Role of Prdx6 in the activation of NADPH oxidase

描述(由申请人提供)：本提案的目标是评估Prdx6在NADPH氧化酶(NOX2)激活中的新作用。我们的假设是，作为对刺激的反应，Prdx6被磷酸化并移位到质膜，在那里它产生溶血磷脂酰胆碱(LysoPC)，从而激活NOX2。这一假设将通过(共5个)具体目标中的3个来检验。目的1评价Prdx6基因敲除对体外培养的肺泡巨噬细胞(AM)和肺微血管内皮细胞中NOX2激动剂诱导活化的影响。激活是由细胞质成分转移到细胞膜和产生活性氧物种(ROS)来决定的。特定目的2将评估Prdx6介导的NOX2的激活机制；我们认为Prdx6的磷脂酶A2(PLA2)活性产生溶酶PC是相关的。具体目标3将评估Prdx6磷酸化在其转位到质膜和NOX2激活过程中的需求。我们进一步认为，胞浆组分之一(P67Phox)与Prdx6的结合抑制了其PLA2活性，并取消了NOX2激活信号。目的4研究Prdx6在完整细胞内与p67Phox的相互作用以及在体外与重组蛋白的相互作用，重点研究Prdx6的磷脂酶A2活性及其与蛋白质相互作用的动力学。最后，特异靶5将研究Prdx6 PLA2抑制剂MJ33在体内预防离体肺缺血氧化应激和急性肺缺血再灌注损伤中的作用。我们推测，该制剂在保持Prdx6的保护性过氧化物酶活性的同时，抑制了NOX2的激活。这些研究将为研究Prdx6的这一新的作用提供协调的努力，并将为开发新的方法来抑制NOX2酶复合体的激活和改善ROS介导的肺损伤提供基本的见解。 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。一种通过该途径抑制ROS产生的新型无毒药物(MJ33)将被评估其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.