EFFECTS OF BIOLOGICAL OXIDANTS ON THE CATALYTIC ACTIVITY AND STRUCTURE OF GROUP

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Group VIA phospholipase A2 (iPLA2) is expressed in phagocytes, vascular cells, pancreatic islet -cells, neurons, and other cells and plays roles in transcriptional regulation, cell proliferation, apoptosis, secretion, and other events. A bromoenol lactone (BEL) suicide substrate used to study iPLA2 functions inactivates iPLA2 by alkylating Cys thiols. Because thiol redox reactions are important in signaling and some cells that express iPLA2 produce biological oxidants, iPLA2 might be subject to redox regulation. We report that biological concentrations of H2O2, NO, and HOCl inactivate iPLA2, and this can be partially reversed by dithiothreitol (DTT). Oxidant-treated iPLA2 modifications were studied by LC-MS/MS analyses of tryptic digests and included DTT-reversible events, e.g., formation of disulfide bonds and sulfenic acids, and others not so reversed, e.g., formation of sulfonic acids, Trp oxides, and Met sulfoxides. W460 oxidation could cause irreversible inactivation because it is near the lipase consensus sequence (463GTSTG467), and site-directed mutagenesis of W460 yields active mutant enzymes that exhibit no DTT-irreversible oxidative inactivation. Cys651-sulfenic acid formation could be one DTT-reversible inactivation event because Cys651 modification correlates closely with activity loss and its mutagenesis reduces sensitivity to inhibition. Intermolecular disulfide bond formation might also cause reversible inactivation because oxidant-treated iPLA2 contains DTT-reducible oligomers, and oligomerization occurs with time- and temperature-dependent iPLA2 inactivation that is attenuated by DTT or ATP. Subjecting insulinoma cells to oxidative stress induces iPLA2 oligomerization, loss of activity, and subcellular redistribution and reduces the rate of release of arachidonate from phospholipids. These findings raise the possibility that redox reactions affect iPLA2 functions.