Holdings: Structure, Function and Regulation of the Hsp90 Machinery

Bibliographic Details
Main Authors:	Jing Li, Johannes Buchner
Format:	Article
Language:	English
Published:	Elsevier 2013-06-01
Series:	Biomedical Journal
Subjects:	Click here to view optimized website for mobile devices Journal is indexed with MEDLINE/Index Medicus and PubMed Share on facebookShare on twitter Share on citeulike Share on googleShare on linkedin More Sharing Services Table of Contents REVIEW ARTICLE Year : 2013 \| Volume : 36 \| Issue : 3 \| Page : 106-117 Structure Function and Regulation of the Hsp90 Machinery Jing Li1 Johannes Buchner2 1 Division of Biology California Institute of Technology Pasadena California USA 2 Center for Integrated Protein Science Department of Chemistry Technische Universität München Munich Germany Date of Submission 05-Sep-2012 Date of Acceptance 02-Nov-2012 Date of Web Publication 10-Jun-2013 Correspondence Address: Johannes Buchner Center for Integrated Protein Science Technical University of Munich. Lichtenbergstrasse 4 85747 Garching Germany Login to access the Email id Crossref citations 19 PMC citations 11 DOI: 10.4103/2319-4170.113230 PMID: 23806880 Get Permissions Abstract Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction intracellular transport and protein degradation it became an interesting target for cancer therapy. Structurally Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function Hsp90 works together with a large group of cofactors termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90 which facilitate the maturation of client proteins. In addition posttranslational modifications of Hsp90 such as phosphorylation and acetylation provide another level of regulation. They influence the conformational cycle co-chaperone interaction and inter-domain communications. In this review we discuss the recent progress made in understanding the Hsp90 machinery. Keywords: ATPase clients co-chaperones conformational cycle Hsp90 posttranslational modifications How to cite this article: Li J Buchner J. Structure Function and Regulation of the Hsp90 Machinery. Biomed J 2013;36:106-17 How to cite this URL: Li J Function and Regulation of the Hsp90 Machinery. Biomed J [serial online] 2013 [cited 2014 Dec 31];36:106-17. Available from: http://www.biomedj.org/text.asp?2013/36/3/106/113230 Heat shock protein 90 (Hsp90) one of the most abundant and conserved molecular chaperones is essential in eukaryotic cells. [1] [2] Different from other well-known molecular chaperone like Hsp70 and GroEL/ES Hsp90 is not required for de novo folding of most proteins but facilitates the final maturation of a selected clientele of proteins. [3] Hsp90 clients include protein kinases transcription factors such as p53 and steroid hormone receptors (SHRs). [4] [5] [6] [7] Therefore Hsp90 does not only function in protein folding but also contribute to various cellular processes including signal transduction and protein degradation. Interestingly while bacteria possess an Hsp90 protein called HtpG in Escherichia More Details coli no Hsp90 gene has been found in archea. [8] [9] [10] However bacterial Hsp90 is not essential and its precise function remains to be investigated. Recent studies suggest that it collaborates with the DnaK (Hsp70) system in substrate remodeling and may function against oxidative stress. [11] [12] In yeast there are two Hsp90 isoforms in the cytosol Hsc82 and Hsp82 of which Hsp82 is up-regulated up to 20 times under heat stress. [2] Hsp90α and Hsp90β are the two major isoforms in the cytoplasm of mammalian cells. Hsp90α is inducible under stress conditions while Hsp90β is constitutively expressed. [13] Hsp90 analogues also exist in other cellular compartments such as Grp94 in the endoplasmic reticulum Trap-1 in the mitochondrial matrix and ch-Hsp90 in the chloroplast. [14] [15] [16] Interestingly Hsp90 can be secreted as well and it promotes tumor invasiveness. Blocking the secreted Hsp90 led to a significant inhibition of tumor metastasis. [17] Structure of Hsp90 Top Structurally Hsp90 is a homodimer and each protomer contains three flexibly linked regions an N-terminal ATP-binding domain (N-domain) a middle domain (M-domain) and a C-terminal dimerization domain (C-domain) [Figure 1]. [18] Except for the charged linker located between the N- and M-domains in eukaryotic Hsp90 this domain organization is conserved from bacteria to man. Hsp90 is a member of a special class of structurally related evolutionarily conserved split ATPases the so-called Gyrase Histindine Kinase MutL (GHKL) domain ATPases which contain a Bergerat ATP-binding fold. [19] Another interesting feature of the ATP binding region is that several conserved amino acid residues form a "lid" that closes over the nucleotide binding pocket in the ATP-bound state but is open during the ADP-bound state. [18] The M-domain of Hsp90 is involved in ATP hydrolysis as it contains crucial catalytic residues for forming the composite ATPase site. Moreover the M-domain contributes to the interaction sites for client proteins and some co-chaperones. [20] The C-domain is essential for the dimerization of Hsp90. Interestingly in eukaryotic Hsp90 the opening of the C-domains is anti-correlated to the closing of the N-domain. [21] A conserved MEEVD motif at the C-terminal end serves as the docking site for the interaction with co-chaperones which contain a tetratricopeptide repeat (TPR) clamp. [22] Figure 1: Open and closed conformation of Hsp90. Crystal structures of full-length Hsp90 from E. coli (HtpG) in the open conformation (left PDB 2IOQ) and nucleotide-bound yeast Hsp90 in the closed conformation (right PDB 2CG9). The N-domain is depicted in green the M-domain in blue and the C-domain in orange. Click here to view Conformational dynamics of Hsp90 Top Hsp90 is a weak ATPase and the turnover rates are very low with 1 min–1 for yeast Hsp90 and 0.1 min–1 for human Hsp90. [23] [24] [25] Structural studies revealed that Hsp90 spontaneously adopts structurally distinct conformations which seem to be in a dynamic equilibrium [Figure 1]. [9] [26] Nucleotide binding induces directionality and a conformational cycle. [9] [27] [28] In the apo state Hsp90 adopts a "V"- shaped form termed "open conformation" [Figure 1]. ATP binding triggers a series of conformational changes including repositioning of the N-terminal lid region and a dramatic change in the N-M domain orientation. Finally Hsp90 reaches a more compact state termed "closed conformation" in which the N-domains are dimerized [Figure 1]. [9] [18] Recent biophysical studies using ensemble and single molecule fluorescence resonance energy transfer (FRET) assays allowed to further dissect the ATP-induced conformational changes [Figure 2]. [26] [28] After fast ATP binding Hsp90 slowly reaches the first intermediate state (I1) in which the ATP lid is closed but the N-domains are still open. The N-terminal dimerization leads to the formation of the second intermediate state (I2) in which the M-domain repositions and interacts with the N-domain. Then Hsp90 reaches a fully closed state in which ATP hydrolysis occurs. After ATP is hydrolyzed the N-domains dissociate release ADP as well as inorganic phosphate (Pi) and Hsp90 returns to the open conformation again. [28] Figure 2: Conformational cycle of Hsp90. After fast ATP binding in which the ATP lid is closed but the N-domains are still open. Then the N-terminal dimerization leads to the formation of the second intermediate state (I2) in which the M-domain repositions and interacts with the N-domain. Then Hsp90 reaches a fully closed state in which ATP hydrolysis occurs. After ATP is hydrolyzed release ADP and Pi and Hsp90 returns to the open conformation. Click here to view Notably nucleotide binding is not the only determinant for Hsp90 conformation. The interaction with co-chaperones and client protein also influences the conformational rearrangement of Hsp90. [29] [30] These results suggest that there may be a dynamic equilibrium between the different conformations of Hsp90 and this conformational plasticity is functionally important since it may allow Hsp90 to adapt to different client proteins. Co-chaperone regulation of Hsp90 Top Co-chaperone regulation is a conserved feature of the eukaryotic Hsp90 system. To date more than 20 co-chaperones have been identified. [1] [31] They regulate the function of Hsp90 in different ways such as inhibition and activation of the ATPase of Hsp90 as well as recruitment of specific client proteins to the cycle. Interestingly different co-chaperones work together to facilitate the maturation of Hsp90 clients. [32] The composition of co-chaperone complexes seems to depend to some degree on the presence of a specific client protein. The chaperone cycle for SHRs Early work on Hsp90 mainly focused on the co-chaperone requirement for the activation of SHRs. [32] [33] The maturation of most SHRs strictly depends on the interaction with Hsp90. Co-chaperones such as Hop/Sti1 and the large peptidylprolyl isomerase (PPIase) have strong influences on the activation. [32] [34] Research on the assembly of Hsp90 with SHRs had shown that several distinct complexes are formed during the maturation processes. [32] [35] [36] According to reconstitution experiments SHRs must pass through three complexes with different co-chaperone compositions chronologically to reach their active conformation. Hsp70/Hsp40 were identified as components in the "early complex." [32] After association with Hsp90 through the adaptor protein Hop the "intermediate complex" is formed. [37] [38] In addition to the intermediate complex a third complex that contains a PPIase and the co-chaperone p23 had been found as the last step of the cycle. [39] [40] [41] Notably similar heterocomplexes can be found from yeast to man even in the absence of client protein. [32] Recent studies [using FRET analytical ultracentrifugation (aUC) nuclear magnetic resonance (NMR) spectroscopy and electron microscopy] provided insight into how the exchange of co-chaperones is regulated. [42] [43] [44] Based on these results a new model of the chaperone cycle emerges [Figure 3]A in which first one Hop/Sti1 binds to the Hsp90 dimer and stabilizes its open conformation. As a result the Hsp90 ATPase activity is inhibited. The other TPR-acceptor site is then preferentially occupied by a PPIase leading to an asymmetric Hsp90 intermediate complex. After the binding of ATP and p23/Sba1 Hsp90 adopts the "closed" conformation which weakens the binding of Hop/Sti1 and therefore promotes its exit. Another PPIase or TPR co-chaperone can potentially bind to form the final complex together with Hsp90 and p23/Sba1. Following ATP hydrolysis p23/Sba1 PPIase and the folded client are released from Hsp90. [42] Figure 3: Hsp90 chaperone cycles. (A) Hsp90 chaperone cycle for SHRs. Hsp70 Hsp40 and a client protein form an "early complex." The client protein is transferred from Hsp70 to Hsp90 through the adaptor protein Hop/Sti1. One Hop/Sti1 bound is sufficient to stabilize the open conformation of Hsp90. The other TPR-acceptor site is preferentially occupied by a PPIase leading to an asymmetric intermediate complex. Hsp90 adopts the ATPase-active (closed) conformation after binding of ATP. p23/Sba1 stabilizes the closed state of Hsp90 which weakens the binding of Hop/Sti1 and promotes its exit from the complex. Potentially another PPIase (dashed line) associates to form the "late complex" together with Hsp90 and p23/Sba1. After the hydrolysis of ATP p23/Sba1 and the folded client are released from Hsp90. (B) Hsp90 chaperone cycle for kinases. In the early stage Hsp70 and Hsp40 interact with newly synthesized kinases. Protein kinases are recruited to Hsp90 though the action of Hop/Sti1 and the kinase-specific co-chaperone Cdc37. Both are able to stabilize the Hsp90/kinase complex. Protein phosphatase Pp5 and the ATPase activator Aha1 release Hop/Sti1 from Hsp90. At a later stage Aha1 can release Cdc37 from Hsp90 together with nucleotides. (C) Hsp90 chaperone cycle for NLRs. Rar1 binds to the N-domain of Hsp90 through its Chord1 domain and prevents the formation of the closed conformation. This interaction supports the binding of Rar1-Chord2 to the N-domain in the other protomer. With the association of Rar1-Chord2 Sgt1 interacts with Hsp90 as well as with an NLR protein. In the stable ternary complex the lid segment promotes ATP hydrolysis. Once ATP is hydrolyzed Rar1 Sgt1 and the NLR protein may dissociate from Hsp90. (D) Hsp90-R2TP complex. Model of the R2TP complex in yeast. Pih1 interacts with Rvb1/2 with the M-domain of Hsp90 and the C-domain of Tah1. Tah1 binds to the C-terminal MEEVD motif of Hsp90 through its TPR domain. Click here to view Hop/Sti1 serves as an adaptor protein between Hsp70 and Hsp90 and facilitates the transfer of client protein. [37] [38] Therefore it is indispensable for maintaining the hormone binding activity of the glucocorticoid receptor (GR) and progesterone receptor (PR). [45] [46] Notably Hop/Sti1 is a member of the large group of TPR co-chaperones. They contain a specialized conserved TPR-clamp domain which consists of three TPR motifs and recognizes the C-terminal MEEVD motif in Hsp90. [22] Besides Hop/Sti1 the protein phosphatase PP5 (yeast homologue Ppt1) and members of the PPIase family like Fkbp52 Fkbp51 and Cyp40 (yeast homologues Cpr6/Cpr7) belong to this group. The TPR-containing PPIases contain a PPIase domain which catalyzes the interconversion of the cis-trans isomerization of peptide bonds prior to proline residues [47] and a TPR domain for the interaction with Hsp90. Most of these large PPIases show independent chaperone activity. [48] [49] [50] However the function of PPIases in SHR complexes is not well understood. They may be selected by specific client proteins. For example Cyp40 is most abundant in estrogen receptor (ER) complexes [51] and Fkbp52 mediates potentiation of GR through increasing GR hormone-binding affinity. [34] Interestingly the potentiation effects do not strictly depend on the PPIase activity of Fkbp52 as PPIase-deficient mutants are also able to potentiate GR transactivation which suggests a noncatalytic role of PPIases in the regulation of SHR signaling. [52] In contrast to Hop/Sti1 and the TPR-PPIases p23 is a conformation-specific co-chaperone which binds exclusively to the closed conformation of Hsp90. [53] [54] This small acidic protein contains an unstructured C-terminal tail which is essential for its intrinsic chaperone activity. [55] [56] p23 was identified as a component in SHR complexes together with Hsp90 and a PPIase. [57] It facilitates the maturation of client proteins by stabilizing the closed conformation of Hsp90. [58] As a result the ATP hydrolysis which is indispensable for the release of the client protein [59] [60] [61] is partially inhibited in the presence of p23/Sba1. [41] [62] Chaperone cycle for protein kinases Similar to SHRs the maturation of protein kinases also requires the Hsp70 chaperone machinery [Figure 3]B. [63] In the early stage Hsp70 and Hsp40 interact with newly synthesized kinases. Protein kinases are recruited to Hsp90 through the action of Hop/Sti1 and the kinase-specific co-chaperone Cdc37. Both are able to stabilize the Hsp90/kinase complex. [64] At a later stage the ATPase activator Aha1 can release Cdc37 from Hsp90 together with nucleotides [65] which leads to the final activation of protein kinases. Cdc37 is specific for chaperoning kinases. [66] [67] It was originally identified in Saccharomyces cerevisiae as a gene essential for cell cycle progression. [68] [69] Cdc37 interacts with kinases through its N-terminal domain and binds to the N-domain of Hsp90 via its C-terminal part. Similar to Hop/Sti1 the interaction of Cdc37 with Hsp90 leads to the stabilization of the open conformation and the inhibition of Hsp90 ATPase activity. [70] In contrast to the co-chaperones discussed above Aha1 is the most powerful ATPase activator of Hsp90. [71] It binds the N- and M-domains of Hsp90. [20] [30] Binding of Aha1 induces a partially closed Hsp90 conformation and accelerates the progression of the ATPase cycle dramatically. [28] [30] The presence of Aha1 enables Hsp90 to bypass the I1 state and to directly reach I2 in the ATPase cycle. [28] The activation of specific clients such as viral Src kinase (v-Src) and SHRs is severely affected in Aha1 knockout cells. [72] Moreover Aha1 plays a critical role in the inherited misfolding disease cystic fibrosis (CF) through participating in the quality control pathway of the cystic fibrosis transmembrane conductance regulator (CFTR). Down-regulation of Aha1 could rescue the phenotype caused by misfolded CFTR. [73] Recent research highlighted the function of Aha1 in the progression of the Hsp90 cycle. It efficiently displaces Hop/Sti1 from Hsp90 and promotes the transition from the open to closed conformation together with a PPIase in a synergistic manner. [74] Pp5/Ppt1 is a protein phosphatase which is involved in this cycle through regulating the phosphorylation states of Cdc37. It associates with Hsp90 through its N-terminal TPR domain. Binding to Hsp90 results in the abrogation of the intrinsic inhibition of Pp5/Ppt1. [75] Pp5/Ppt1 specifically dephosphorylates Hsp90 and Cdc37 in Hsp90 complexes. [76] [77] In Ppt1 knockout strains the activity of Hsp90-specific clients is significantly reduced which implies that the tight regulation of the Hsp90 phosphorylation state is necessary for the efficient processing of client proteins. [76] Chaperone cycle for nucleotide-binding site and leucine-rich repeat domain containing (NLR) proteins NLRs are conserved immune sensors which recognize pathogens. [78] Accumulating evidence indicates that Hsp90 and its co-chaperones Sgt1 and Rar1 are involved in the maturation of these proteins. [79] Sgt1 interacts with the N-domain of Hsp90 through its CS domain which is structurally similar to p23/Sba1. [80] [81] However Sgt1 has no inherent Hsp90 ATPase regulatory activity due to differences in interaction. [81] Interestingly although a TPR domain is present in Sgt1 as well it is not involved in the interaction with Hsp90. [82] Functionally Hsp90 and Sgt1 form a ternary complex with the co-chaperone Rar1 which acts as a core modulator in plant immunity. [78] During the recruitment and activation of NLRs Rar1 binds to the N-domain of Hsp90 through its Chord1 domain and prevents the formation of the closed conformation [Figure 3]C. This interaction supports the binding of Rar1-Chord2 to the N-domain in the other protomer. With the association of Rar1-Chord2 Sgt1 is promoted to interact with Hsp90 as well as with an NLR protein. In the stable ternary complex the lid segment is very flexible thus permitting access by a catalytic arginine residue of the M-domain to the ATP binding site and promoting ATP hydrolysis. Once ATP is hydrolyzed and the NLR protein may dissociate from Hsp90. [83] Hsp90 complexes in RNA processing Recent studies showed that Hsp90 is also involved in the assembly of small nucleolar ribonucleoproteins (snoRNPs) and RNA polymerase. [84] [85] [86] The chaperone cycle is not completely understood yet. However the central player in this process the R2TP complex (consisting of Tah1 Pih1 and the AAA+ ATPase Rvb1 and Rvb2) has been extensively investigated [Figure 3]D. [86] [87] The co-chaperone Tah1 interacts with Hsp90 through its TPR domain and its C-terminal region binds Pih1 an unstable non-TPR co-chaperone of Hsp90 [Figure 3]D. During the maturation of snoRNP the Hsp90-Tah1 complex stabilizes Pih1 in vivo and prevents its aggregation in vitro. [84] The Tah1-Pih1 heterodimer is able to inhibit the ATPase activity of Hsp90. [88] Tah1 and Pih1 are then transferred to the Rvb1/2 complex leading to the formation of the R2TP complex [Figure 3]D. Together Hsp90 and the R2TP complex are involved in the biogenesis and assembly of snoRNPs. Notably neither Hsp90 nor R2TP are components of the mature snoRNP complex. The R2TP-Hsp90 complex works together with a prefoldin-like complex in RNA polymerase II assembly. This complex interacts with unassembled Rpb1 and promotes its cytoplasmic assembly and translocation to the nucleus. [85] In addition to the activation of client protein co-chaperones are also involved in other physiological processes such as mitochondrial/chloroplast protein import (Tom70/Toc64) [89] [90] nuclear migration (NudC) [91] and melanoma progression (TTC4). [92] The above examples provide a glimpse on Hsp90 co-chaperone cycles. For some cycles we have obtained a full picture with detailed information; for others we just start to understand their contributions to client protein activation. Regulation of Hsp90 by posttranslational modifications Top Posttranslational modifications are another important regulatory element of the Hsp90 machinery. Different posttranslational modifications such as phosphorylation acetylation nitrosylation and methylation tightly control the function of Hsp90 and thus influence the maturation of client proteins. [93] Phosphorylation Phosphorylation is the most frequently detected posttranslational modification of Hsp90. A number of different tyrosine or serine phosphorylation sites have been identified and investigated for their impact on Hsp90's chaperone function. [94] For example only phosphorylated Hsp90 stimulates the activity of the Hsp90 client protein heme-regulated inhibitor kinase (HRI); dephosphorylation eliminated the ability of Hsp90 to activate this client protein. [95] Interestingly hyperphosphorylation also leads to a decreased Hsp90 activity. In yeast the protein phosphatase Ppt1 deletion compromised the activation of specific clients. [76] Therefore the phosphorylation states of Hsp90 must be precisely regulated in order to maintain the proper function of Hsp90. In addition phosphorylation also modulates the interaction with co-chaperones and thus exerts further influence on the Hsp90 machinery. [96] For example tyrosine phosphorylation on Hsp90 disrupts the interaction with Cdc37 and promotes the recruitment of Aha1. [97] C-terminal phosphorylation of Hsp90 regulates alternate binding to co-chaperones Chip and Hop which determine cellular protein folding/degradation balances. [98] Furthermore phosphorylation affects the conformational cycle of Hsp90 such as formation of the active sites general flexibility and inter-domain communication. [96] [99] A number of different kinases can phosphorylate Hsp90 such as double-stranded DNA protein kinase c-Src kinase protein kinase A (PKA) CK2 protein kinase and Swe1Wee1 kinase. [100] [101] [102] [103] Interestingly many of them are at the same time Hsp90 client proteins. This indicates that the change of phosphorylation states of Hsp90 may influence the folding and activation of certain groups of client proteins. Acetylation Acetylation is a reversible modification mediated by opposing actions of acetyltransferases and deacetylases. [104] Hsp90 acetylation and its influence on the chaperone machinery have been extensively investigated in recent years. In the case of Hsp90 p300 was reported to be the acetyltransferase and HDAC6 acts as a deacetylase which removes the acetyl group from the protein. [105] [106] Deacetylation of Hsp90 drives the formation of Hsp90 client complexes and promotes the maturation of the client protein GR. Hsp90 can be acetylated at different sites. [107] A study from Necker's lab pointed out that K294 an acetylation site in the M-domain strongly influences the binding between Hsp90 and its client protein. In general acetylation weakens Hsp90-client interaction and thus Hsp90 fails to support the activation of the client protein. [108] Nitrosylation S-nitrosylation is a reversible covalent modification of reactive cysteine thiols in proteins by nitric oxide (NO). [109] [110] Mammalian Hsp90 is a target of S-nitrosylation mediated by NO produced by its client protein endothelial nitric oxide synthase (eNOS). [111] S-nitrosylation was reported as a negative regulator which inhibits the ATPase activity of Hsp90. [111] In addition the activation of its client protein eNOS was also reduced consistent with the notion thatHsp90 acts as an NO sensor. [111] This provides a feedback mechanism to inhibit further eNOS activation. Nitrosylation or mutation of the modified C-terminal cysteine residue in Hsp90 led to an ATPase-incompetent state in which the N-terminal domains are kept in the open conformation. [112] The result indicates that nitrosylation has a profound impact on the inter-domain communication in the Hsp90 dimer. Hsp90 client protein recognition Top To date more than 200 Hsp90 client proteins have been identified (see http://www.picard.ch/downloads/Hsp90interactors.pdf ). Besides the well-studied clients such as protein kinases and SHRs many others related to for example viral infection innate immunity and RNA modification have been discovered in recent years. [84] [113] [114] The interaction with the Hsp90 machinery enables their correct folding activation transport and even degradation. [89] [115] [116] [117] Our understanding of the Hsp90 machinery has been greatly advanced by research of the last decades. However some fundamental questions related to client proteins still remained unanswered such as the location of the client-binding sites on Hsp90. Current evidence suggests that binding sites could be localized in each of the domains of Hsp90. [8] Another intriguing question unsolved so far is how Hsp90 recognizes its clients. Hsp90 clients belong to different families and do not share common sequences or structural motifs. Although some regions were identified which are important for the recognition of certain group of clients the αC-β4 loop in kinases [118] [119] this is not the only determinant for the interaction as other regions adjacent to the kinase domain also influence the binding to Hsp90. [120] [121] It is reasonable to assume that Hsp90 recognizes certain conformations or the stability of the client protein rather than its primary sequence. Src kinase is a prominent example here. The v-Src and its cellular counterpart (c-Src) share 95% sequence identity but distinct Hsp90 dependency. [122] The activation of v-Src strictly depends on Hsp90 while c-Src is largely independent of Hsp90. [122] Notably v-Src is an aggregation-prone protein and much more sensitive to thermal and heat denaturation than c-Src. [123] In the case of p53 biochemical experiments suggest that p53 interacts with Hsp90 in a rather folded state. [124] [125] [126] However recent results imply that p53 may be destabilized by Hsp90 [127] and NMR-based approaches suggested that for heat-treated p53 Hsp90 binds the largely unfolded protein. [128] Park et al. proposed that Hsp90-bound p53 is in a molten globule state. [129] In contrast Hagn et al. reported a native-like structure of p53 interaction with Hsp90. [130] Further analysis seems to be required to resolve this conundrum and to determine the molecular mechanism for client recognition. Hsp90 and protein degradation Top Although in general Hsp90 stabilizes and promotes the correct folding of its client proteins it was also found to facilitate protein degradation. In addition to soluble cytosolic proteins several reports have shown that Hsp90 is also required for the degradation of ER membrane proteins such as cytochrome p450 2E1 mutant CFTRΔF508 and apolipoprotein B. [131] [132] [133] Another aspect which supports the idea that Hsp90 may be involved in the ubiquitin-proteasome pathway is the discovery of a protein called carboxyl terminus of Hsp70-interacting protein (CHIP). [134] As an E3 ubiquitin ligase CHIP can ubiquitinate unfolded proteins. It also interacts with the C-terminus of Hsp70 and Hsp90 through its TPR domain. [135] [136] The CHIP knockdown is known to stabilize some Hsp90 clients ATPase posttranslational modifications
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