Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker.
A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Kristin M
KM
Allan
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Matthew A
MA
Loberg
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Juliet
J
Chepngeno
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Jennifer E
JE
Hurtig
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Susmit
S
Tripathi
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Min Goo
MG
Kang
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Jonathan K
JK
Allotey
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Afton H
AH
Widdershins
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Jennifer M
JM
Pilat
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Herbert J
HJ
Sizek
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Wesley J
WJ
Murphy
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Matthew R
MR
Naticchia
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States.
Joseph B
JB
David
Department of Microbiology & Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States.
Kevin A
KA
Morano
Biochemistry & Molecular Biology Program, Departments of Biology and Chemistry, The College of Wooster, Wooster, OH, United States. Electronic address: jwest@wooster.edu.
James D
JD
West
D003432:Cross-Linking Reagents / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005979:Glutathione Peroxidase / Q000737:chemistry; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry; D016877:Oxidants / Q000737:chemistry / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry; D054464:Peroxiredoxins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000187:drug effects; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013450:Sulfones / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry; D020122:tert-Butylhydroperoxide / Q000737:chemistry / Q000494:pharmacology
C515936:AHP1 protein, S cerevisiae; D003432:Cross-Linking Reagents; D004220:Disulfides; D016877:Oxidants; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; D013450:Sulfones; C516110:TRX2 protein, S cerevisiae; D013879:Thioredoxins; C009873:divinyl sulfone; D020122:tert-Butylhydroperoxide; D054464:Peroxiredoxins; D005979:Glutathione Peroxidase; D050862:Oxidoreductases Acting on Sulfur Group Donors; C479426:SRX1 protein, S cerevisiae; D051150:Methionine Sulfoxide Reductases
Cross-linker; Disulfide; Electrophile; Glutathione peroxidase; Glutathionylation; Methionine sulfoxide reductase; Peroxiredoxin; Sulfiredoxin; Thiol; Thioredoxin