The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.     

Sgt1p is a unique co-chaperone that acts as a client adaptor to link Hsp90 to Skp1p

Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.     

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone-substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.     

Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70 x Hop x Hsp90 complexes

Protein-protein interaction modules containing so-called tetratricopeptide repeats (TPRs) mediate the assembly of Hsp70/Hsp90 multi-chaperone complexes. The TPR1 and TPR2A domains of the Hsp70/Hsp90 adapter protein p60/Hop specifically bind to short peptides corresponding to the C-terminal tails of Hsp70 and Hsp90, respectively, both of which contain the highly conserved sequence motif EEVD-COOH. Here, we quantitatively assessed the contribution of TPR-mediated peptide recognition to Hsp70.Hop.Hsp90 complex formation. The interaction of TPR2A with the C-terminal pentapeptide of Hsp90 (MEEVD) is identified as the core contact for Hop binding to Hsp90. (In peptide sequences, italics are used to highlight residues specific for Hsp70 or Hsp90.) In contrast, formation of the Hsp70.Hop complex depends not only on recognition of the C-terminal Hsp70 heptapeptide (PTIEEVD) by TPR1 but also on additional contacts between Hsp70 and Hop. The sequence motifs for TPR1 and TPR2A binding were defined by alanine scanning of the C-terminal octapeptides of Hsp70 and Hsp90 and by screening of combinatorial peptide libraries. Asp0 and Val-1 of the EEVD motif are identified as general anchor residues, but the highly conserved glutamates of the EEVD sequence, which are critical in Hsp90 binding by TPR2A, do not contribute appreciably to the interaction of Hsp70 with TPR1. Rather, TPR1 prefers hydrophobic amino acids in these positions. Moreover, the TPR domains display a pronounced tendency to interact preferentially with hydrophobic aliphatic and aromatic side chains in positions -4 and -6 of their respective peptide ligands. Ile-4 in Hsp70 and Met-4 in Hsp90 are most important in determining the specific binding of TPR1 and TPR2A, respectively.     

Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine

The adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR-peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70-Hsp90 multichaperone complexes.     

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.     

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.     

The multiple functions of the co-chaperone stress inducible protein 1

Stress inducible protein 1 (STI1) is a co-chaperone acting with Hsp70 and Hsp90 for the correct client proteins’ folding and therefore for the maintenance of cellular homeostasis. Besides being expressed in the cytosol, STI1 can also be found both in the cell membrane and the extracellular medium playing several relevant roles in the central nervous system (CNS) and tumor microenvironment. During CNS development, in association with cellular prion protein (PrP^c), STI1 regulates crucial events such as neuroprotection, neuritogenesis, astrocyte differentiation and survival. In cancer, STI1 is involved with tumor growth and invasion, is undoubtedly a pro-tumor factor, being considered as a biomarker and possibly therapeutic target for several malignancies. In this review, we discuss current knowledge and new findings on STI1 function as well as its role in tissue homeostasis, CNS and tumor progression.     

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.     

Exploring ligand recognition,selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 from Arabidopsis thaliana

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C‐termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C‐terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C‐termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.     

Tetratricopeptide repeat motif-mediated Hsc70-mSTI1 interaction. Molecular characterization of the critical contacts for successful binding and specificity

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys(8) or Asn(12) did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn(43) lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys(8) or Asn(12) reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys(8) or Asn(12) define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.     

Multiple domains of the co-chaperone Hop are important for Hsp70 binding

The Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction.     

Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae

Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed. In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain. Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone. Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell. Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain. Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function. Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90. EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90. Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.     

The Non-canonical Hop Protein from Caenorhabditis elegans Exerts Essential Functions and Forms Binary Complexes with Either Hsc70 or Hsp90

Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.     

Structural studies on the co-chaperone Hop and its complexes with Hsp90

The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90.Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure.Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, ‘open’ state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

Ppp5c 和TTC16 基因的TPR 结构域与Hsp70、Hsp90 蛋白 的相互作用及对细胞周期的影响

目的:研究Ppp5c及TTC16基因的TPR(tetratricopeptide repeat)结构域短片段和Hsp70及Hsp90家族蛋白的相互做用,及其过表达对细胞周期的影响。方法:通过生物信息学的分析及PCR的方法,克隆Ppp5c及TTC16基因的TPR结构域以及HSPA1A、HSP90AA1的全长基因,并连入酵母双杂交载体,通过ClonTech的酵母双杂交实验体系研究蛋白和蛋白之间的相互作用。把Ppp5c及TTC16基因的TPR结构域克隆入真核表达载体,构架稳定表达Ppp5c及TTC16基因的TPR结构域的MCF-7细胞系,并通过流式细胞实验观察细胞周期。结果:Ppp5c及TTC16基因的TPR结构域能与HSPA1A或HSP90AA1发生相互作用。Ppp5c及TTC16基因的TPR结构域在MCF-7中的过表达能严重影响细胞周期,引起细胞凋亡和S期阻滞。结论:本实验初步揭示了不同蛋白的TPR结构域在与Hsp70及Hsp90蛋白的相互作用性质的异同点以及其过表达对细胞周期的影响,为全面理解TPR结构域的功能、Ppp5c以及TTC16蛋白在细胞内的功能奠定了前期实验基础。     

Effect of mutation of the tetratricopeptide repeat and asparatate-proline 2 domains of Sti1 on Hsp90 signaling and interaction in Saccharomyces cerevisiae

Through simultaneous interactions with Hsp70 and Hsp90 via separate tetratricopeptide repeat (TPR) domains, the cochaperone protein Hop/Sti1 has been proposed to play a critical role in the transfer of client proteins from Hsp70 to Hsp90. However, no prior mutational analysis demonstrating a critical in vivo role for the TPR domains of Sti1 has been reported. We used site-directed mutagenesis of the TPR domains combined with a genetic screen to isolate mutations that disrupt Sti1 function. A single amino acid alteration in TPR2A disrupted Hsp90 interaction in vivo but did not significantly affect function. However, deletion of a conserved residue in TPR2A or mutations in the carboxy-terminal DP2 domain completely disrupted Sti1 function. Surprisingly, mutations in TPR1, previously shown to interact with Hsp70, were not sufficient to disrupt in vivo functions unless combined with mutations in TPR2B, suggesting that TPR1 and TPR2B have redundant or overlapping in vivo functions. We further examined the genetic and physical interaction of Sti1 with a mutant form of Hsp90, providing insight into the importance of the TPR2A domain of Sti1 in regulating Hsp90 function.