title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Truman
authors.affiliation: Department of Biomedical Sciences, Midwestern University, Downers Grove, IL 60515, USA.
authors.forename: Kolbrun
authors.initials: K
authors.lastname: Kristjansdottir
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Donald
authors.initials: D
authors.lastname: Wolfgeher
authors.affiliation: Departament de Ciències Bàsiques, Universitat Internacional de Catalunya, Barcelona, Catalunya, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Ricco
authors.affiliation: Computation Institute, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Anoop
authors.initials: A
authors.lastname: Mayampurath
authors.affiliation: Computation Institute, The University of Chicago, Chicago, IL 60637, USA; Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Volchenboum
authors.affiliation: Departament de Ciències Bàsiques, Universitat Internacional de Catalunya, Barcelona, Catalunya, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Clotet
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA. Electronic address: skron@uchicago.edu.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Kron
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004249:DNA Damage; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D018840:HSP70 Heat-Shock Proteins; C074510:HSP82 protein, S cerevisiae; D018841:HSP90 Heat-Shock Proteins; D009363:Neoplasm Proteins; D029701:Saccharomyces cerevisiae Proteins; D012262:Ribonucleoside Diphosphate Reductase; C523202:Rnr4 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae
keywords: Cancer; DNA damage; Global interactome; Molecular chaperones; Yeast