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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 磷脂酶A2(IPLA2)通过磷脂酶A2在吞噬细胞、血管细胞、胰岛细胞、神经元等细胞中表达，在转录调控、细胞增殖、细胞凋亡、分泌等事件中发挥作用。一种用于研究iPLA2功能的溴烯醇内酯(BEL)自杀底物通过烷基化半胱氨酸硫醇来灭活iPLA2。由于硫醇氧化还原反应在信号转导中起重要作用，一些表达iPLA2的细胞产生生物氧化剂，iPLA2可能受到氧化还原调节。我们报告了生物浓度的H_2O_2、NO和HOCl使iPLA2失活，这可以被二硫苏糖醇(DTT)部分逆转。通过对胰酶消化的LC-MS/MS分析，研究了氧化剂处理的iPLA2的修饰，包括DTT可逆事件，例如二硫键和磺酸的形成，以及其他不那么可逆的事件，例如磺酸、色氨酸氧化物和甲硫醚的形成。W460氧化可能导致不可逆失活，因为它接近脂肪酶共同序列(463GTSTG467)，而W460的定点突变产生活性突变酶，没有DTT不可逆氧化失活。Cys651-磺酸的形成可能是一种DTT可逆的失活事件，因为Cys651的修饰与活性丧失密切相关，其突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iPLA2含有DTT可还原的寡聚体，而寡聚发生时iPLA2的失活依赖于时间和温度，而DTT或ATP减弱了iPLA2的失活。使胰岛素瘤细胞承受氧化应激诱导iPLA2寡聚、活性丧失和亚细胞重新分布，并降低从磷脂中释放花生四烯酸的速率。这些发现增加了氧化还原反应影响iPLA2功能的可能性。