ phenotype. 3. Chaperoning stress responses in the cytosol Upon heat shock, transcription of heat shock and stress-inducible genes is rapidly stimulated by heat

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shock factor (HSF). HSF is synthesized constitutively in a latent form in normal conditions and binds to strong heat shock elements (HSEs), located upstream from heat shock gene promoters, but does not stimulate transcription. Upon heat shock, HSF is converted to an active form by trimerization, binds to additional weaker HSEs and activates transcription. In very recent years it has become evident that other proteins also regulate the activity of HSF. There were several talks that discussed the potential for other gene products to regulate HSF function. HSF is a winged/helix/turn/helix protein that contains from the amino terminus an activation domain, a DNA binding domain, a trimerization domain and a C-terminal activation domain. The structure of the DNA-binding domain bound to DNA was identi¢ed several years ago by H. Nelson. H. Nelson described a new regulatory domain between the DNA-binding and trimerization domain that is required for peak activation by the N-terminal activation domain. These studies indicate the importance of understanding the structure of the full-length protein. To approach this understanding, Dr. Nelson performed alanine scanning mutagenesis of surface residues. Of these mutants, 33 had increased activity, seven had dramatically increased activity. Mutation Lys237 Ala had a major increase that was dependent on the C-terminal activation domain. Other mutants were speci¢c to a¡ect activation by the N-terminal activation domain suggesting domain-domain interactions. The properties of HSF activation by trimerization using a puri¢ed in vitro system were described by C. Wu. In this system the equilibrium between monomer and trimer can be a¡ected by protein concentration and time. However, the magnitude of the trimerization was very small and indicates that there must be additional modulators in vivo. Heat, oxidants (H2 O2 ), and low pH (6.6) all synergize to increase the degree of trimerization. In contrast, sodium salicylate, dinitrophenol, ethanol, and indomethacin, all inducers of the heat shock response, did not a¡ect the trimerization reaction. Finally, lethal mutations were isolated in the Drosophila HSF gene. A conditional allele was used to show an essential role in survival from extreme heat stress and an additional role under normal growth conditions for oogenesis and early larval development.

The chaperone complement in a cell determines the speed and duration of the heat shock response. Attenuation of the response is mediated by molecular chaperones Hsp70 and Hdj1. Over-expression of either chaperone represses the activation of HSF1. Both Hsp70 and Hdj1 associate with the activation domain of HSF1, thereby providing a mechanism by which the stress-induced activator senses the cellular levels of these chaperones. To elucidate how HSF1 is negatively regulated, R. Morimoto's laboratory searched for proteins that bind its trimerization heptad repeat using the yeast two-hybrid system. A 76 amino acid nuclear protein, heat shock binding protein 1 (HSBP1), was identi¢ed as a novel, conserved, and constitutively expressed protein that contains two heptad repeats and binds HSF1 via heptad-heptad interactions. HSBP1 negatively regulates stressinduced HSF DNA binding and transcriptional activity in transfected mammalian cells [6]. HSBP1 selectively binds the HSF1 trimer and converts it to a monomer. HSBP1 also binds Hsp70 after it dissociates from HSF1. Deletion of the HSBP1 gene in C. elegans improved survival to stress, although the worms did not live very long. Over-expression of HSBP1 elicited hypersensitivity to stress where there was a 50% decrease in viability after stress. These results indicate that HSF1 activity is under the control of at least two classes of trans-regulatory proteins which bind speci¢c regions of HSF1 and repress HSF1 activation and also titrate the level of non-native proteins that accumulate during heat stress. J. Lis' laboratory found a domain in the middle of HSF that represses the activation of HSF in nonstressed cells. Phage display and selection for binding to the HSF repression domain identi¢ed the GAC1 gene that encodes the regulatory subunit for a type 1 serine/threonine phosphoprotein phosphatase, Glc7p. Glc7p is required for activation of glycogen synthase. These proteins interacted in vitro in pull-down assays. A glc7 deletion strain cannot respond to heat shock to induce the CUP1 gene and a gac1 deletion strain has a 60% reduced response. However, the inducibility of other heat shock genes, such as SSA4, were not a¡ected. The results suggest that the Glc7p and Gac1p proteins are involved in activating HSF transcriptional activity for selective heat shock-responsive genes.

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Although Hsp70 is thought to be a primary regulator of HSF1 activation, experiments presented by R. Voellmy indicate this may not be the case and implicate HSP90 as the primary negative regulator of HSF1 activation. In vitro studies demonstrated that Hsp/c70, Hsp40, Hsp90, and Hsp90-associated proteins Hop, p23, and Cyp-40 speci¢cally interact with HSF1 and these interactions are disrupted by stress. An in vitro activation assay demonstrated that HSF1 can be activated by heat, denatured proteins and geldanamycin, a molecule that binds to and inhibits Hsp90. The addition of Hsp90 prevented HSF1 activation and depletion of Hsp90 resulted in massive HSF1 activation in this in vitro assay. In contrast, depletion of Hsp/c70, Hop, Hip, p23, Cyp-40, or Hsp40 did not activate HSF1. HSP90 holds HSF1 as a monomer. Disruption of Hsp90 function leads to HSF1 activation. For example, either geldanamycin or over-expression of steroid receptors, which bind and possibly sequester Hsp90, activate HSF1 in vivo. The investigators conclude that Hsp90, alone or in combination with other chaperones, is the negative regulator of HSF1 activation and stress-induced HSP expression. In vertebrates, heat shock transcription factors (HSF1^4) regulate stress-inducible heat shock gene expression. I. Benjamin described the e¡ects of knocking out the HSF-1 gene in murine embryonic stem cells [7]. Although the synthesis of the constitutively expressed and inducible members of the heat shock family of stress proteins correlates with increased cellular protection, their relative contributions in acquired cellular resistance or `thermotolerance' in mammalian cells is presently unknown. Constitutive expression of multiple HSPs in cultured embryonic cells was una¡ected by disruption of the murine HSF1 gene. In contrast, thermotolerance was not attainable in hsf1(3/3) mouse embryo ¢broblasts, and this response was required for protection against heat-induced apoptosis [7]. Homozygous mice die at day 13.5 likely due to a placental defect. In addition, they exhibit growth retardation and female mice do not ovulate, similar characteristics to those observed in the de¢ciency of HSF in Drosophila. These studies indicate that: (1) HSF1 displays unexpected defects in mammalian development, growth, and female reproduction, (2) constitutively and inducibly expressed HSPs exhibit distinct phys-

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iological functions for cellular maintenance and adaptation, respectively, and (3) other mammalian HSFs or distinct evolutionarily conserved stress response pathways do not compensate for HSF1 in the physiological response to heat shock and other stresses. 4. Chaperoning stress responses in the endoplasmic reticulum Cells respond to the accumulation of unfolded protein in the lumen of the endoplasmic reticulum (ER) by activating transcription of genes encoding ER resident proteins that include chaperones such as BiP/GRP78, GRP94, and protein disul¢de isomerase (PDI). P. Walter described a novel signal transduction pathway in S. cerevisiae that involves three gene products IRE1, HAC1, and RLG1 (Fig. 1). Initially, a transmembrane protein kinase Ire1p is activated by the presence of unfolded protein in the ER to transmit a signal across the ER membrane by dimerization and trans-autophosphorylation of the Ire1p cytoplasmic domain. HAC1 encodes a bZIP transcription factor that regulates transcription of genes controlled by this pathway. The activity of Hac1p is controlled by regulated cleavage and ligation of its mRNA. This `splicing' reaction is uniquely di¡erent from typical tRNA and mRNA splicing and removes a 252 base intron in a reaction that is initiated by Ire1p. Activation of Ire1p kinase elicits an endonuclease activity that cleaves the 5P and 3P splice site junctions of HAC1 mRNA. Then tRNA ligase (encoded by RLG1) joins the 5P and 3P exon fragments. Whereas unspliced HAC1 mRNA is not e¤ciently translated, spliced HAC1 mRNA is very e¤ciently translated, thus increasing the levels of Hac1p in the cell [8,9]. P. Walter presented data that show Ire1p can bind and cleave two stem loop structures, one at the 5P splice site and one at the 3P splice site, independent of the HAC1 mRNA. In addition, a label transfer experiment demonstrated the reaction proceeds through a 2P,3P cyclic phosphate intermediate. M.-J. Gething screened for multicopy suppressors of the signaling defect for a mutant UPRE (the unfolded protein response element upstream from the yeast BiP (KAR2) promoter) and identi¢ed Bck1p, a

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Fig. 1. The unfolded protein response pathway in S. cerevisiae. Upon accumulation of unfolded or misfolded proteins in the ER, a transmembrane receptor kinase Ire1p undergoes dimerization and autophosphorylation. This process activates the endonuclease activity that cleaves HAC1 mRNA at two speci¢c sites. The 5P and 3P ends of cleaved HAC1 mRNA are ligated together by tRNA-ligase RLG1. Whereas unspliced HAC1 mRNA is on polysomes, the protein product is not detected, most likely due to a translation elongation block. Spliced HAC1 mRNA is e¤ciently translated and yields a protein product that has a greater transcriptional activation due to the presence of 18 new amino acid residues (indicated in red) that are encoded at the carboxy terminus of the newly spliced HAC1 mRNA. Hac1p then translocates to the nucleus to activate genes encoding ER chaperones. These genes have an unfolded protein response element upstream from their promoter.

component of the cell integrity (CI) MAP kinase module that is required for cell wall synthesis and cell proliferation, and can be activated in response to low osmolarity in a Rho1p and protein kinase Cdependent process. HAC1 mRNA splicing was increased in hypotonic medium and decreased in hypertonic medium. Strains deleted of PKC1 were not able to respond to hypotonic stress. The proposal was put forth that PKC might phosphorylate the N-terminal cytosolic regulatory domain of Ire1p and the purpose of this interaction is to link the CI response to the UPR pathway to coordinate and

optimize cell wall biosynthesis and the synthesis of plasma membrane components. K. Mori described the characterization of the DNA sequence requirements for the yeast UPRE. In S. cerevisiae, ¢ve UPR target genes (KAR2, PDI1, EUG1, FKB2, and LHS1) contain a single UPRE sequence that is necessary and su¤cient for induction. This sequence binds speci¢cally to Hac1p in vitro. All of the ¢ve functional UPRE sequences identi¢ed contain a partially palindromic sequence (CAGCGTG) that has, in four cases, a spacer of one nucleotide C [10]. This unique property of the

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UPRE sequence suggests a basis for the speci¢city of the UPR and why only a speci¢c set of proteins are induced to cope with ER stress. H. Yoshida described the DNA sequence requirements for the mammalian ER stress response element (ERSE). Previous studies by A. Lee and colleagues suggested that two cis-acting elements CORE and C1 bind transcription factors YY1 and CBF/NF-Y, respectively, and mediate induction of the BiP/GRP78 promoter. Yoshida and colleagues noticed that these two elements share a common 19 nucleotide sequence motif (CCAAT(N)9 CCACGT), indicating that GRP78 expression may be mediated by a single mechanism. They demonstrated that transcriptional induction of GRP78, GRP94, and calreticulin is mediated by a novel cis-acting element termed ERSE with the consensus sequence CCAAT(N)9 CCACG. In addition, a bZIP protein was cloned that binds this element and its expression was immediately increased upon ER stress, without a change in its mRNA level. This factor may represent the mammalian homologue of yeast Hac1p. R. Kaufman described the isolation of the human homologue of yeast IRE1 [11]. The protein expressed in mammalian cells displayed autophosphorylation activity and an endoribonuclease activity that was able to cleave the 5P splice site, but not the 3P splice site, of yeast HAC1 mRNA. These results suggest signi¢cant divergence in cleavage speci¢city from the yeast Ire1p. It was proposed that another gene product for which there is an EST in the database may represent another component that may cleave the 3P splice site junction in mammalian substrates. hIre1p over-expressed in mammalian cells was localized to the ER membranes that were juxtaposed with the nuclear envelope, with some co-localization with the nuclear pore complex. Expression of hIre1p was autoregulated through a process that required a functional hIre1p kinase activity, suggesting the nuclease activity of hIre1p may digest its own mRNA. Over-expression of wild-type hIre1p constitutively activated a reporter gene under control of the rat BiP promoter, and expression of a catalytically inactive hIre1p acted in a trans-dominant negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress. The results indicate that hIre1p is a crucial component that acts as a proximal sensor of the UPR in mammalian cells.

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5. The structure and function of cytosolic chaperones The classical view of protein folding kinetics relies on phenomenological models, and regards folding intermediates in a structural way that are on a linear pathway to the ¢nal structure of lowest energy. The classical view invokes pathways to solve the problem of searching for the needle in the haystack. The pathway idea con£icts with An¢nsen's experiments showing that folding is pathway-independent. K. Dill presented a model of protein folding that resolves this contradiction known as Levinthal's paradox, i.e. how do proteins fold so fast when they can have so many conformations [12]. This new view considers protein folding as parallel microscopic multiple pathways that traverse along an energy landscape funnel. The classical model views intermediates as productive and necessary for successful folding, whereas in the new model they are `kinetic traps' with local minimums in the energy landscape and are not absolute minimums. The new view eliminates the inherent paradox because it eliminates the pathway idea: folding can funnel to a single stable state by multiple routes in conformational space. The general energy landscape picture provides a conceptual framework for understanding both two-state and multi-state folding kinetics. The landscape perspective suggests that speeding up protein folding need not require molecular recognition of transition states. Rather, acceleration of protein folding could also occur by unfoldase proteins. This may be a more useful model for some protein chaperone functions. 5.1. The GroEL-GroES family of chaperonins One fundamental question regarding the mechanism of chaperone action is whether the chaperone catalyzes unfolding to prevent accumulation of nonproductive intermediates that are trapped in the folding landscape. The analysis of the structure and function of the GroEL-GroES complex has elucidated that partial structure is present both in conformers recognized by GroEL and in stably bound substrate proteins; however, it is unclear whether the act of binding promotes partial unfolding [13]. M. Shtilerman and S. Englander have utilized hydrogen-tritium exchange to demonstrate that highly protected amide protons in misfolded Rubisco are completely depro-

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Fig. 2. The GroEL-GroES chaperonin. The apical end of the cis cavity of the asymmetric GroEL-GroES complex binds the unfolded polypeptide or kinetically trapped intermediates in the folding process. This trans ternary complex is in equilibrium between two states, one which binds seven molecules of ATP (T) and GroES at the cis cavity with release of seven molecules of ADP (D) and GroES from the trans cavity. Binding of GroES and ATP induces a conformational change involving an expansion of the cis cavity and polypeptide release from the apical binding sites is triggered as folding begins. ATP hydrolysis at the cis cavity primes release of GroES and the folding of the polypeptide continues. Binding of seven molecules of ATP at the trans cavity induces release of GroES and polypeptide from the cis cavity. The released polypeptide is either completely folded or partially folded. Partially folded products require another round of binding and release. Time (15 s) is required from polypeptide binding at the cis cavity to release of the polypeptide. Modi¢ed from [13].

tected in a chaperonin-dependent manner. Unfolding was measured by the release of tritium label and was shown to depend on the presence of GroEL, GroES, and MgATP or MgAMP-PNP. Complete tritium release was accomplished is a single round of ATP hydrolysis (15 s). Using sub-stoichiometric amounts of GroEL or GroES with respect to Rubisco, it was demonstrated that the substrate leaves the cavity `An¢nsen's cage' after each round of ATP hydrolysis, regardless of whether the substrate reaches its native conformation. Continued turnover of unfolded protein required the continuing hydrolysis of ATP. These experiments provide the ¢rst direct evidence of ATP-dependent unfolding of a substrate protein by the complete chaperonin machine. The generation of an enclosed hydrophilic cis cavity is a requirement in the polypeptide folding activity mediated by GroEL and GroES. This hydrophilic cavity is formed upon GroES and ATP binding to a substrate occupied ring of GroEL (Fig. 2). Subsequently, hydrolysis of seven molecules of cis-bound ATP and binding of seven molecules of ATP to the trans ring is required to release GroES and substrate. Rye and Horwich have elucidated how the dis-

charged GroEL tetradecamer resets for the next round of substrate binding. They used a ATP hydrolysis-de¢cient 398A GroEL and £uorescence resonance energy transfer between an IAEDANS probe engineered into GroEL and a £uorescein probe attached to either GroES or substrate. The results show that ATP turnover in the cis cavity is necessary for destabilization of the folding cage and ATP occupancy of the cis binding sites prevents any ligand (nucleotide, GroES, or polypeptide) from binding at the trans site. Once ATP in the cis cavity is hydrolyzed, loading of ATP, substrate, and GroES can occur. In addition, binding of substrate at the trans ring of an ADP bullet signi¢cantly accelerates disassembly events on the cis ring and provides directionality for the GroEL folding reaction. The GroEL-GroES chaperonin cannot facilitate the folding of proteins larger than 60^70 kDa because they cannot ¢t into the cis cavity. One way around the size constraint is to provide a co-chaperonin with a longer mobile loop and a taller dome, thereby enlarging the cis cavity. C. Georgopoulos and S. Landry described that bacteriophage T4 does this through its own co-chaperonin gp31 that

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uniquely facilitates folding of its large capsid protein gp23. Gp31 acts as a functional homologue of GroES in its ability to facilitate protein folding with GroEL. In addition, Gp31 can replace GroES in vivo to support both bacterial and phage V growth. The Hsp60/chaperonin family of molecular chaperones can be divided into two large groups. The GroEL-Gro-ES family have sevenfold symmetry and exist in bacteria and eukaryotic cellular organelles. The second group includes the eukaryotic TRiC/CCT complex that folds actin, tubulin and various other proteins. The second group has mostly eightfold symmetry and exist in archaea and the eukaryotic cytosol. The second group has conserved the equatorial domain of GroEL but has no homology with the apical domain which is the substrate binding domain. In addition, the group II chaperonins do not require co-chaperonins like GroES. In contrast to TRiC/CCT, which has eight types of subunits, the chaperonin from Thermoplasma acidophilum has only two subunits. L. Essen and W. Bauî structure of the meister presented the 2.3 A substrate-binding domain of the K-subunit from the chaperonin in T. acidophilum [14]. The core of the group II apical domain is similar to GroEL; however, it lacks the hydrophobic residues implicated in binding substrates. In contrast, a large hydrophobic surface exists in a novel helix-turn-helix motif and likely represents the substrate binding site. This novel structure is incompatible with binding to a GroESlike co-chaperonin. A model was presented where the helical protrusion functions in substrate binding as well as controlling access to the central cavity and may explain why group II chaperonins function without a co-chaperonin. The majority of bacterial proteins are relatively small, less than 55 kDa, and they are synthesized rapidly and folded independently of the chaperonin GroEL in a post-translational manner. Analysis by F.U. Hartl and associates demonstrated that 10^15% of the total cytosolic proteins in Escherichia coli interact with GroEL under normal growth conditions [15]. Eukaryotes have a proportionally larger number of multi-domain proteins than bacteria. The individual domains of these proteins can be folded co-translationally and sequentially. The use of this mechanism explains how large proteins fold independently

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of a chaperonin [16]. F.U. Hartl asked whether chaperone pathways of protein folding exist in the cell. To address this issue, an ATPase defective GroEL Asp87Lys was expressed in yeast. This mutant can bind non-native polypeptides and trap them. Therefore, if unfolded polypeptides exist free in the cell, the mutant GroEL should bind irreversibly to them. GroEL was expressed at a tenfold greater level than endogenous TRiC/CCT. Very few polypeptides were observed to be bound to the trap. This trap was able to bind unfolded actin in vitro. However, there was no change in the kinetics of actin folding where the T1=2 was 1.3 min, indicating the trap did not have access to actin molecules in the process of folding. To further validate that the GroEL is able to trap unfolded polypeptides, studies were performed in yeast defective in GIM, which is a six subunit complex that interacts with TRiC/CCT and is involved in microtubule biogenesis [17]. Deletion of GIM subunits delays actin folding where the T1=2 is prolonged to 6^7 min. In this mutant strain, non-native actin can be trapped by the mutant GroEL. When a similar mutant in TCP1 (a subunit of TRiC/CCT) was made, it was able to bind non-native actin in vivo, indicating that actin does have access to the endogenous TRiC/CCT. These results suggest that protein interactions with chaperones are channeled and directed, where unfolded intermediates are not freely exposed. J. Frydman characterized chaperone assisted folding in mammalian cells. Approx. 20% of cytosolic protein interacted with Hsp70 and chased out. In addition, approx. 16% of cellular protein associated with TRiC/CCT and chased out. The molecular mass of these proteins associated with TRiC/CCT was between 30 and 60 kDa. J. Frydman also performed an experiment described by F.U. Hartl in trying to identify whether folding intermediates can be trapped by the mutant GroEL. Newly synthesized proteins did not bind either wild-type GroEL or the mutant Asp87 Lys GroEL. When azacytidine, a proline analogue, was used to disrupt protein folding, the mutant GroEL was able to bind misfolded proteins and the spectrum of polypeptides bound was similar to those bound to Hsp70. These experiments indicate that the trap can bind misfolded proteins in vivo, but cannot bind proteins on the normal folding pathway.

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Fig. 3. Model of DnaJ- and DnaK-dependent folding. Association of DnaJ (J) with an unfolded substrate (red) transfers the substrate to the ATP-bound form (T) of DnaK. This transfer is coupled with ATP hydrolysis and release of DnaJ. This process locks the peptide in the peptide binding pocket. Then ADP (D) is released and ATP binds. Exchange of ATP for ADP promotes release of the peptide substrate. The ATP-bound DnaK displays low substrate a¤nity and fast exchange of substrate, whereas the ADP-bound form of DnaK displays high substrate a¤nity and slow exchange of substrate.

5.2. The DnaK/DnaJ/GrpE family of chaperones ATP is used by DnaK to drive conformational changes that alter its a¤nity for peptide substrates. The ATP bound state has low a¤nity and fast exchange rates for polypeptide substrates, whereas the ADP bound state has high a¤nity and low exchange rates for substrates (Fig. 3). Conformational changes between these two states involve opening (ATPbound) and closing (ADP-bound) of the substrate binding pocket. This cycle is regulated by DnaJ, which stimulates the ATPase activity, and GrpE, which stimulates nucleotide dissociation [13]. There were a number of reports in this meeting concerning the mechanism of action and physiological signi¢cance of the DnaJ family members. T. Laufen and B. Bukau described the role of DnaJ in the DnaK chaperone cycle. Data from the

Bukau lab and the McMacken lab [18] have led to a model that couples DnaJ with DnaK. First, DnaJ transiently and rapidly associates with substrates and then DnaK-ATP binds with high a¤nity to the DnaJ-substrate complex in a process requiring two steps. The ¢rst step involves transient interaction of DnaK-ATP with the J domain of DnaJ and the second step involves the transfer of substrate from DnaJ to the peptide binding site in DnaK-ATP. The two steps together signal activation of the ATPase activity of DnaK which stabilizes the DnaK-substrate complex. The lab of R. McMacken demonstrated that DnaJ stimulates the ATPase activity of DnaK by a surprising 15 000-fold. This model is supported by the similar sequence motif in substrates for binding to DnaJ and to DnaK and the observation that DnaK mutants with lower a¤nity for substrates are de¢cient in the DnaJ interaction. In addition, DnaK

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mutants defective in coupling ATPase activity with substrate binding are defective in interaction with DnaJ. S. Landry also presented NMR data that support the J domain interaction is localized to the ATPase domain of DnaK and is likely to be mediated by electrostatic interactions [19]. B. Misselwitz and T. Rapoport described a BIAcor assay to measure interaction of yeast BiP with small peptide substrates and the J domain of Sec63p. BiP bound to the fusion protein GST-J domain only in the presence of ATP. BiP mutants defective in the ATPase cycle did not bind, indicating that ATP hydrolysis is required for the BiP-GST-J interaction. GST-J ¢rst binds BiP-ATP, stimulates ATP hydrolysis, and then binds BiP in the ADP-bound form. Substrate peptides competed with GST-J for the interaction with BiP, but did not stimulate the dissociation of BiP from GST-J. The dissociation of BiP from peptides, as well as from GST-J, was stimulated by ATP. The experiments lead to the conclusion that the BiP interacts with GST-J in part through its peptide binding pocket. Additional experiments using truncation mutants of BiP demonstrated that the peptide binding pocket, without the carboxy-terminal lid, is essential for the BiP-DnaJ interaction, which con£icts somewhat with Landry's results, unless DnaK and BiP have signi¢cantly diverged or unless there are two sites of DnaJ interaction on the DnaK family members. The results from Misselwitz and Rapoport are consistent with a model where BiP has two di¡erent interaction sites, one for J and one for peptide. Binding at both sites is required to stimulate the ATPase activity to maximum level. This model ensures that BiP only binds substrates in the vicinity of J and ensures that J only stimulates the ATPase of BiP only if a substrate is present. SV40 large tumor antigen (T antigen) is a protein necessary and su¤cient for SV40-induced tumorigenesis and viral functions that include viral assembly, viral DNA replication, transcriptional regulation, and transformation. Transformation minimally requires three domains: (1) a C-terminal domain that mediates binding to p53, (2) the LxCxE motif (residues 103^107) that is necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related proteins p107 and p130, and (3) an Nterminal domain homologous to the J domain of DnaJ chaperones [20]. J. Zalvide and J. DeCaprio

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demonstrated that the J domain in SV40 T antigen is required to inactivate RB-related proteins. The J domain perturbs phosphorylation status of p107 and p130 by promoting the degradation of p130. In addition, this domain is required for transformation of cells that express either p107 of p130. The J domain of T antigen can inactivate the ability of each RB family member to induce a G1 arrest in Saos-2 cells. In addition, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. Thus, although the LxCxE is a binding site for the pRB family members, the J domain is required to inactivate their function. J. Brodsky and J. Pipas demonstrated that the SV40 DnaJ domain has properties of a DnaJ protein. In addition they demonstrated that the SV40 T antigen J domain can replace the corresponding domain from E. coli DnaJ to allow growth of bacteriophage lambda and replication of E. coli in a dnaJ deletion strain. In addition, the SV40 T antigen J domain can replace the corresponding domain in S. cerevisiae. In contrast, replacement of the T antigen J domain with the corresponding domain from bacterial DnaJ yields SV40 defective for infection. Therefore, the SV40 J domain contains one or more elements that are selectively required for function in mammalian cells. The double-stranded RNA activated protein kinase PKR, a previously described tumor suppressor gene product, is activated upon viral infection and a variety of cellular stresses. PKR inhibits translation initiation through phosphorylation of the K-subunit of heterotrimeric eIF-2. In£uenza virus irreversibly inhibits PKR activation in infected cells by recruiting the cellular protein P58IPK . M. Melville and M. Katze described how inhibition of PKR by P58IPK can transform NIH3T3 cells. Hsp40, a mammalian DnaJ homologue, inhibits P58IPK in uninfected cells. A heterotrimeric complex of Hsp40, Hsp70, and P58IPK can be detected in vitro. A model was put forth that the trimeric complex is in an inactive, but ready, state. Upon cellular stress, Hsp40 is released and Hsp70 is targeted to PKR by P58IPK and refolds the kinase into an inactive form. The targeting step is proposed to be mediated by the sixth tetratricopeptide domain of P58IPK . P58IPK stimulates the ATPase activity of Hsp70, a property of DnaJ homologues. Therefore, P58IPK may function

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as a co-chaperone for Hsp70. It is speculated that P58IPK -induced tumorigenesis is mediated by speci¢c targeting of Hsp70 to PKR or potentially other tumor suppressor gene products, resulting in their inactivation. E. Craig described experiments that elucidate the role of the cytosolic Hsp70 family member Ssb. In S. cerevisiae there are two classes of cytosolic Hsp70 family members. The SSA proteins are involved in protein translocation into organelles and regulation of the heat shock response. The SSB class, encoded by SSB1 and SSB2, appear important in translational elongation. Disruption of both SSB1 and SSB2 resulted in sensitivity to certain protein synthesis inhibitors and slow growth, especially at low temperature. The majority of the SSB proteins were associated with translating ribosomes. Analysis of the Ssb association with the ribosome demonstrated that it is released with the nascent chain upon puromycin treatment. The association with the nascent chain was stable to high salt bu¡ers. In contrast, the interaction of Ssb with the ribosome in the absence of a nascent chain was sensitive to salt. There are approximately two to four molecules of Ssb per ribosome. It is proposed that Ssb binding to the ribosome targets Ssb to the nascent chain and thereby prevents misfolding of nascent polypeptides. Hsp70 family members frequently act in concert with a DnaJ homologue as a co-chaperonin. The phenotype of yeast deleted of the DnaJ-related protein Zuotin (Zuo1) is similar to those lacking Ssb (sensitive to protein synthesis inhibition, high NaCl, low temperature). Zuo1 binds tRNA and zDNA and is a ribosomal associated protein. Zuo1 mutants deleted of a positively charged region are defective in RNA binding and also do not associate with the ribosome. Craig et al. proposed that Zuo1 binds to ribosomes by interaction with ribosomal RNA and it functions with Ssb as a chaperone on the ribosome. 5.3. The Hsp90 family of chaperones B. Panaretou and P. Piper demonstrated that an active ATPase catalytic center within the amino-terminal domain of Hsp90 is needed for essential in vivo functions. Previous studies have yielded con£icting results concerning the role of ATP in the mechanism of protein folding and activation by Hsp90. It

has been suggested that the ATPase activities measured on puri¢ed Hsp90 represent contaminating activities. Piper et al. recently described the 3D structure of the amino-terminal domain of heat Hsp90 [21]. The structure revealed that the most highly conserved part of the protein forms a ATP/ADP binding site with strong similarities to the ATPase catalytic center of DNA gyrase. This ATP/ADP binding site is where the ansamycin antibiotics, such as geldanamycin, bind to Hsp90. This indicates that these drugs are acting as ATP/ADP mimetics. Recent studies identi¢ed a geldanamycin sensitive ATPase activity for yeast Hsp90. In addition, mutagenesis of key residues that are implicated in ADP/ATP binding and ATP hydrolysis and that should not disorder the protein, yield mutant Hsp90s that cannot provide essential Hsp90 functions in yeast cells. These studies identify an essential requirement for the ATPase activity of Hsp90. J. Buchner described that two di¡erent chaperone sites within Hsp90 di¡er in their substrate speci¢city and ATP dependence. Under normal conditions, a speci¢c set of proteins are substrates for Hsp90, and under stress conditions Hsp90 performs more general functions. J. Buchner characterized the function of two conserved domains of Hsp90: an N-terminal fragment (amino acids 1^210) and a C-terminal fragment (amino acids 262^709) [21]. The Cterminal fragment bound to partially folded proteins in an ATP-independent manner potentially regulated by co-chaperones. In contrast, the N-terminal domain contains a peptide binding site that prefers longer peptides (greater that ten amino acids) and peptide dissociation was induced by ATP binding. In addition, geldanamycin inhibited the weak ATPase and stimulated peptide release. A model was suggested where the two functionally di¡erent sites allow Hsp90 to guide the folding of a subset of target proteins and at the same time to display general chaperone functions. 6. The structure and function of ER chaperones Two homologous protein chaperones that share 42^78% identity and prevent transport of unfolded glycoproteins from the ER are calnexin (CNX), a highly conserved integral ER membrane protein,

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and calreticulin (CRT), an ER lumenal protein. The sequence similarity between CNX and CRT suggests that these distinct ER proteins may have common functions. CNX and CRT associate transiently and selectively with newly synthesized glycoprotein folding intermediates, thereby preventing their transit through the secretory compartment. In addition, both CNX and CRT bind p57, a homologue of protein disul¢de isomerase, and promote disul¢de bond formation in vitro [22]. Prolonged association of a glycoprotein with CNX or CRT is observed when the glycoprotein is misfolded or unable to assemble with its partners. The CNX and CRT recognition motifs within newly synthesized proteins are at least partly determined by the structures of asparaginelinked oligosaccharides. Upon translocation into the lumen of the ER, a core unit of 14 saccharides (GlcNAc2 Man9 Glc3 ) is added to selective asparagine residues. Immediately after, trimming of the three terminal glucose residues occurs by sequential action of glucosidase I that cleaves the terminal K(1,2)-glucose and glucosidase II that cleaves the two internal K(1,3)-glucose residues present on the core oligosaccharide structure. Although many glycoproteins bind both CNX and CRT, the overall spectrum of glycoproteins that bind CNX are signi¢cantly di¡erent than those that bind CRT. CRT binds to a di¡erent and larger set of glycoproteins. Binding to either CNX or CRT requires monoglucosylated asparagine-linked oligosaccharides. D. Williams demonstrated that CNX and CRT have similar relative binding a¤nities for the monoglucosylated oligosaccharide [23]. Deletion mutagenesis demonstrated that both chaperones utilize a similar lectin binding site that is composed of two tandem repeats. In addition, CRT or a truncated soluble form of CNX can replace the requirement for CNX in enhancing secretion, promoting subunit assembly, and preventing rapid degradation of mouse class I histocompatibility molecules in a Drosophila expression system. However, complexes of class I molecules with soluble CNX are much less stable than with full-length CNX. Surprisingly, a CNX molecule deleted of the cytosolic and transmembrane to create a soluble form changed its spectrum of glycoprotein binding to that similar to CRT. These experiments suggest that the transmembrane segment restricts the specificity for glycoprotein interaction.

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J. Weissman performed a large scale screen in yeast to identify a number of proteins required for e¤cient folding in the ER. Two screens involved selection for high copy number resistance to dithiothreitol (DTT) or mutagenesis and selection for DTT sensitivity. One gene identi¢ed in both screens (named ERO1) was previously unidenti¢ed and is an essential resident ER protein that is conserved from yeast to humans. Mutations in ERO1 cause extreme sensitivity to the reducing agent DTT and over-expression confers resistance to DTT. Compromised Ero1p function results in ER retention of disul¢de stabilized proteins in a reduced, non-native form, while not a¡ecting structural maturation of a disul¢de free protein. Weissman concludes that Ero1p is an essential component of a cellular redox machinery that is required for disul¢de bond formation in the ER. Recent studies from several laboratories have shown that improperly folded secretory proteins within the ER are exported to the cytoplasm for degradation by the cytosolic 26S proteasome. In addition, defects in the yeast chaperones CNX and BiP compromise their degradation in yeast. J. Brodsky and A. McCracken demonstrated that yeast deleted of CNX and harboring a mutant BiP (one of which is kar2-133 and is defective in promoting folding) are defective for the degradation of a mutated form of the yeast mating pheromone pro-K factor. One of these mutant BiP molecules is competent for translocation of protein into the ER, but defective for degradation, indicating indicate that BiP plays di¡erent roles in protein import into the ER and protein export out of the ER. In contrast, mutation or reduced expression of the cytosolic chaperone Ssa1p, a member of the Hsp70 family required for post-translational co-translocation, did not interfere with degradation of proteins in the ER in an in vitro degradation system. Proteins destined for the cell surface are translocated into the lumen of the ER through an aqueous pore in the translocon, although the permeability barrier is maintained by a tight ribosome-membrane junction. The luminal end of the pore is also blocked early during translocation. L. Hendershot described £uorescence collisional quenching experiments performed with B. Hamman and A. Johnson [24]. Microsomes were extracted by high pH wash to remove soluble luminal proteins and they were reconstituted

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with puri¢ed proteins. Studies with membranes reconstituted with di¡erent components demonstrated that BiP was required to seal the luminal end of the î diameter). Translocons not engaged in pore (40^60 A î diameter) were also translocation (having a 9^15 A sealed by BiP. Either ADP or ATP, but not AMPPNP, supported sealing by BiP. Sealing required stoichiometric levels of BiP and translocon. Therefore, BiP maintains the permeability barrier by sealing both non-translocating and newly targeted translocons. This provides the ¢rst evidence for a role of BiP in the mammalian translocation process. S. Nishikawa described the role of JEM1 (DnaJlike protein of the ER membrane) which encodes a novel DnaJ-like protein that is peripherally associated with the luminal side of the ER membrane. Jem1p stimulated the ATPase activity of BiP. Mutation of His566 Gln, in the conserved and functionally required His-Pro-Asp region of DnaJ-related proteins, destroyed this activity. Disruption of the JEM1 gene had no e¡ect on vegetative growth; however, the mutant was defective in nuclear membrane fusion during mating, suggesting Jem1p plays an essential role in nuclear membrane fusion. Since Sec63p, another DnaJ-like protein of the yeast ER membrane, forms a complex with Sec71p and Sec72p that is also required for nuclear membrane fusion, the structures of the jem1 and sec71 disrupted strains were analyzed by electron microscopy [25]. These investigators studied sec71 deletion because sec63 deletion is not viable. In strains disrupted in JEM1 the two haploid nuclei fused to each other at the outer nuclear membrane, but not at the inner membrane. In the SEC71 deleted strain, the two haploid nuclei were closely juxtaposed but did not fuse, even at the outer membrane. These ¢ndings suggest that Jem1p and Sec63p complex are involved in distinct steps in nuclear membrane fusion. 6.1. Chaperones in medicine G. Perdrizet described that heat shock can provide protection to ischemia/reperfusion injury to preserve microvascular integrity in kidneys isolated from donors for transplants and this is dependent upon new heat shock gene expression in the kidney. Rat kidneys stored for 48 h do not maintain viability upon

transplant into recipient animals. A 15 min hyperthermia and a 6 h recovery provided immediate reperfusion after transplant and total recovery. In contrast, 15 min whole body hyperthermia without recovery did not provide any recovery, indicating a recovery period is required, presumably to allow new heat shock protein synthesis. It is also possible to induce heat shock gene expression with SnCl2 treatment and this has the same protective e¡ect, but does not have the side e¡ect of paralysis. C. Nicchitta described the ability for ER chaperones to elicit host cell speci¢c T-cell responses and induce tumor immunity in mice. Previous studies from Srivastava demonstrated that GRP94 is identical to the previously described tumor rejection antigen [26]. The basis for this property stems from the ability of GRP94 to bind peptides speci¢c to the tumor cell and present them in an immunocompetent manner to the host. To elucidate the mechanism for this response, C. Nicchitta performed studies with GRP94 derived from ovalbumin expressing thymoma cells and melanoma tumors. In these studies, ER chaperones were delivered (using DOTAP) to spleen-derived dendritic cells (DC) and the uptake and exchange of associated peptides onto DC MHC class I molecules were measured by CTL assay (for thymoma cells secreting ovalbumin) or by in vivo tumor regression studies (for melanoma tumor cells). Animals vaccinated with melanoma tumor-derived GRP94, but not spleen-derived GRP94, displayed a dramatic reduction in metastatic tumor burden, assayed as mean lung weight. Surprisingly, calreticulin was as e¡ective as GRP94 in eliciting a T-cell response in the T-cell assay. Microbial and parasitic heat shock proteins are powerful antigenic targets of anti-microbial immune responses. Bacterial Hsps can be used as adjuvantfree carriers to elicit strong immune responses to proteins that are fused to them. J. Richmond and R. Young described studies that suggest Hsp70 can carry proteins and peptides into mammalian cells and they can enter antigen processing and presentation pathways [27]. Mice injected with mycobacterial Hsp70 covalently linked to ovalbumin as a soluble fusion protein without adjuvant mounted a long-lasting cellular CD8 T-cell response which recognized an ovalbumin-derived peptide, SIINFEKL, in association with host MHC class I. This peptide is known

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to arise from natural processing of ovalbumin in murine cells. The response required the antigen to be conjugated with Hsp70, where injection of either alone or injection of the two proteins together did not elicit the response. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, Hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHCdisparate individuals. Cystic ¢brosis is caused by mutations in the gene encoding the cystic ¢brosis transmembrane conductance regulator (CFTR). CFTR is a PKA-regulated, ATP-dependent chloride channel that modulates the function of other ion channels. D. Cyr described the role of cytosolic chaperones in the folding of CFTR. CFTR contains two transmembrane domains (TMD), two cytosolic nucleotide binding folds (NBF) and a regulatory domain (R domain). The ability of Hsp70, Hdj-1 and Hdj-2 to bind CFTR fragments was studied in mammalian cells and in a reticulocyte lysate system. TMD1 could be immunoprecipitated with Hdj-1 and Hsp70, but not Hdj-2. Hdj-2 could bind to a fragment of TMD1 and NBF1. However, expression of the R domain, which is adjacent to NBF1, greatly reduced the association with Hdj-2. Complex formation was ATP dependent and ATP depletion caused CFTR to aggregate. Hdj-2 was much more e¤cient than Hdj-1 in interacting with unfolded polypeptides to assist Hsp70mediated folding. Therefore, Hdj-1/Hsp70 and Hdj2/Hsp70 chaperone pairs do not provide redundant functions and are capable of facilitating early but di¡erent steps in CFTR biogenesis. These studies suggest that each step in biogenesis of a protein may be facilitated by di¡erent chaperones.

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