G Protein Regulation of the Neutrophil NADPH Oxidase

描述（由申请人提供）：吞噬性白细胞形成的活性氧（ROS）在宿主防御中起主要作用，但也使急性和慢性细胞炎症反应持续存在。ROS在非吞噬细胞中的作用包括细胞内信号传导、增殖、凋亡和先天免疫。已经确定，人中性粒细胞ROS生成NADPH氧化酶（Nox 2）的活性受小GT受体Rac 2调节，Rac 2作为粘附中性粒细胞中ROS形成的关键“分子开关”。我们以前的工作提出了一个两步机制，通过该机制，Rac2调节电子转移的过程中，从NADPH分子氧在这个多组分系统。更详细的了解NADPH氧化酶的调控Rac GTdR的分子基础是至关重要的了解ROS形成的生理和病理调节在免疫和非免疫cells.The分子机制调节ROS形成的非吞噬细胞Nox酶仍然不清楚。我们最近的数据表明，Nox 1，血管和胃肠道上皮细胞中丰富的Nox，是由激酶依赖性途径调节。我们已经表明，在各种结肠癌细胞系中，存在活性Src水平与Nox 1活性之间存在相关性。初步数据表明，Src可能通过独特的“组织者”分子起作用，这些分子在结构上与已知的Nox调节蛋白p40、p47和Nox 1相关。我们建议研究Src激酶用于调节Nox 1活性的分子机制，更一般地说，探索Nox调节组织者家族新成员的生物学作用。为了实现这些目标，我们将使用生物化学，遗传学，生物物理学和细胞方法的组合。1.0我们将研究Rac GT3在调节Nox活性中的作用。基于我们对Nox蛋白上的Rac结合位点的初步表征，我们将定义Rac 2在调节对吞噬细胞NADPH氧化酶（phox）功能至关重要的电子转移反应中的活性。将对Nox蛋白上的Rac调控位点进行详细的遗传分析。基于荧光的方法和其他生物物理方法将用于评估Rac GTdR对Nox 2的调节。2.0我们将剖析Src酪氨酸激酶调控Nox 1的分子基础。我们将使用生物化学，细胞和遗传学手段来研究Src介导的NoxA1磷酸化在Nox1调控中的作用。新的p47相关的“组织者”在调节氮氧化物的活动，本地化和对刺激的反应的作用将确定使用生化，细胞，遗传和成像方法。 公共卫生相关性：由NADPH氧化酶形成的活性氧起着重要的生物学作用。我们将研究Rac GT3与NADPH氧化酶（Nox）的结合如何控制它们的活性。Nox 1在肠道和脉管系统中丰富，也受Src激酶调节。我们将研究一组新发现的Src靶蛋白如何组织Nox组装和局部活性。了解氮氧化物的调节将对疾病治疗有重要意义。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) formed by phagocytic leukocytes play a primary role in host defense, but also perpetuate both acute and chronic cellular inflammatory responses. Roles for ROS in non-phagocytic cells include intracellular signaling, proliferation, apoptosis, and innate immunity. It has been established that the activity of the human neutrophil ROS-generating NADPH oxidase (Nox2) is regulated by the small GTPase Rac2, and that Rac2 acts as a key "molecular switch" for ROS formation in adherent neutrophils. Our previous work has suggested a two-step mechanism through which Rac2 regulates the process of electron transfer from NADPH to molecular oxygen in this multi-component system. A more detailed knowledge of the molecular basis for NADPH oxidase regulation by Rac GTPase is critical for understanding the physiological and pathological regulation of ROS formation in both immune- and non-immune cells.The molecular mechanisms regulating ROS formation by non-phagocyte Nox enzymes remain poorly defined. Our recent data indicates that Nox1, an abundant Nox in the vascular and gastrointestinal epithelium, is regulated by kinase-dependent pathways. We have shown that in various colon cancer cell lines there is a correlation between the levels of active Src present and Nox1 activity. Preliminary data suggest that Src may act through unique "organizer" molecules that are structurally related to p40, p47, and Nox1, the known Nox regulatory proteins. We propose to investigate the molecular mechanisms used by Src kinases to regulate Nox1 activity and, more generally, to explore the biological role(s) of the new members of the Nox regulatory organizer family. To accomplish these goals, we will use a combination of biochemical, genetic, biophysical and cellular approaches. 1.0 We will investigate the role of Rac GTPase in regulation of Nox activity. Based upon our initial characterization of a Rac-binding site on Nox proteins, we will define the activity of Rac2 in regulating electron transfer reactions critical to phagocyte NADPH oxidase (phox) function. A detailed genetic analysis of the Rac-regulatory site on Nox protein(s) will be carried out. Fluorescence-based methods and other biophysical approaches will be used to evaluate Nox2 regulation by Rac GTPase. 2.0 We will dissect the molecular basis for Nox1 regulation by Src tyrosine kinase. We will use biochemical, cellular, and genetic means to investigate the role of Src-mediated NoxA1 phosphorylation in Nox1 regulation. The roles of novel p47-related "organizers" in modulating Nox activities, localization, and responsiveness to stimuli will be determined using biochemical, cellular, genetic, and imaging approaches. PUBLIC HEALTH RELEVANCE: The formation of reactive oxygen by enzymes known as NADPH oxidases plays important biological roles. We will investigate how the binding of Rac GTPase to NADPH oxidases (Nox) controls their activity. Nox1, abundant in the gut and vasculature, is also regulated by the Src kinase. We will study how a newly identified group of Src target proteins act to organize Nox assembly and localized activity. Understanding Nox regulation will have important implications for disease therapy.