Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.     

Structural and functional analysis of the Hsp70/Hsp40 chaperone system

As one of the most abundant and highly conserved molecular chaperones, the 70‐kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.     

NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems

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Interaction of BAG1 and Hsp70 mediates neuroprotectivity and increases chaperone activity

It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAGDeltaC) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAGDeltaC compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAGDeltaC was expressed exclusively in the cytosol. Furthermore, cells expressing BAGDeltaC were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.     

Modulation of the chaperone activities of Hsc70/Hsp40 by Hsp105alpha and Hsp105beta

Hsp105alpha and Hsp105beta are stress proteins found in various mammals including human, mouse, and rat, which belong to the Hsp105/Hsp110 protein family. To elucidate their physiological functions, we examined here the chaperone activity of these stress proteins. Hsp105alpha and Hsp105beta prevented the aggregation of firefly luciferase during thermal denaturation, whereas the thermally denatured luciferase was not reactivated by itself or by rabbit reticulocyte lysate (RRL). On the other hand, Hsp105alpha and Hsp105beta suppressed the reactivation of thermally denatured luciferase by RRL and of chemically denatured luciferase by Hsc70/Hsp40 or RRL. Furthermore, although Hsp105alpha and Hsp105beta did not show ATPase activity, the addition of Hsp105alpha or Hsp105beta to Hsc70/Hsp40 enhanced the amount of hydrolysis of ATP greater than that of the Hsp40-stimulated Hsc70 ATPase activity. These findings suggest that Hsp105alpha and Hsp105beta are not only chaperones that prevent thermal aggregation of proteins, but also regulators of the Hsc70 chaperone system in mammalian cells.     

Modulation of chaperone activities of Hsp70 and Hsp70-2 by a mammalian DnaJ/Hsp40 homolog, DjA4

Type I DnaJs comprise one type of Hsp70 cochaperones. Previously, we showed that two type I DnaJ cochaperones, DjA1 (HSDJ/Hdj-2/Rdj-1/dj2) and DjA2 (cpr3/DNAJ3/Rdj-2/dj3), are important for mitochondrial protein import and luciferase refolding. Another type I DnaJ homolog, DjA4 (mmDjA4/dj4), is highly expressed in heart and testis, and the coexpression of Hsp70 and DjA4 protects against heat stress-induced cell death. Here, we have studied the chaperone functions of DjA4 by assaying the refolding of chemically or thermally denatured luciferase, suppression of luciferase aggregation, and the ATPase of Hsp70s, and compared these activities with those of DjA2. DjA4 stimulates the hydrolysis of ATP by Hsp70. DjA2, but not DjA4, together with Hsp70 caused denatured luciferase to refold efficiently. Together with Hsp70, both DjA2 and DjA4 are efficient in suppressing luciferase aggregation. bag-1 further stimulates ATP hydrolysis and protein refolding by Hsp70 plus DjA2 but not by Hsp70 plus DjA4. Hsp70-2, a testis-specific Hsp70 family member, behaves very similarly to Hsp70 in all these assays. Thus, Hsp70 and Hsp70-2 have similar activities in vitro, and DjA2 and DjA4 can function as partner cochaperones of Hsp70 and Hsp70-2. However, DjA4 is not functionally equivalent in modulating Hsp70s.     

Kinetics of chaperone activity of proteins Hsp70 and Hdj1 in human leukemia U-937 cells after preconditioning with thermal shock or compound U-133

Kinetics of the chaperone activity of proteins Hsp70 and Hdj1 were analyzed in human U-937 promonocytes during their response to heat shock or to treatment with the echinochrome triacetyl glucoside derivative U-133. To measure the chaperone activity of both proteins, a special test was developed for their recognition and binding of a denatured protein. Using this test, the chaperone activity could be concurrently estimated in large numbers of cellular or tissue extracts. We also estimated the contents of both chaperones in cells by immunoblotting. The values for contents of Hsp70 and Hdj1 obtained by two independent test systems coincided, and this suggested that the substrate-binding activity could change proportionally to the chaperone content in the protein mixture. Therefore, the test developed by us can be employed for high throughput screening of drugs activating cellular chaperones. The analysis of quantity and activity of two cellular chaperones during the cell response to heat stress or to the drug-like substance U-133 showed that both factors caused the accumulation of chaperones with similar kinetics. We conclude that the efficiency of drug preconditioning could be close to the efficiency of hyperthermia and that the high activity of chaperones could be retained in human cells for no less than 1.5 days.     

The role of Hsp70 chaperone in the reaction of human leukemic cells to anticancer drugs

Most of anti-tumor factors are designed to kill selectively cancer cells; in most cases this action is related to the ability of the above substances to induce apoptosis. One of potent anti-apoptotic mechanisms is based on Hsp70 protein. Since the level of this protein is often higher in malignant tumors than in normal tissues, the aim of this study was to establish whether the elevated Hsp70 content may influence the process of apoptosis induced by anti-tumor drugs in cancer cells population. The increase of intracellular content of Hsp70 in human leukemia U-937 cells was attained by a mild heat stress or by transfection of cells with the human hsp70 gene. The elevation of Hsp70 quantity, irrespective of the way it was performed, leads to the inhibition of apoptosis in cells treated with two substances, etoposide or adriamycin. The inhibition of apoptosis was accompanied with the reducing of the share of cells with fragmented nuclei and with the delay in caspase activation. It is suggested that in addition to the previously discovered targets, whose activity is suppressed by the Hsp70 chaperone, this protein can inhibit the activity of caspase-3 and -7; this delays the onset of apoptosis in part of a cancer cell population.     

FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70

Some pan-histone-deacetylase (HDAC) inhibitors have recently been reported to exert their anti-leukemia effect by inhibiting the activity of class IIB HDAC6, which is the deacetylase of Hsp90 and α-tubulin, thereby leading to hyperacetylation of Hsp90, disruption of its chaperone function and apoptosis. In this study, we compared the effect of a class I HDAC inhibitor FK228 with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the Hsp90 chaperone function of K562 cells. We demonstrated that, although having a weaker inhibitory effect on HDAC6, FK228 mediated a similar disruption of Hsp90 chaperone function compared to SAHA. Unlike SAHA, FK228 did not mediate hyperacetylation of Hsp90, instead the acetylation of Hsp70 was increased and Bcr-Abl was increasingly associated with Hsp70 rather than Hsp90, forming an unstable complex that promotes Bcr-Abl degradation. These results indicated that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70 and inhibition of its function.     

Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange

The Hsp70 chaperone activity in protein folding is regulated by ATP-controlled cycles of substrate binding and release. Nucleotide exchange plays a key role in these cycles by triggering substrate release. Structural searches of Hsp70 homologs revealed three structural elements within the ATPase domain: two salt bridges and an exposed loop. Mutational analysis showed that these elements control the dissociation of nucleotides, the interaction with exchange factors and chaperone activity. Sequence variations in the three elements classify the Hsp70 family members into three subfamilies, DnaK proteins, HscA proteins and Hsc70 proteins. These subfamilies show strong differences in nucleotide dissociation and interaction with the exchange factors GrpE and Bag-1.     

Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells

Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K., Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272, 33283-33289). We now provide further evidence for a functional interaction between Hsp70 and the J-domain of Hsp40 with denatured luciferase resulting in reactivation of heat-denatured luciferase within living mammalian cells. The stimulating effect of Hsp40 on the Hsp70-mediated refolding is lost when the proteins cannot interact as accomplished by their expression in different intracellular compartments. Likewise, the cooperation between Hsp40 and Hsp70 is lost by introduction of a point mutation in the conserved HPD motif of the Hsp40 J-domain or by deletion of the four C-terminal amino acids of Hsp70 (EEVD motif). Most strikingly, co-expression of a truncated protein restricted to the J-domain of Hsp40 had a dominant negative effect on Hsp70-facilitated luciferase reactivation. Taken together, these experiments indicate for the first time that the Hsp70/Hsp40 chaperones functionally interact with a heat-denatured protein within mammalian cells. The dominant negative effect of the Hsp40 J-domain on the activity of Hsp70 demonstrates the importance of J-domain-containing proteins in Hsp70-dependent processes.     

Subcellular localization and chaperone activities of Borrelia burgdorferi Hsp60 and Hsp70.

Subcellular locations and chaperone functions of Hsp60 and Hsp70 with flagellin were investigated in Borrelia burgdorferi. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of fractionated cells showed Hsp60 to be present in the soluble fractions and the Triton X-100 detergent-soluble membrane fraction at growth temperatures ranging from 20 to 37 degrees C. The relative amount of Hsp60 associated with the membrane increased with growth temperature. Hsp70 was found in soluble fractions at growth temperatures between 28 and 37 degrees C, but at 20 degrees C it was also present in the Triton X-100-insoluble membrane fraction. Immunoelectron microscopy revealed that the majority of Hsp60 was localized in the cytoplasm but a detectable fraction (approximately 30%) was associated with the cell envelope. The chaperone functions of Hsp60 and Hsp70 were analyzed by immunoprecipitation of [35S]methionine-labeled cell lysates under nondenaturing conditions in the presence or absence of ATP. Hsp70 was found to bind flagellin at all temperatures tested between 33 and 41 degrees C. This association could be decreased with ATP when cells had been incubated at 41 degrees C during radioactive labeling but not at lower temperatures. Both flagellin and Hsp70 were found to associate with Hsp60, forming a complex of the three proteins. Hsp70 association with this complex could be decreased with ATP, but flagellin binding to Hsp60 was ATP independent at all temperatures studied. Both Hsp70 and flagellin were inaccessible to monoclonal antibodies against them when bound to Hsp60. These studies suggest that in B. burgdorferi, a major function of Hsp60 and Hsp70 is in the molecular processing of flagellin.     

Homodimerization of human mu-opioid receptor overexpressed in Sf9 insect cells

In this study, we demonstrate that human mu-opioid receptors do form SDS-resistant homodimers and examine the ability of human mu-opioid receptors to dimerize and the role of agonists in the dimerization. Increasing concentrations and longer exposure of agonists reduce the levels of dimmer with a corresponding increase in the levels of monomer. This effect is achieved with both peptide and alkaloid opioid agonists and it is antagonist reversible. These results suggest that human mu-opioid receptors are present as receptor oligomers and interconversion between dimeric and monomeric forms may be important for biological activity.     

Prion-impairing mutations in Hsp70 chaperone Ssa1: effects on ATPase and chaperone activities

We previously described many Hsp70 Ssa1p mutants that impair [PSI⁺] prion propagation in yeast without affecting cell growth. To determine how the mutations alter Hsp70 we analyzed biochemically the substrate-binding domain (SBD) mutant L483W and the nucleotide-binding domain (NBD) mutants A17V and R34K. Ssa1^L483W ATPase activity was elevated 10-fold and was least stimulated by substrates or Hsp40 co-chaperones. Ssa1^A17V and Ssa1^R34K ATPase activities were nearly wild type but both showed increased stimulation by substrates. Peptide binding and reactivation of denatured luciferase were enhanced in Ssa1^A17V and Ssa1^R34K but compromised in Ssa1^L483W. The nucleotide exchange factor Fes1 influenced ATPase of wild type Ssa1 and each mutant differently. Partial protease digestion uncovered similar and distinct conformational changes of the substrate-binding domain among the three mutants. Our data suggest that prion-impairing mutations of Ssa1 can increase or decrease substrate interactions, alter the Hsp70 reaction cycle at different points and impair normal NBD-SBD cooperation.     

The Hsp70 and Hsp40 chaperones influence microtubule stability in Chlamydomonas

Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70-Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex.     

Cooperative interaction of Hsp40 and TPR1 with Hsp70 reverses Hsp70-HspBp1 complex formation

Hsp40 and TPR1 are chaperone adaptors that regulate Hsp70-dependent folding processes by interacting with the amino terminal and carboxy terminal domains of Hsp70, respectively. In this study, we report cooperative interactions involving Hsp70, Hsp40, and TPR1 that enhance Hsp70-dependent folding of chemically denatured substrates. Hsp40 and Hsp70 dependent folding of chemically denatured luciferase was enhanced by up to 80% when TPR1 was also present. HspBp1, a negative modulator of Hsp70, completely inhibited Hsp70-dependent folding in the presence of Hsp40. However, when TPR1 was included in the reaction, the inhibitory effect of HspBp1 was reversed. To analyze the interactions, Kd analysis and competition assays were carried out. The Kds of the interactions of Hsp40, TRP1, and HspBp1 with Hsp70 were 0.5, 0.6, and 0.04 mM, respectively. Interestingly, the Hsp70/HspBp1 complex could only be dissociated in the presence of both Hsp40 and TPR1, suggesting cooperative interaction between Hsp70, Hsp40 and TPR1. To examine these interactions in vivo, we established a tetracycline-regulatable Hela cell line that expresses Hsp70 in the absence of doxycycline. Expression of HspBp1 inhibited Hsp70-dependent folding of heat-denatured luciferase, and this effect was only reversed in the presence of Hsp40 and TPR1. Our findings reveal a novel mechanism of positive regulation of Hsp70-dependent folding.     

Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum

Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes. Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.     

Hsp70 chaperone and the prospects of its application in anticancer therapy

Major stress protein Hsp70 is known to possess two important properties: ATP-dependent activity and protective activity; these two are thought to play a significant role in anticancer therapy. Many malignant tumors contain high amounts of intracellular Hsp70. Moreover, many anticancer drugs themselves are able to elevate Hsp70 expression in tumor cells. Since Hsp70 was found to disturb many signal pathways of apoptosis in many points, the high chaperone expression may lead to an increased resistance of tumor cells to anticancer drugs. On the other hand, when overexpressed by a certain mechanism, Hsp70 is able to emerge at the cell surface by itself or together with tumor antigens and present these to immune cells T-lymphocytes and natural killers, in such a manner that makes cancer cell recognized and abolished. These properties make Hsp70 very promising instrument in designing some novel anticancer vaccines.