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A direct interplay between the co-chaperones Hop and p23 regulates cycle progression and client folding in the Hsp70/Hsp90 chaperone machineries

ABSTRACT: Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfolds a stringent client, the glucocorticoid receptor ligand binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. To proceed to client folding, current concepts assume that Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 directly interacts with Hop, relieves the stalling Hsp90-Hop interaction and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle. To study the interaction between the different proteins, especially p23 and the DP2 domain of Hop, we performed crosslinking-MS.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Moritz Mühlhofer

LAB HEAD: Johannes Buchner

PROVIDER: PXD027413 | Pride | 2022-02-24

REPOSITORIES: Pride

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			Action	DRS
	Kojak_conf.txt	Txt
	V_1.kojak.txt	Txt
	V_1.raw	Raw
	V_2.kojak.txt	Txt
	V_2.raw	Raw

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Hsp multichaperone complex buffers pathologically modified Tau

Project description:Alzheimer's disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery forming a 710 kDa client-loading complex. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau’s aggregation-prone repeat region. The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative disorders.

2022-08-11 | PXD032037 | Pride

Molecular determinants of Hsp90 recognition of Src kinase revealed by deep mutational scanning

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A specialized Hsp90 co-chaperone network regulates steroid hormone receptor response to ligand

Project description:Heat shock protein-90 chaperone machinery is involved in the stability and activity of its client proteins. The chaperone function of Hsp90 is regulated by co-chaperones and post-translational modifications. Although structural evidence exists for Hsp90 interaction with clients, our understanding of the impact of Hsp90 chaperone function towards client activity in cells remains elusive. Here, we dissected the impact of newly identified co-chaperones in higher eukaryotes, FNIP1/2 (FNIPs) and Tsc1, towards Hsp90 client activity. Our data show that Tsc1 and FNIP2 form mutually exclusive complexes with FNIP1 and that unlike Tsc1, FNIP1/2 interact with the catalytic residue of Hsp90. Functionally, these co-chaperone complexes increase the affinity of the steroid hormone receptors glucocorticoid receptor and estrogen receptor to their ligands in vivo. Here, we provide a model for the responsiveness of the steroid hormone receptor activation upon ligand binding as a consequence of their association with specific Hsp90:co-chaperone subpopulations.

2023-05-30 | PXD030486 | Pride

The Quantitative Changes in the Yeast Hsp70 and Hsp90 Interactomes upon DNA Damage

Project description:The molecular chaperones Hsp70 and Hsp90 participate in many important cellular processes, including how cells respond to DNA damage. In this study, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand the protein network through which Hsp70 and Hsp90 exert their effects on the DNA damage response (DDR). We characterized the interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. We identified 256 chaperone interactors, 146 of which are novel. Although the majority of chaperone interaction remained constant under DNA damage, 5 proteins (Coq5, Ast1, Cys3, Ydr210c and Rnr4) increased in interaction with Ssa1 and/or Hsp82. This data is related to article “Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperone-dependent regulation of ribonucleotide reductase”.

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Mechanism of client kinase dephosphorylation by the HSP90-CDC37-PP5 complex

Project description:Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, on the kinase specific co-chaperone CDC37, and the client itself. Removal of regulatory phosphorylation from client kinases and release from the HSP90-CDC37 system depends on a Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here we present the cryoEM structure of the oncogenic client kinase BRAFV600E bound to HSP90-CDC37, and structures of complexes of PP5 with that. Together with proteomic analysis of its phosphatase activity, our results reveal how PP5 is activated by recruitment to HSP90 complexes to dephosphorylate client proteins.

2023-03-11 | PXD033678 | Pride

Mechanism of client kinase dephosphorylation by the HSP90-CDC37-PP5 complex

2023-03-11 | PXD035934 | Pride

Cross-linking Analysis (HSP70-HSP90-CHIP)

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2019-03-06 | PXD009777 | Pride

Chaperone Activation and Client Binding of a 2-Cysteine Peroxiredoxin

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2019-02-19 | PXD010281 | Pride

Dynamic interplay between Ydj1 and Hsp90 for interaction with nucleotide binding domain of Hsp70 regulates client maturation

Project description:Hsp90 constitutes one of the major chaperone machinery in the cell. The Hsp70 assists Hsp90 in its client maturation though the underlying basis of the Hsp70 role remains to be explored. In the present study we identified novel mutations in the nucleotide-binding domain of yeast Ssa1 Hsp70 (Ssa1-T175N and Ssa1-D158N) that adversely affect the maturation of Hsp90 clients v-Src and Ste11. The identified Ssa1 amino acids critical for Hsp90 function were also found to be conserved across species such as in E.coli DnaK and the constitutive Hsp70 isoform (HspA8) in humans. These mutations are distal to the C-terminus of Hsp70 that primarily mediates Hsp90 interaction through the bridge protein Sti1. Intriguingly, we found that the bridge protein Sti1 is critical for cellular viability in cells expressing Ssa1-T175N (A1-T175N) or Ssa1-D158N (A1-D158N) as sole Ssa Hsp70. The growth defect was specific for sti1Δ, as deletion of none of the other Hsp90 co-chaperones showed lethality in A1-T175N or A1-D158N. Mass-spectrometry based whole proteome analysis of A1-T175 cells lacking Sti1 showed an altered abundance of various kinases and transcription factors suggesting compromised Hsp90 activity. Further proteomic analysis showed that pathways involved in signaling, signal transduction, and protein phosphorylation are markedly downregulated in the A1-T175N upon repression of Sti1 under DOX regulated promoter. In contrast to Ssa1, the homologous mutations in Ssa4 (Ssa4-T175N/D158N) supported cell growth even in the absence of Sti1. Overall, our data suggest that Ydj1 competes with Hsp90 for binding to Hsp70, and thus regulates Hsp90 interaction with the nucleotide-binding domain of Hsp70. The study thus provides new insight into the Hsp70-mediated regulation of Hsp90 and broadens our understanding of the intricate complexities of the Hsp70-Hsp90 network. This project was done under Deepak Sharma, CSIR-IMTECH, deepaks@imtech.res.in as a PI of the project.

2022-10-20 | PXD034983 | Pride

Native SEC-MS of the human HT29 colon adenocarcinoma cell line treated with the HSP90 inhibitor tanespimycin (17-AAG)

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