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Mechanisms of assembly and function of the Hsp70-Hsp40 chaperone machinery

ABSTRACT: The orchestration of protein folding and maintenance by molecular chaperones is crucial for cellular homeostasis. Among them, Hsp70 and its co-chaperone Hsp40 form a central machinery that remodels client proteins involved in numerous biological processes. Despite their essential role, how Hsp70 and Hsp40 assemble into a functional complex has remained elusive. Here, we integrated cryo-electron microscopy and nuclear magnetic resonance spectroscopy to determine the architecture of the full-length Hsp70-Hsp40 machinery. The structure of the complex in a physiological inactive state reveals distinct inhibitory mechanisms. In the active state, a new set of interactions is formed wherein the Hsp40 G/F-rich region acts as a pseudo-substrate and binds to the Hsp70, thereby directly modulating the refolding process. The G/F region maintains an Hsp40 autoinhibited state; upon binding to Hsp70, this state is disrupted, exposing a cryptic client-binding site and enabling interaction with protein clients to facilitate their refolding. The transition between these states is central to regulating the machinery’s refolding efficiency. Disrupting the Hsp40 autoinhibited state or the G/F–Hsp70 interaction leads to functional impairments that trigger a pronounced heat shock response in cells as a compensatory mechanism. Our findings provide crucial insights into the regulatory dynamics of this essential chaperone machinery, with broad implications for understanding protein homeostasis and the cellular response to stress.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE305556 | GEO | 2025/10/15

REPOSITORIES: GEO

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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

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2022-02-24 | PXD027413 | Pride

Rapid acetylation of yeast Hsp70 mediates the cellular response to heat stress

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A central chaperone-like role for 14-3-3 proteins in human cells [RNA-seq]

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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

Gene expression profiles mas5D, ssa2D, hsf1-overexpressing and heat-stressed wild type Schizosaccharomyces pombe cells

2013-09-20 | E-GEOD-50156 | biostudies-arrayexpress

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Project description:NRAS-mutant melanoma is currently a challenge to treat. This is due to an absence of inhibitors directed against NRAS, along with acquired and adaptive resistance of this tumor type to inhibitors in the MAPK pathway. Inhibitors to MEK (mitogen-activated protein kinase kinase) have shown some promise for this tumor type. In this work we explored the use of MEK inhibitors for NRAS-mutant melanoma, and at the same time investigated the impact of the brain micro-environment, specifically astrocytes, on the response of a melanoma brain metastatic cell line to MEK inhibition. This led to the surprising finding that astrocytes enhance the sensitivity of melanoma tumors to MEK inhibitors (MEKi). We show that MEKi cause an upregulation of the transcription factor ID3, but this is blocked by conditioned media from astrocytes. We show that silencing ID3 enhances the sensitivity of melanoma to MEK inhibitors, thus mimicking the effect of the brain microenvironment. Moreover, we report that ID3 is a client protein of the chaperone HSP70, and that HSP70 inhibition causes ID3 to misfold and accumulate in a detergent-insoluble fraction in cells. We show that HSP70 inhibitors synergize with MEK inhibitors against NRAS-mutant melanoma, and that this combination significantly enhances the survival of mice in two different models of NRAS-mutant melanoma. These studies highlight ID3 as a mediator of adaptive resistance, and support the combined use of MEK and HSP70 inhibitors for the therapy of NRAS-mutant melanoma.

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