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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 VIA族磷脂酶A2（iPLA 2）在吞噬细胞、血管细胞、胰岛细胞、神经元和其它细胞中表达，并在转录调节、细胞增殖、凋亡、分泌和其它事件中起作用。用于研究iPLA 2功能的溴烯醇内酯（BEL）自杀底物通过烷基化Cys硫醇使iPLA 2失活。由于巯基氧化还原反应在信号传导中很重要，并且一些表达iPLA 2的细胞产生生物氧化剂，因此iPLA 2可能受到氧化还原调节。我们报告说，生物浓度的H2 O2，NO和HOCl抑制iPLA 2，这可以部分逆转二硫苏糖醇（DTT）。通过胰蛋白酶解酶的LC-MS/MS分析来研究氧化剂处理的iPLA 2修饰，并包括DTT可逆事件，例如，二硫键和次磺酸的形成，以及其他不这样逆转的，例如，形成磺酸、Trp氧化物和Met亚砜。W 460氧化可能导致不可逆失活，因为它靠近脂肪酶共有序列（463 GTSTG 467），W 460的定点诱变产生不表现出DTT不可逆氧化失活的活性突变酶。Cys 651-次磺酸的形成可能是一种DTT可逆失活事件，因为Cys 651修饰与活性丧失密切相关，并且其诱变降低了对抑制的敏感性。分子间二硫键的形成也可能导致可逆失活，因为氧化剂处理的iPLA 2含有DTT可还原的寡聚体，并且寡聚化随着时间和温度依赖性iPLA 2失活而发生，其被DTT或ATP减弱。使胰岛素瘤细胞经受氧化应激诱导iPLA 2寡聚化、活性丧失和亚细胞再分布，并降低花生四烯酸从磷脂的释放速率。这些发现提高了氧化还原反应影响iPLA 2功能的可能性。

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.