Role of Prdx6 in the activation of NADPH oxidase

Prdx6 在 NADPH 氧化酶激活中的作用

基本信息

批准号：
8212032
负责人：
Aron B. FISHER
金额：
$ 51.41万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8212032
关键词：
Acute Lung Injury Agonist Alveolar Macrophages Angiotensin II Applications Grants Binding Biochemical Pathway Cell membrane Cell-Free System Cells Concanavalin A Cysteine Data Development Endothelial Cells Endothelium Family Generations Goals Grant Human In Vitro Inflammation Inflammatory Injury Ischemia Kinetics Knock-out Knockout Mice Lung Lung diseases Lysophosphatidylcholines Measurement Mediating Membrane Methods Modeling Molecular Multienzyme Complexes Mus NADPH Oxidase Null Lymphocytes Oxidants Oxidative Stress Pathway interactions Peroxidases Phospholipase A2 Phospholipids Phosphorylation Phosphotransferases Physiological Production Proteinase 3 Proteins Publications Reactive Oxygen Species Recombinant Proteins Reperfusion Therapy Role Signal Transduction Stimulus Testing Tissues cell injury cell type in vivo inhibitor/antagonist insight lung injury lung ischemia member neutrophil cytosol factor 67K novel peroxiredoxin phospholipase A2 inhibitor prevent protein protein interaction public health relevance reconstitution response

项目摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to evaluate a novel role for Prdx6 in the activation of NADPH oxidase (NOX2). Our hypothesis is that in response to a stimulus, Prdx6 is phosphorylated and translocates to the plasma membrane where it generates lysophosphatidylcholine (lysoPC) resulting in activation of NOX2. This hypothesis will be tested by 3 (of 5) specific aims. Specific Aim 1 will evaluate the effect of Prdx6 "knockout" on agonist-induced activation of NOX2 in alveolar macrophages (AM) and pulmonary microvascular endothelial cells in culture and in the isolated perfused lung. Activation is determined by translocation of cytosolic components to the membrane and generation of reactive oxygen species (ROS). Specific Aim 2 will evaluate the mechanism for Prdx6-mediated activation of NOX2; we propose that generation of lysoPC by the phospholipase A2 (PLA2) activity of Prdx6 is responsible. Specific Aim 3 will evaluate the requirement for Prdx6 phosphorylation in its translocation to the plasma membrane and NOX2 activation. We further propose that binding of one of the cytosolic components (p67phox) to Prdx6 inhibits its PLA2 activity and abrogates the NOX2-activation signal. Specific Aim 4 will study the interaction of Prdx6 with p67phox in intact cells and with recombinant protein in vitro with specific focus on the PLA2 activity of Prdx6 and the kinetics of the protein-protein interaction. Finally, Specific Aim 5 will investigate the effect of a Prdx6 PLA2 inhibitor, MJ33, in preventing oxidative stress with ischemia in the isolated lung and acute lung injury with ischemia-reperfusion in vivo. We postulate that this agent will maintain the protective peroxidase activity of Prdx6 while inhibiting the activation of NOX2. The proposed studies will provide a coordinated effort to investigate this novel role of Prdx6 and will provide the basic insights for development of new methods to inhibit the activation of the NOX2 enzyme complex and ameliorate ROS-mediated lung injury. PUBLIC HEALTH RELEVANCE: This grant application will evaluate the biochemical pathway associated with the production of strong oxidants (ROS) by lung endothelial and inflammatory cells. A novel and non-toxic agent (MJ33) that inhibits the production of ROS by the pathway will be evaluated for its ability to prevent lung injury associated with ischemia and reperfusion.

描述（由申请人提供）：该提案的目的是评估PRDX6在NADPH氧化酶激活中的新作用（NOX2）。我们的假设是，为了响应刺激，PRDX6被磷酸化，并易位到质膜，在该刺激中产生溶血磷脂酰胆碱（lysopc），从而导致NOX2的激活。该假设将通过3（5个）特定目的进行检验。具体目标1将评估PRDX6“基因敲除”对激动剂诱导的肺泡巨噬细胞（AM）和肺微血管内皮细胞在培养和孤立的灌注肺中的作用。激活是通过将胞质成分转移到膜上和活性氧（ROS）的产生来确定的。具体目标2将评估pRDX6介导的NOX2激活的机制；我们提出，PRDX6的磷脂酶A2（PLA2）活性的产生是负责的。特定的目标3将评估PRDX6磷酸化的需求，以易位到质膜和NOX2激活。我们进一步提出，一种胞质成分（p67phox）的结合以抑制其PLA2活性并消除NOX2激活信号。具体目标4将研究PRDX6与完整细胞中P67Phox的相互作用以及在体外重组蛋白质中的相互作用，并特别关注PRDX6的PLA2活性和蛋白质蛋白质相互作用的动力学。最后，具体的目标5将研究PRDX6 PLA2抑制剂MJ33的作用，以防止分离的肺部缺血和急性肺损伤在体内氧化应激和急性肺损伤。我们假设该药物将在抑制NOX2的激活的同时维持PRDX6的保护性过氧化物酶活性。拟议的研究将提供协调的努力来研究PRDX6的新作用，并为开发新方法提供基本见解，以抑制NOX2酶复合物的激活和改善ROS介导的肺损伤。公共卫生相关性：该赠款应用将评估与肺内皮和炎症细胞产生强氧化剂（ROS）相关的生化途径。一种新型和无毒的剂（MJ33）通过途径抑制ROS产生的一种新型和无毒剂，将评估其防止与缺血和再灌注相关的肺损伤的能力。