REDOX REGULATION OF TRANSCRIPTION FACTOR FUNCTION

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Reactive oxygen species (ROS) are a by-product of a normal cellular metabolism. Inappropriate levels of these ROS has been implicated in the pathophysiology of a number of important human health concerns, ranging from cancer to the effects of aging. We are employing the yeast Saccharomyces cerevisiae as a model system for the ability of eukaryotic cells to detoxify ROS. Work from our laboratory and others has demonstrated that the S. cerevisiae yAP-1 transcriptional regulatory protein is crucial for this organism to survive oxidant exposure. The mammalian c- Jun transcription, a yAP-1 homolog, has been shown to be involved in the response of animal cells to oxidative stress, suggesting the possible evolutionary conservation of this role of yAP-1 and c-Jun. Our preliminary experiments have demonstrated that mutant forms of yAP-1 behave differently depending on the type of oxidant used to challenge the cells. Truncated mutant forms of yAP-1 are hyper-resistant to diamide stress but hypersensitive to H2O2. We have demonstrated that control of yAP-1 by oxidative stress occurs at a posttranslational step that requires cysteine residues in the C-terminus of the factor. To explore role of these cysteine residues as potential redox sensors, we will evaluate their reactivity using a chemical probe for cysteine modification. Peptide mapping will be used to detect post- translational modifications of the protein. The influence of oxidants on subcellular localization of wild-type and mutant forms of Yap1p will be examined using green fluorescent protein fusions to the transcription factor. We hypothesize that the failure of truncated forms of yAP-1 to confer H2O2 resistance while providing greater-than-normal diamide tolerance is due to a defect in these mutant factors to activate target gene expression in the face of H2O2 challenge. In support of this idea, the Yap1p-dependent H2O2 resistance gene TRX2 is not normally regulated by mutant forms of Yap1p. Experiments are proposed to determine the features of the TRX2 promoter that are required to confer its characteristic response to oxidants and Yap1p. To identify proteins that act to influence the function of yAP-1 at H2O2 resistance genes, second- site suppressors will be isolated that allow the truncated mutant forms of yAP-1 to confer H2O2 resistance. The successful completion of this set of experiments will delineate the events that lead to activation of yAP-1 by oxidative stress and provide new insight into the mechanisms behind redox control of protein function.