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

Abstract
The highly conserved molecular chaperones Hsp90 and Hsp70 are indispensible for folding and maturation of a significant fraction of the proteome, including many proteins involved in signal transduction and stress response. 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.
BIOLOGICAL 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.
AuthorsAndrew W Truman, Kolbrun Kristjansdottir, Donald Wolfgeher, Natalia Ricco, Anoop Mayampurath, Samuel L Volchenboum, Josep Clotet, Stephen J Kron
JournalJournal of proteomics (J Proteomics) Vol. 112 Pg. 285-300 (Jan 01 2015) ISSN: 1876-7737 [Electronic] Netherlands
PMID25452130 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
CopyrightCopyright © 2014 Elsevier B.V. All rights reserved.
Chemical References
  • DNA, Fungal
  • HSP70 Heat-Shock Proteins
  • HSP82 protein, S cerevisiae
  • HSP90 Heat-Shock Proteins
  • Neoplasm Proteins
  • Saccharomyces cerevisiae Proteins
  • Ribonucleoside Diphosphate Reductase
  • Rnr4 protein, S cerevisiae
  • Adenosine Triphosphatases
  • SSA1 protein, S cerevisiae
Topics
  • Adenosine Triphosphatases (genetics, metabolism)
  • Breast Neoplasms (genetics, metabolism, pathology)
  • Cell Line, Tumor
  • DNA Damage
  • DNA, Fungal (genetics, metabolism)
  • Female
  • HSP70 Heat-Shock Proteins (genetics, metabolism)
  • HSP90 Heat-Shock Proteins (genetics, metabolism)
  • Humans
  • Neoplasm Proteins (genetics, metabolism)
  • Proteomics
  • Ribonucleoside Diphosphate Reductase (genetics, metabolism)
  • Saccharomyces cerevisiae (genetics, metabolism)
  • Saccharomyces cerevisiae Proteins (genetics, metabolism)

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