Pro-oxidant conditions lead, among other effects, to lipid peroxidation. The ensuing chain reaction yields, for each initiating event, hundreds of lipid hydroperoxide molecules. Some of these give rise to highly electrophilic 4-hydroxyalkenals, including the predominant 4-hydroxynonenal (4-HNE). 4-HNE is toxic at high levels but has signaling functions at physiological concentrations. Generally, 4-HNE has anti-proliferative differentiating, and pro-apoptotic effects on cells. Both the signaling and toxic outcomes of 4-HNE an thought to be mediated by the compound's ability to cause covalent modifications of proteins, including key regulatory proteins whose function is altered upon 4-HNE adduct formation. Thus, 4-HNE needs to be metabolized to prevent its toxicity (which contributes to the etiology of degenerative diseases such a atherosclerosis or Alzheimer's disease), and to terminate signaling (which may contribute to control of cell division and cell death, and thus be relevant to tumor biology). The major mode of 4-HNE metabolism is glutathione conjugation, catalyzed by specialized glutathione S-transferases. We have previously studied the biochemical and structural properties of mGSTA4-4, a prototypical member of this group of enzymes. In continuation of this project, we propose to shift emphasis to the biological properties and physiologic significance of mGSTA4-4. Accordingly, we propose to generate a mGSTA4-4 knockout mouse and stud its phenotype, especially in response to oxidative stress. We further propose to transfect cells with mGSTA4-4 variants which are selectively altered in their partial catalytic properties, and to define the resulting phenotypes. On the subcellular level, we determined that mGSTA4-4 is associated with the plasma membrane, in agreement with the enzyme's function in the metabolism of lipid-derived substrates. We now propose to determine whether the intracellular localization is affected by oxidative stress, and conversely - whether experimental manipulation of localization affects function. Finally, we propose t continue structure-function relationship studies on mGSTA4-4, particularly on a mutant with six-fold increased catalytic efficiency. The proposed work is intended to advance the understanding of the physiological and toxicological consequences of oxidative stress, as mediated by 4-HNE.
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