Protective Role of the Yeast Peroxiredoxin Tsa1 against Electrophile-Mediated Stress

Andrew Lamade, The College of Wooster

Abstract

ABSTRACT The Saccharomyces cerevisiae peroxiredoxin, Tsa1, is responsible for the thiol-dependent detoxification of peroxides. Similar to other typical 2-Cys peroxiredoxins, Tsa1's catalytic mechanism is dependent upon disulfide bond formation between its two cysteine residues. Site-directed mutagenesis of these residues followed by treatment with electrophilic protein cross-linkers has shown that they are the primary targets of modification. Therefore, exposure to electrophiles precludes disulfide bond formation and inhibits Tsa1's peroxidase activity. Despite this apparent inactivation, Tsa1 protects yeast against the toxic effects of electrophiles. To ascertain the extent of this protection, Tsa1 was expressed at varying levels in S. cerevisiae lacking endogenous Tsa1 and its closely related homolog Tsa2. Reintroduction of Tsa1 at intermediate and high levels conferred enhanced survival of cultures exposed to H2O2 or bifunctional electrophiles, such as divinyl sulfone (DVSF) and butane-1,4-diisothiocyanate (BDITC). Past studies have suggested hyperoxidation of Tsa1 is accompanied by high molecular weight (HMW) complex formation which is indicative of a transition from peroxidase to molecular chaperone. Confocal fluorescent microscopy live cell imaging was used to confirm that Tsa1-GFP treated with H2O2 or electrophiles [DVSF, diethyl acetylenedicarboxylate (DAD)] undergoes a relocalization event following modification. Moreover, we found that Tsa1 undergoes a transition from a low molecular weight species to a HMW complex upon electrophile exposure both in vitro and in vivo. Further experimentation is required to elucidate the HMW complex structure and mechanism of action underlying its proposed role in mitigating electrophilic toxicity. If Tsa1 is able to transition from a peroxidase to a chaperone upon electrophilic modification, as hypothesized, this would represent a novel protective mechanism against a broad range of small molecule stressors.