Oxidative Stress Causes Modifications and Decreased Function of System xc-

Oxidative stress represents an imbalance between the production of reactive oxygen species, and their removal by antioxidants. System xc-­ is a plasma membrane transport system that exchanges intracellular glutamate for extracellular cystine, a precursor to cysteine, the limiting reagent for glutathione production. As glutathione is an endogeneous antioxidant, system xc-­ plays a key role in combating oxidative stress. Previously, our lab has observed using western blot analysis that a fraction of xCT shifts to a higher molecular weight form after hydrogen peroxide exposure. As hydrogen peroxide produces free radicals, we believe xCT is oxidized, therefore decreasing its function. Two methods are being taken to observe oxidation, biochemical strategies in our lab and mass spectroscopy done at the Fred Hutchinson Cancer Research Center Proteomics Core. Biochemical strategies include DNPH assays to detect the presence of carbonyls after oxidative stress, and glutathiolation assays to detect the presence of glutathiolated proteins. Results indicate that xCT is oxidized. In addition to oxidation, we found that xCT is glutathiolated, which could have a variety of effects. xCT may be glutathionylated on cysteine residues and form high molecular weight multimers as a result of cross-­linking by reactive cyteine residues. It is also possible that reactive cysteinyl thiols may undergo glutathiolation reactions to act as an on/off switch in response to oxidative insult with the effect of initiating and depleting signal transduction pathways. Mass spectroscopy will help identify specific locations of oxidation and glutathiolation. Once targets are identifies, a combination of site-directed mutagenesis and mass spectroscopy will be used to identify critical residues that are modified after oxidative stress