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.     

Hsp104 interacts with Hsp90 cochaperones in respiring yeast

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi(+)] prions in the budding yeast Saccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.     

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.     

The M-Domain Controls Hsp104 Protein Remodeling Activity in an Hsp70/Hsp40-Dependent Manner

Yeast Hsp104 is a ring-forming ATP-dependent protein disaggregase that, together with the cognate Hsp70 chaperone system, has the remarkable ability to rescue stress-damaged proteins from a previously aggregated state. Both upstream and downstream functions for the Hsp70 system have been reported, but it remains unclear how Hsp70/Hsp40 is coupled to Hsp104 protein remodeling activity.Hsp104 is a multidomain protein that possesses an N-terminal domain, an M-domain, and two tandem AAA⁺ domains. The M-domain forms an 85-Å long coiled coil and is a hallmark of the Hsp104 chaperone family. While the three-dimensional structure of Hsp104 has been determined, the function of the M-domain is unclear. Here, we demonstrate that the M-domain is essential for protein disaggregation, but dispensable for Hsp104 ATPase- and substrate-translocating activities. Remarkably, replacing the Hsp104 M-domain with that of bacterial ClpB, and vice versa, switches species specificity so that our chimeras now cooperate with the noncognate Hsp70/DnaK chaperone system. Our results demonstrate that the M-domain controls Hsp104 protein remodeling activities in an Hsp70/Hsp40-dependent manner, which is required to unleash Hsp104 protein disaggregating activity.     

Hsp10, Hsp70, and Hsp90 immunohistochemical levels change in ulcerative colitis after therapy

Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) characterized by damage of large bowel mucosa and frequent extra-intestinal autoimmune comorbidities. The role played in IBD pathogenesis by molecular chaperones known to interact with components of the immune system involved in inflammation is unclear. We previously demonstrated that mucosal Hsp60 decreases in UC patients treated with conventional therapies (mesalazine, probiotics), suggesting that this chaperonin could be a reliable biomarker useful for monitoring response to treatment, and that it might play a role in pathogenesis. In the present work we investigated three other heat shock protein/molecular chaperones: Hsp10, Hsp70, and Hsp90. We found that the levels of these proteins are increased in UC patients at the time of diagnosis and decrease after therapy, supporting the notion that these proteins deserve attention in the study of the mechanisms that promote the development and maintenance of IBD, and as biomarkers of this disease (e.g., to monitor response to treatment at the histological level).     

Hsp70 in oncology

Hsp70 classes of molecular chaperones are highly conserved in all organisms and play an essential role in the maintenance of cellular homeostasis. Hsp70s assist nascent chain protein folding and denatured proteins, as well as the import of proteins to the organelles, and solubilization of aggregated proteins. ATPase function is required for Hsp70 function. Hsp70s use ATP hydrolysis driven mechanism for substrate protein binding and release. Various Hsps are unregulated in cancers but their significance for tumor growth is poorly understood. Studies have linked Hsp70 to several types of carcinoma. Human Hsp70s allow proliferation of cancer cells and suppress apoptotic and senescence pathways. This review presents Hsp70s role for growth of transformed cells and the current state of Hsp70 as a drug target along with recent patents in humans in this particular area.     

Hsp40s Specify Functions of Hsp104 and Hsp90 Protein Chaperone Machines

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI⁺] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN⁺] prions was less sensitive than [URE3], but more sensitive than [PSI⁺]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.     

Intermolecular Interactions between Hsp90 and Hsp70

Members of the Hsp90 and Hsp70 families of molecular chaperones are imp\ortant for the maintenance of protein homeostasis and cellular recovery following environmental stresses, such as heat and oxidative stress. Moreover, the two chaperones can collaborate in protein remodeling and activation. In higher eukaryotes, Hsp90 and Hsp70 form a functionally active complex with Hop (Hsp90–Hsp70 organizing protein) acting as a bridge between the two chaperones. In bacteria, which do not contain a Hop homolog, Hsp90 and Hsp70, DnaK, directly interact during protein remodeling. Although yeast possesses a Hop-like protein, Sti1, Hsp90, and Hsp70 can directly interact in yeast in the absence of Sti1. Previous studies showed that residues in the middle domain of Escherichia coli Hsp90 are important for interaction with the J-protein binding region of DnaK. The results did not distinguish between the possibility that (i) these sites were involved in direct interaction and (ii) the residues in these sites participate in conformational changes which are transduced to other sites on Hsp90 and DnaK that are involved in the direct interaction. Here we show by crosslinking experiments that the direct interaction is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, J-protein promotes the Hsp70–Hsp90 interaction in the presence of ATP, likely by converting Hsp70 into the ADP-bound conformation. The identification of the protein–protein interaction site is anticipated to lead to a better understanding of the collaboration between the two chaperones in protein remodeling.     

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.     

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.     

Hsp70 regulates the doxorubicin-mediated heart failure in Hsp70-transgenic mice

The aim of this study was to investigate the potential protective effect of the Hsp70 protein in the cardiac dysfunction induced by doxorubicin (DOX) and the mechanisms of its action. For this purpose, we used both wild-type mice (F1/F1) and Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70. Both types were subjected to chronic DOX administration (3 mg/kg intraperitoneally every week for 10 weeks, with an interval from weeks 4 to 6). Primary cell cultures isolated from embryos of these mice were also studied. During DOX administration, the mortality rate as well as weight reduction were lower in Tg/Tg compared to F1/F1 mice (P < 0.05). In vivo cardiac function assessment by transthoracic echocardiography showed that the reduction in left ventricular systolic function observed after DOX administration was lower in Tg/Tg mice (P < 0.05). The study in primary embryonic cell lines showed that the apoptosis after incubation with DOX was reduced in cells overexpressing Hsp70 (Tg/Tg), while the apoptotic pathway that was activated by DOX administration involved activated protein factors such as p53, Bax, caspase-9, caspase-3, and PARP-1. In myocardial protein extracts from identical mice with DOX-induced heart failure, the particular activated apoptotic pathway was confirmed, while the presence of Hsp70 appeared to inhibit the apoptotic pathway upstream of the p53 activation. Our results, in this DOX-induced heart failure model, indicate that Hsp70 overexpression in Tg/Tg transgenic mice provides protection from myocardial damage via an Hsp70-block in p53 activation, thus reducing the subsequent apoptotic mechanism.     

Independent regulation of Hsp70 and Hsp90 chaperones by Hsp70/Hsp90-organizing protein Sti1 (Hop1)

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many "client" proteins. The co-chaperone Sti1p coordinates functions of Hsp70 and Hsp90 in this pathway. Sti1p has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a binds Hsp90, and the ligand for TPR2b is unknown. Although Sti1p is thought to be dedicated to the client folding pathway, we earlier showed that Sti1p regulated Hsp70, independently of Hsp90, in a way that impairs yeast [PSI+] prion propagation. Using this prion system to monitor Sti1p regulation of Hsp70 and an Hsp90-inhibiting compound to monitor Hsp90 regulation, we identified Sti1p mutations that separately affect Hsp70 and Hsp90. TPR1 mutations impaired Sti1p regulation of Hsp70, but deletion of TPR2a and TPR2b did not. Conversely, TPR2a and TPR2b mutations impaired Sti1p regulation of Hsp90, but deletion of TPR1 did not. All Sti1p mutations variously impaired the client folding pathway, which requires both Hsp70 and Hsp90. Thus, Sti1p regulated Hsp70 and Hsp90 separately, Hsp90 is implicated as a TPR2b ligand, and mutations separately affecting regulation of either chaperone impair a pathway that is dependent upon both. We further demonstrate that client folding depended upon bridging of Hsp70 and Hsp90 by Sti1p and find conservation of the independent regulation of Hsp70 and Hsp90 by human Hop1.     

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs.     

Hsp110 is a nucleotide-activated exchange factor for Hsp70

Hsp110 proteins constitute a subfamily of the Hsp70 chaperones and are potent nucleotide exchange factors (NEFs) for canonical Hsp70s of the eukaryotic cytosol. Here, we show that the NEF activity of the yeast Hsp110 homologue Sse1 itself is controlled by nucleotide. Nucleotide binding results in formation of a stabilized conformation of Sse1 that is required for association with the yeast Hsp70 Ssa1. The interaction triggers release of bound ADP from Ssa1, but nucleotide persists bound to Sse1 in the complex. Surprisingly, removal of this nucleotide does not affect the integrity of the complex. Instead, rebinding of ATP to the Hsp70 prompts the dissociation of the complex. Our data demonstrate that in contrast to previously characterized NEFs for Hsp70 chaperones, the NEF activity of Sse1 requires nucleotide binding and let us propose a new model for Hsp110 function.     

Antagonistic Interactions between Yeast Chaperones Hsp104 and Hsp70 in Prion Curing

The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions.     

J-domain Proteins form Binary Complexes with Hsp90 and Ternary Complexes with Hsp90 and Hsp70

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

Mechanisms for regulation of Hsp70 function by Hsp40

The Hsp70 family members play an essential role in cellular protein metabolism by acting as polypeptide-binding and release factors that interact with nonnative regions of proteins at different stages of their life cycles. Hsp40 cochaperone proteins regulate complex formation between Hsp70 and client proteins. Herein, literature is reviewed that describes the mechanisms by which Hsp40 proteins interact with Hsp70 to specify its cellular functions.