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. 2016 Dec:101:356-366.
doi: 10.1016/j.freeradbiomed.2016.10.506. Epub 2016 Nov 2.

Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker

Kristin M Allan  1 , Matthew A Loberg  1 , Juliet Chepngeno  1 , Jennifer E Hurtig  1 , Susmit Tripathi  1 , Min Goo Kang  1 , Jonathan K Allotey  1 , Afton H Widdershins  1 , Jennifer M Pilat  1 , Herbert J Sizek  1 , Wesley J Murphy  1 , Matthew R Naticchia  1 , Joseph B David  1 , Kevin A Morano  2 , James D West  3
Affiliations

Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker

Kristin M Allan et al. Free Radic Biol Med. 2016 Dec.

Abstract

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.

Keywords: Cross-linker; Disulfide; Electrophile; Glutathione peroxidase; Glutathionylation; Methionine sulfoxide reductase; Peroxiredoxin; Sulfiredoxin; Thiol; Thioredoxin.

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Figures

Figure 1
Figure 1. Cross-Linking of Multiple Oxidant-Sensitive Proteins in Cells Treated with DVSF
(A) Scheme of DVSF-mediated thiol cross-linking. (B) Log-phase cells expressing FLAG-tagged forms of oxidant-sensitive proteins were treated with vehicle (DMSO) or 1 mM DVSF for 1 h at 30°C. Protein lysates (10-20 μg) were resolved by SDS-PAGE, transferred to PVDF membrane, and detected with an antibody against the FLAG-tag or Pgk1 (loading control) via Western blot. An asterisk (*) represents a background band that appears with the FLAG-antibody in certain blots. A double asterisk (**) represents a species of the protein that electrophoreses more quickly, suggesting intramolecular cross-linking. Results are representative of three independent experiments.
Figure 2
Figure 2. Involvement of Redox-Active Cys Residues in Cross-Linking of DVSF Targets
Log-phase cells expressing wild-type (wt) proteins or ones with individual Cys-to-Ala substitutions were treated with DMSO (vehicle) or 1 mM DVSF for 1 h at 30°C. Protein lysates (10-20 μg) were resolved by SDS-PAGE, transferred to PVDF membrane, and detected with an antibody against the FLAG-tag or Pgk1 (loading control) via Western blot. An asterisk (*) represents a background band that appears with the FLAG-antibody in certain blots. A double asterisk (**) indicates potential intramolecular cross-linking in the protein. Results are representative of three independent experiments.
Figure 3
Figure 3. Decreased Cross-Linking of DVSF Targets in Cells Pretreated with H2O2
Log-phase cells expressing DVSF targets were incubated for 10 min with either vehicle (H2O or DMSO) or oxidant (1 mM H2O2 or 0.5 mM TBHP, respectively) prior to treatment for 1 h at 30°C with DMSO (vehicle) or 1 mM DVSF. Protein lysates (10 μg) were resolved by SDS-PAGE, transferred to PVDF membrane, and detected with an antibody against the FLAG-tag or Pgk1 (loading control) via Western blot. An asterisk (*) represents a background band that appears with the FLAG-antibody in certain blots. Results are representative of three independent experiments.
Figure 4
Figure 4. Cross-Linking of Selected DVSF Targets to Thioredoxin
(A) Scheme depicting cross-linking between Trx2 and its interaction partners. (B) Indicated proteins were expressed in wild-type (BY4741), trx1Δ trx2Δ (labeled as trxΔ) or, in the case of Srx1, tsa1Δ tsa2Δ (labeled as tsaΔ) cells and treated with DMSO (vehicle) or 1 mM DVSF for 1 h at 30°C. Protein lysates (10-20 μg) were resolved by SDS-PAGE, transferred to PVDF membrane, and detected with an antibody against the FLAG-tag or Pgk1 (loading control) via Western blot. Dots (•) represent potential intermolecular cross-links to Trx1 or Trx2, whereas an arrow head (<) indicates potential cross-links between Srx1 and Tsa1 or Tsa2. (C) FLAG-tagged proteins were co-expressed in yeast containing a TAP-tagged Trx2 (Trx2-TAP) and treated with DMSO (vehicle) or 1 mM DVSF for 1 h at 30°C. FLAG-tagged proteins were immunoprecipitated from cell lysates (100 μg) and detected, along with Trx2-TAP isolated in cross-linked complexes, via Western blot. Results are representative of three independent experiments.
Figure 5
Figure 5. Disruption of the Redox Center at the Dimer Interface of Ahp1 Compromises Cellular Defense against Organic Peroxides and Impairs Cross-Linking by DVSF
(A) Structure of Ahp1 depicting catalytic Cys residues (C62 (peroxidatic Cys) and C31 (resolving Cys)) and conserved Phe residues at the dimer interface. Protein structures were generated with Chimera (https://www.cgl.ucsf.edu/chimera/) using PDB 4DSR. (B) Purified recombinant Ahp1 (wild-type (wt) or dimer interface variants, 20 μM) were resolved on native-PAGE and detected with Coomassie blue to determine oligomeric state. (C) Wild-type (wt) or mutant forms of Ahp1 were expressed in ahp1Δ yeast. Serial dilutions of these cultures and corresponding controls were grown on YPD medium containing 2 mM TBHP for 48 h at 30°C to determine the effect of dimer interface disruption on oxidant defense. (D) Scheme depicting proposed inter-subunit cross-linking in Ahp1 by DVSF. (E) Purified Ahp1 proteins (10 μM) were treated with increasing concentrations of DVSF for 3 h at 37°C, prior to electrophoresis on SDS-PAGE and detection by staining with Coomassie blue. (F) Log-phase yeast cells expressing Ahp1 variants were treated with 1 mM DVSF for 1 h at 30°C. Protein lysates (10-20 μg) were resolved by SDS-PAGE, transferred to PVDF membrane, and detected with an antibody against the FLAG-tag or Pgk1 (loading control) via Western blot. Results for all experiments are representative of three independent trials.
Figure 6
Figure 6. Cross-Linking of GSH to Redox-Active Proteins in Cells Treated with DVSF
(A) Scheme depicting GSH cross-linking to putative DVSF targets. (B) Log-phase yeast cells (BY4741) were treated with increasing doses of DVSF for 1 h at 30°C. Protein lysates (10 μg) were resolved by SDS-PAGE, transferred to PVDF membrane, and detected with an antibody against the FLAG-tag or Pgk1 (loading control) via Western blot. (C) Log-phase yeast cells (BY4741 or gsh1Δ) were treated with 1 mM DVSF for 1 h at 30°C. Protein lysates (10 μg) were resolved by SDS-PAGE, transferred to PVDF membrane, and detected with an antibody against the FLAG-tag or Pgk1 (loading control) via Western blot. (D and E) Cells expressing FLAG-tagged proteins were treated with 1 mM DVSF for 1 h at 30°C. FLAG-tagged proteins were immunoprecipitated from cell lysates (100 μg). Western blots were conducted to determine isolation of FLAG-tagged protein and its conjugation to GSH. Arrows point to protein-glutathione conjugates. (F) Purified Ahp1 variants (10 μM) were incubated with varying concentrations of GSSG for 20 min, prior to alkylation of free cysteines with N-ethylmaleimide and resolution by non-reducing SDS-PAGE. S-glutathionylation was monitored by Western blot. Ahp1 levels and oxidation were visualized with Coomassie blue. Results are representative of two-three independent experiments.
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