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

生物氧化剂对催化活性和基团结构的影响

• 批准号: 7721510
• 负责人: HAOWEI SONG
• 金额: $ 0.44万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721510
• 关键词: 6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; Affect; Apoptosis; Attenuated; Biological; Blood Vessels; Cell Proliferation; Cells; Computer Retrieval of Information on Scientific Projects Database; Consensus Sequence; Dithiothreitol; Enzymes; Event; Exhibits; Funding; Grant; Group Structure; Hydrogen Peroxide; Institution; Islet Cell; Islets of Langerhans; Lipase; Modification; Mutagenesis; Neurons; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Phagocytes; Phospholipase A2; Phospholipids; Rate; Reaction; Regulation; Reporting; Research; Research Personnel; Resources; Role playing therapy; Signal Transduction; Site-Directed Mutagenesis; Source; Sulfenic Acids; Sulfhydryl Compounds; Sulfonic Acids; Sulfoxide; Temperature; Time; Transcriptional Regulation; United States National Institutes of Health; arachidonate; disulfide bond; insulinoma; mutant; oxidation; suicide substrates

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 通过磷脂酶A2（IPLA2）组在吞噬细胞，血管细胞，胰岛 - 细胞，神经元和其他细胞中表达，并在转录调节，细胞增殖，凋亡，分泌和其他事件中发挥作用。用于研究IPLA2功能的溴苯醇内酯（BEL）自杀底物通过烷基化Cys硫醇使IPLA2失活。由于硫醇氧化还原反应在信号传导中很重要，并且某些表达IPLA2产生生物氧化剂的细胞可能会受到氧化还原调节的影响。我们报告说，H2O2，NO和HOCL灭活IPLA2的生物浓度可以通过Dithiothreitol（DTT）部分逆转。通过对胰蛋白酶消化的LC-MS/MS分析研究了经过氧化剂处理的IPLA2修饰，包括DTT可逆事件，例如，二硫键键和硫酸酸的形成，以及其他并未如此逆转的形成，例如，硫酸，TRP，TRP氧化物和硫化物的形成，硫代氧化物和硫代氧化物。 W460氧化可能会导致不可逆的失活，因为它接近脂肪酶共有序列（463GTSTG467），并且W460的位置为定向的诱变会产生活性突变酶，没有表现出DTT-IRRVERVERESIBLIBIBIBLE氧化氧化失活。 CYS651-硫酸形成可能是一种DTT可逆的失活事件，因为CYS651的修饰与活性丧失紧密相关，其诱变可降低对抑制的敏感性。分子间二硫键的形成也可能导致可逆的失活，因为氧化剂处理的IPLA2含有DTT可还原寡聚体，并且寡聚发生在时间和温度依赖性IPLA2灭活的情况下会导致DTT或ATP减弱。氧化应激会导致胰岛素瘤细胞诱导IPLA2寡聚，活性丧失和亚细胞重新分布，并降低磷脂中蛛网膜酸盐的释放速率。这些发现增加了氧化还原反应影响IPLA2功能的可能性。