Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveal chaperone-dependent regulation of ribonucleotide reductase.

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To examine the dynamics of chaperone-client interactions after DNA damage, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to characterize interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. Of 256 proteins identified and quantified via (16)O(/18)O labeling and LC-MS/MS, 142 are novel Hsp70/90 interactors. Nearly all interactions remained unchanged or decreased after DNA damage, but 5 proteins increased interactions with Ssa1 and/or Hsp82, including the ribonucleotide reductase (RNR) subunit Rnr4. Inhibiting Hsp70 or 90 chaperone activity destabilized Rnr4 in yeast and its vertebrate homolog hRMM2 in breast cancer cells. In turn, pre-treatment of cancer cells with chaperone inhibitors sensitized cells to the RNR inhibitor gemcitabine, suggesting a novel chemotherapy strategy. All MS data have been deposited in the ProteomeXchange with identifier PXD001284.\nBIOLOGICAL SIGNIFICANCE: This study provides the dynamic interactome of the yeast Hsp70 and Hsp90 under DNA damage which suggest key roles for the chaperones in a variety of signaling cascades. Importantly, the cancer drug target ribonucleotide reductase was shown to be a client of Hsp70 and Hsp90 in both yeast and breast cancer cells. As such, this study highlights the potential of a novel cancer therapeutic strategy that exploits the synergy of chaperone and ribonucleotide reductase inhibitors.", "claims": [{"id": "claim-target-result", "statement": "MMS-induced DNA damage increased chaperone–client interactions, with Rnr4 significantly increasing its association with both Ssa1 and Hsp82; the study used quantitative affinity-purification mass spectrometry with 16O/18O labeling and LC-MS/MS, followed by statistical filtering and ANOVA, and the source proteomics data are available through ProteomeXchange/PRIDE under identifier PXD001284.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:25452130", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Nearly all interactions remained unchanged or decreased after DNA damage, but 5 proteins increased interactions with Ssa1 and/or Hsp82, including the ribonucleotide reductase (RNR) subunit Rnr4."}, {"asset": "source:pmid:25452130", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "All MS data have been deposited in the ProteomeXchange with identifier PXD001284."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "MMS-induced DNA damage increased chaperone–client interactions, with Rnr4 significantly increasing its association with both Ssa1 and Hsp82; the study used quantitative affinity-purification mass spectrometry with 16O/18O labeling and LC-MS/MS, followed by statistical filtering and ANOVA, and the source proteomics data are available through ProteomeXchange/PRIDE under identifier PXD001284.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:25452130"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. 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