Group VIA
phospholipase A(2) (
iPLA(2)beta) is expressed in phagocytes, vascular cells, pancreatic islet beta-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
iPLA(2)beta functions inactivates
iPLA(2)beta by alkylating Cys
thiols. Because
thiol redox reactions are important in signaling and some cells that express
iPLA(2)beta produce
biological oxidants,
iPLA(2)beta might be subject to redox regulation. We report that
biological concentrations of H(2)O(2), NO, and HOCl inactivate
iPLA(2)beta, and this can be partially reversed by
dithiothreitol (DTT).
Oxidant-treated
iPLA(2)beta 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. W(460) oxidation could cause irreversible inactivation because it is near the
lipase consensus sequence ((463)GTSTG(467)), and site-directed mutagenesis of W(460) 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
iPLA(2)beta contains DTT-reducible oligomers, and oligomerization occurs with time- and temperature-dependent
iPLA(2)beta inactivation that is attenuated by DTT or
ATP. Subjecting
insulinoma cells to oxidative stress induces
iPLA(2)beta 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
iPLA(2)beta functions.