Role of Prdx6 in the activation of NADPH oxidase

描述（由申请人提供）：本提案的目的是评估Prdx6在NADPH氧化酶（NOX2）激活中的新作用。我们的假设是，在对刺激的反应中，Prdx6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NOX2的激活。这个假设将通过3个（或5个）具体目标来检验。特异性目的1将评估Prdx6“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中NOX2在培养和离体灌注肺中的激活作用。激活是由细胞质成分向膜的易位和活性氧（ROS）的产生决定的。特异性目的2将评估prdx6介导的NOX2激活的机制；我们提出，通过Prdx6的磷脂酶A2 （PLA2）活性产生lysoPC是负责任的。特异性Aim 3将评估Prdx6转运到质膜和NOX2激活过程中磷酸化的必要性。我们进一步提出其中一种细胞质成分（p67phox）与Prdx6结合可抑制其PLA2活性并消除nox2激活信号。Specific Aim 4将在完整细胞中研究Prdx6与p67phox的相互作用，并在体外研究Prdx6与重组蛋白的相互作用，重点研究Prdx6的PLA2活性以及蛋白-蛋白相互作用的动力学。最后，Specific Aim 5将研究Prdx6 PLA2抑制剂MJ33在体内对离体肺缺血氧化应激和急性肺损伤缺血再灌注的预防作用。我们假设该制剂将维持Prdx6的保护性过氧化物酶活性，同时抑制NOX2的激活。拟议的研究将为研究Prdx6的新作用提供协调的努力，并将为开发抑制NOX2酶复合物激活和改善ros介导的肺损伤的新方法提供基本见解。

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.