Binding of ATP to Heat Shock Protein 90

Garnier, Cyrille; Lafitte, Daniel; Tsvetkov, Philipp O.; Barbier, Pascale; Leclerc-Devin, Jocelyne; Millot, Jean-Marc; Briand, Claudette; Makarov, Alexander А.; Catelli, Maria-Grazia; Peyrot, Vincent

doi:10.1074/jbc.m111874200

Cited by 131 publications

(63 citation statements)

References 41 publications

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“…ATPase activity specifically inhibited by geldanamycin and radicicol, implied at least in some of the chaperone activities described for the protein, is well characterized in the N-terminal domain of Hsp90 (32), although a putative secondary ATP binding site has also been described to exist in the C-terminal domain (33)(34)(35). No cysteine is located in the N-terminal domain, and Cys 597, which we have identified as a target for S-nitrosylation, is located in the C-terminal domain, in a strand belonging to a ␤-sheet separated from the dimerization helices described to be involved in the putative secondary ATP-binding site (11).…”

Section: Discussionmentioning

confidence: 99%

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

Martínez‐Ruiz

Villanueva²,

Orduña³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

285

221

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Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-L-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.atherosclerosis ͉ nitrosation ͉ vascular wall ͉ chaperone R ecent years have witnessed an increasing interest in the roles of nitric oxide (NO) in signal transduction pathways other than its activation of the cGMP pathway. Many of these roles rely on NO's ability to alter protein function through posttranslational modifications. Among these modifications, S-nitrosylation has emerged as a potential and fundamental regulator of protein function. S-nitrosylation is a covalent modification of thiol groups by formation of a thionitrite (-S-NϭO) group, facilitated by the formation of higher nitrogen oxides (1, 2). To date, several dozens of proteins have been shown to become S-nitrosylated and, in many cases, this modification was accompanied by altered function (see table S1 of ref. 1 for review).Nitric oxide, synthesized in the endothelium by endothelial nitric oxide synthase (eNOS), plays crucial roles in the vascular wall, including the maintenance of vascular tone. The possibility that NO might modify eNOS, or elements of the complex system involved in its activation, is an attractive hypothesis, suggesting a potential autoregulatory feedback mechanism. The eNOS enzyme is regulated by several posttranslational modifications including myristoylation, palmitoylation, and phosphorylation (3). This enzyme is also tightly regulated by specific interactions with inhibitory proteins such as caveolin-1 and by positive modulation by the scaffolding protein Hsp90. These interactions have been described in detail, and a relatively complete picture is beginning to emerge (4).We have previously used a proteomic approach to identify several proteins that were S-nitrosylated after exposure of vascular endothelial cells to the physiological nitrosothiol, Snitroso-L-cysteine (CSNO) (5). Further work led to the identification of Hsp90 as a protein susceptible to S-nitrosylation. This chaperone protein, known for its functions in protein folding, degradation, and scaffolding, has attracted renewed ...

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Section: Discussionmentioning

confidence: 99%

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

Martínez‐Ruiz

Villanueva²,

Orduña³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

285

221

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“…There is now accumulating evidence that the conformational effects of novobiocin on the Hsp90 C terminus are communicated to the N-terminal domain (23,54). ATP binding in the Hsp90 N terminus leads to the exposure of a cryptic nucleotidebinding site within the C-terminal domain, increasing accessibility to novobiocin (54,59). Drug interaction with the C terminus disrupts nucleotide binding at both C-and N-terminal sites (23,54,59).…”

Section: Discussionmentioning

confidence: 99%

“…ATP binding in the Hsp90 N terminus leads to the exposure of a cryptic nucleotidebinding site within the C-terminal domain, increasing accessibility to novobiocin (54,59). Drug interaction with the C terminus disrupts nucleotide binding at both C-and N-terminal sites (23,54,59). Evidence suggests that novobiocin induces alterations in Hsp90 proteolytic fragmentation patterns caused by structural changes throughout multiple domains of the Hsp90 protein (55).…”

Section: Discussionmentioning

confidence: 99%

Modulation of Chaperone Function and Cochaperone Interaction by Novobiocin in the C-terminal Domain of Hsp90

Allan

Mok

Ward

et al. 2006

Journal of Biological Chemistry

143

141

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The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An ␣-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50 There is now increasing evidence that receptor function is critically dependent on the selection of immunophilin within steroid receptor complexes (2-4). This may be governed, in part, by the selective preference of receptors for specific immunophilins. PP5 has been reported to have a modulating role in glucocorticoid signaling (5), and there are strong indications that FKBP51 inhibits glucocorticoid receptor function. Elevated expression of FKBP51, resulting in greatly increased incorporation of FKBP51 into glucocorticoid receptor complexes, reduces hormone-binding affinity and promotes glucocorticoid resistance in primates (6, 7). FKBP51 also sequesters glucocorticoid receptor within the cytoplasm (8, 9), but hormone binding induces a functional exchange of FKBP52 for FKBP51 in receptor complexes allowing translocation of the complex to the nucleus (8). In a yeast model, FKBP52 was shown to dramatically potentiate glucocorticoid-dependent reporter gene activity through a mechanism that results in increased receptor hormone-binding affinity (10). Consistent with previous findings, coexpression of FKBP51 blocked the potentiating effects of FKBP52. These potentiating properties require FKBP52 catalytic activity as well as a functional interaction of the immunophilin with Hsp90. Receptor function, then, can be directly influenced by the prolyl isomerase activity of a TPR immunophilin. The Smith laboratory has now extended the study of FKBP52 function to a FKBP52 knock-out mouse model (11-13). Male mice exhibit many features in common with partial androgen insensitivity, reflecting loss of FKBP52-mediated potentiation of androgen receptor function (11). Female mice display a maternal defect linked to progesterone ins...

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“…1B, the E42A variant of Hsp90␤ binds ATP more efficiently than WT Hsp90␤, whereas the binding of ATP by D88N Hsp90␤ is weaker. The D88N Hsp90␤ variant still binds ATP more efficiently than BSA (background) probably due to the existence of the second ATP binding site located in the C-terminal domain of Hsp90 (29,30). Both variants, E42A and D88N, do not catalyze the ATP hydrolysis (Fig.…”

Section: Characterization Of Hsp90␤mentioning

confidence: 96%

ATP Binding to Hsp90 Is Sufficient for Effective Chaperoning of p53 Protein

Walerych

Gutkowska

Klejman

et al. 2010

Journal of Biological Chemistry

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Hsp90 is a ubiquitous, ATP-dependent chaperone, essential for eukaryotes. It possesses a broad spectrum of substrates, among which is the p53 transcription factor, encoded by a tumor-suppressor gene. Here, we elucidate the role of the adenine nucleotide in the Hsp90 chaperone cycle, by taking advantage of a unique in vitro assay measuring Hsp90-dependent p53 binding to the promoter sequence. E42A and D88N Hsp90␤ variants bind but do not hydrolyze ATP, whereas E42A has increased and D88N decreased ATP affinity, compared with WT Hsp90␤. Nevertheless, both of these mutants interact with WT p53 with a similar affinity. Surprisingly, in the case of WT, but also E42A Hsp90␤, the presence of ATP stimulates dissociation of Hsp90-p53 complexes and results in p53 binding to the promoter sequence. D88N Hsp90␤ is not efficient in both of these reactions. Using a trap version of the chaperonin GroEL, which irreversibly captures unfolded proteins, we show that Hsp90 chaperone action on WT p53 results in a partial unfolding of the substrate. The ATP-dependent dissociation of p53-Hsp90 complex allows further folding of p53 protein to an active conformation, able to bind to the promoter sequence. Furthermore, in support of these results, the overproduction of WT or E42A Hsp90␤ stimulates transcription from the WAF1 gene promoter in H1299 cells. Altogether, our research indicates that ATP binding to Hsp90␤ is a sufficient step for effective WT p53 client protein chaperoning.Hsp90 is an abundant protein in cells of all known organisms, with the exception of the Archea kingdom. Although in bacteria its presence is not required for survival (1), yeast and higher eukaryotes are fully dependent on its activity (2, 3). In multicellular organisms Hsp90 plays a key role in the activation and stabilization of various protein substrates. Among these are kinases (Raf1, Akt, and Src), telomerase, Rab GDP dissociation inhibitors, glucocorticoid hormone receptors (GR), 3 and transcription factors such as the p53 tumor suppressor protein (4 -6). These Hsp90 substrates belong to different protein families and do not share common sequence or structural motifs. Hence, modes of interaction and chaperoning may possess both common features and specific differences.Hsp90 is functional as a dimer, each monomer consisting of three domains connected with flexible linkers of different length and sequence depending on organism and isoform. The main substrate binding region is proposed to be localized in the middle domain (7), however structural (8) and biochemical studies (9 -11) suggest that at least two distinct surfaces of interaction should exist on Hsp90 chaperone while binding its protein substrate. Repositioning of the domains of the Hsp90 may be translated into conformational rearrangements of a protein substrate, changing its tertiary structure and exposing buried residues, thus enabling interaction with other proteins and ligands, such as hormones or nucleic acids.Despite the initial controversy on the ATP dependence of Hsp90 (12), it was unambig...

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Binding of ATP to Heat Shock Protein 90

Cited by 131 publications

References 41 publications

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

Modulation of Chaperone Function and Cochaperone Interaction by Novobiocin in the C-terminal Domain of Hsp90

ATP Binding to Hsp90 Is Sufficient for Effective Chaperoning of p53 Protein

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