Role of Prdx6 in the activation of NADPH oxidase

描述（由申请方提供）：本提案的目的是评价Prdx 6在NADPH氧化酶（NOX 2）活化中的新作用。我们的假设是，响应于刺激，Prdx 6被磷酸化并易位到质膜，在那里它产生溶血磷脂酰胆碱（lysoPC），导致NOX 2的激活。将通过3个（共5个）具体目标来检验这一假设。具体目标1将评估Prdx 6“敲除”对激动剂诱导的培养物和分离的灌注肺中的肺泡巨噬细胞（AM）和肺微血管内皮细胞中的NOX 2活化的影响。激活是由胞质组分转移到膜和活性氧（ROS）的产生。具体目标2将评估Prdx 6介导的NOX 2活化的机制;我们认为Prdx 6的磷脂酶A2（PLA 2）活性产生lysoPC是负责的。具体目标3将评价Prdx 6磷酸化在其转运至质膜和NOX 2活化中的需求。我们进一步提出，结合的胞质组分之一（p67 phox）Prdx 6抑制其PLA 2活性和废除的NOX 2激活信号。具体目标4将研究Prdx 6与完整细胞中的p67 phox以及与体外重组蛋白的相互作用，特别关注Prdx 6的PLA 2活性和蛋白质-蛋白质相互作用的动力学。最后，具体目标5将研究Prdx 6 PLA 2抑制剂MJ 33在预防离体肺中的缺血性氧化应激和体内缺血-再灌注性急性肺损伤中的作用。我们推测，这种试剂将保持Prdx 6的保护性过氧化物酶活性，同时抑制NOX 2的活化。拟议的研究将提供一个协调的努力来研究Prdx 6的这种新作用，并将为开发新方法提供基本见解，以抑制NOX 2酶复合物的激活并改善ROS介导的肺损伤。 公共卫生相关性：这项拨款申请将评估与肺内皮细胞和炎症细胞产生强氧化剂（ROS）相关的生化途径。将评价一种通过该途径抑制ROS产生的新型无毒剂（MJ 33）预防与缺血和再灌注相关的肺损伤的能力。

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.