Regulation of Oxygen Metabolism in Blood Neutrophils

血液中性粒细胞氧代谢的调节

• 批准号: 6631716
• 负责人: LINDA C. McPHAIL
• 金额: $ 42.28万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6631716
• 关键词: NAD(P)H dehydrogenase; antisense nucleic acid; cell free system; cellular respiration; chimeric proteins; diacylglycerols; enzyme activity; enzyme induction /repression; enzyme mechanism; human subject; isozymes; leukocyte oxidative burst; molecular cloning; neutrophil; phosphatidate; phospholipase D; phosphorylation; protein binding; protein kinase; protein kinase C; protein protein interaction; protein purification; respiratory burst oxidase; site directed mutagenesis

DESCRIPTION (provided by applicant): The phagocyte respiratory burst enzyme, NADPH oxidase, produces superoxide anion. The precursor for other oxygen species (H202, hypochiorous acid) that play important roles in host defense and inflammation and/or act as intracellular signaling molecules. Our long-term goal is to elucidate the signaling pathways and biochemical mechanisms leading to NADPH oxidase activation. Studies in intact cells suggest that the lipid second messengers resulting from the receptor-initiated activation of phospholipase D (PLD) regulate the activation of the respiratory burst. PLD activation increases intracellular levels of phosphatidic acid (PA), which can be converted by the cell to diacyiglycerol (DG). Both PA and DG act as second messengers, but the protein targets involved in NADPH oxidase activation are unclear. DG activates protein kinase C (PKC), which is strongly linked to NADPH oxidase activation. Our results suggest that PA and DG regulate NADPH oxidase activation by indirect and direct mechanisms. The indirect mechanisms involve activation of PKC isoforms and a novel protein kinase (PAPK). These protein kinases phosphorylate the oxidase components p47phax and p22phax and enhance oxidase activation in cell-free systems. The direct mechanisms involve interaction of PA and DG with oxidase component(s). These lipids induce NADPH oxidase activation in a cell-free system when only oxidase proteins are present, hence phosphorylation is not possible. Furthermore, PA binds to p47phax, identifying this protein as a potential functional target of PA. We propose that these indirect (phosphorylation-dependent) and direct (binding of PA and DG to oxidase components) mechanisms mediate PLD-initiated NADPH oxidase activation. The following specific aims are designed to test this hypothesis and to elucidate the biochemical mechanisms involved. In Aim 1, we will test whether PLD-initiated NADPH oxidase activation involves synergy between indirect phosphorylation-dependent and direct lipid-induced mechanisms. We will use several approaches (identification of PKC/PAPK isoforms regulated by PLD, selective reduction of PKC/PAPK expression with antisense oligonucleotides, functional effects of phosphorylation site mutants of p47phax) In Aim 2, we will test whether direct interaction of PA and DG with oxidase proteins is required for PLD initiated NADPH oxidase activation. We will mutate the PA-binding sites in p47phax and characterize the effects of PA binding on p47 function and oxidase activation. Similarly, we will identify the oxidase component(s) that bind(s) DG and assess the role of DG-binding in NADPH oxidase activation. These studies should provide new insights into the regulation of NADPH oxidase activation.

描述（由申请人提供）：吞噬细胞呼吸爆发酶， NADPH氧化酶，产生超氧阴离子。其他氧的前体 种（H202，次氯酸）在宿主防御中发挥重要作用， 炎症和/或作为细胞内信号分子。我们的长期 我们的目标是阐明信号通路和生化机制， NADPH氧化酶激活。对完整细胞的研究表明， 第二信使产生的受体启动激活 磷脂酶D（PLD）调节呼吸爆发的激活。PLD 激活增加细胞内磷脂酸（PA）水平， 被细胞转化为二酰基甘油（DG）。PA和DG作为第二个 信使，但参与NADPH氧化酶激活的蛋白质靶点是 不清楚DG激活蛋白激酶C（PKC），与NADPH密切相关 氧化酶活化我们的结果表明PA和DG调节NADPH氧化酶 通过间接和直接机制激活。间接机制包括 PKC亚型和一种新的蛋白激酶（PAPK）的激活。这些蛋白质 激酶磷酸化氧化酶组分p47 phax和p22 phax， 无细胞系统中的氧化酶活化。直接机制包括 PA和DG与氧化酶组分相互作用。这些脂质诱导NADPH 在无细胞系统中，当只有氧化酶蛋白被 因此，磷酸化是不可能的。此外，PA结合至 p47 phax，鉴定该蛋白为PA的潜在功能靶标。我们 建议这些间接（磷酸化依赖）和直接（结合 PA和DG到氧化酶组分）机制介导PLD启动的NADPH氧化酶 activation.以下具体目标旨在检验这一假设 并阐明所涉及的生化机制。在目标1中，我们将测试 PLD引发的NADPH氧化酶活化是否涉及 间接磷酸化依赖性和直接脂质诱导机制。我们 将使用几种方法（鉴定PKC/PAPK亚型， PLD，选择性降低PKC/PAPK表达与反义 寡核苷酸磷酸化位点突变体的功能效应 在目标2中，我们将测试PA和DG是否与 氧化酶蛋白是PLD启动的NADPH氧化酶活化所必需的。我们 将突变p47 phax中的PA结合位点，并表征PA的作用 结合p47功能和氧化酶活化。同样，我们将确定 结合DG的氧化酶组分，并评估DG结合在NADPH中的作用 氧化酶活化这些研究应该提供新的见解， NADPH氧化酶活化的调节。