The formation of bioactive mediators, prostaglandins, leukotrienes, and HETEs, is initiated by stereoselective oxygenation of arachidonic acid. The goals of this project are to elucidate the function of novel lipoxygenases in mammalian physiology and to elucidate the enzyme-substrate interactions that distinguish the activities of the different lipoxygenase and cyclooxygenase enzymes. Dr. Brash and his colleagues have discovered several new lipoxygenase cDNAs that are the focus of studies in both the physiological function and the structure-function of lipoxygenase proteins. A primary goal is to characterize a new human 15(S) lipoxygenase and its closely related mouse homologue (a phorbol ester-inducible 8S-lipoxygenase) and to determine their function. These enzymes have been detected in skin, and induction of the murine homologue is known to be associated with inflammation and hyperplasia. The investigators will prepare transgenic mice overexpressing the enzymes in skin to examine the effects on inflammation and carcinogenesis. These human and murine lipoxygenases will also be used in studies of protein structure-function to elucidate determinants of positional specificity in these enzymes, initially using a chimera approach. In relation to the structure-function of lipoxygenases, characterization of the primary structures of several novel enzymes with R stereospecificity will provide valuable new insights on structure-function of these proteins. They also propose to elucidate the enzyme responsible for the increased synthesis of 12R-HETE in human proliferative skin disease: they plan to establish whether this is a R-lipoxygenase or a cytochrome P450 and then to characterize the enzyme involved in the production of this distinctive arachidonic acid metabolite that accumulates in psoriases and other dermatoses. The investigators will also examine the mechanisms of oxygenation of the cyclooxygenases, with comparison and contrast to the R and S lipoxygenases, by analysis of reactions of stereoselectivly-labeled 3H-substrate and the enantioselective reactions with a novel series of substrate analogues in the presence and absence of aspirin. The results of this study will help elucidate the molecular basis for the specificity of oxygenation by different lipoxygenases and cyclooxygenases and characterize an important target for known or potential therapeutic interventions.
