Glutathione S-transferases (GSTS) constitute a key component of the cellular detoxication system GSTs catalyze the conjugation to glutathione and, in some cases, the reduction (via glutathione peroxidase activity) of xenobiobc electrophiles, including drugs and environmental toxicants. A novel group of GSTs has been recently identified that acts primarily on endogenous rather than xenobiotic substrates. Specifically, these are electrophilic products of lipid peroxidation (e.g. 4-hydroxynonenal and fatty acid/phospholipid hydroperoxides) which contribute to many toxic, mutagenic, and pathological outcomes, for example atherosclerosis, reperfusion injury, and perhaps aging. The detoxification activity of the above-mentioned specialized GSTs is physiologically relevant, as shown by us in transfection experiments. We have cloned, sequenced, expressed, and - in a collaborative effort - determined the X-ray crystal structure of mGSTA4-4, a prototypical member of the GST group invo.ved in cellular defense against products of lipid peroxidafion. This information is a prerequisite for long-term research into mGSTA4-4 and related enzymes which includes: studies of the structure-function relationship, with emphasis on the specific structural features that impart on the enzyme the ability to detoxify products of lipid peroxidation, and further definition of the toxicological and physiological roles of this group of GSTS.
The specific aims of the proposed research are: Define the residue, or combination of residues, that confers upon mGSTA4-4 its high reactivity with 4-hydroxynonenal and other lipid-derived substrates, by expressing protein forms modified by site-directed mutagenesis and/or chimeric constructs. Localize the active site involved in a distinct functional mode of mGSTA4-4, namely the glutathione-dependent peroxidase activity toward lipid hydroperoxides. Elucidate the role of subunit interactions in the transferase and peroxidase catalytic activities of mGSTA4-4. Continue to document the toxicological and physiological relevance of mGSTA4-4 and related enzymes through characterization of the phenotype of cultured cells transfected with the enzyme, especially in respect to their resistance to oxidative stress.
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