G Protein Regulation of the Neutrophil NADPH Oxidase

描述(申请人提供)：由吞噬的白细胞形成的活性氧物种(ROS)在宿主防御中起主要作用，但也维持急性和慢性细胞炎症反应。ROS在非吞噬细胞中的作用包括细胞内信号、增殖、凋亡和先天免疫。已经证实，人中性粒细胞产生ROS的NADPH氧化酶(NOX2)的活性受小GTP酶rac2的调节，而rac2是中性粒细胞中形成ROS的关键分子开关。我们以前的工作提出了一个两步机制，通过这个机制，在这个多组分体系中，rac2调节电子从NADPH到分子氧的转移过程。更详细地了解RAC GTP酶调节NADPH氧化酶的分子基础对于理解免疫和非免疫细胞中ROS形成的生理和病理调节是至关重要的。我们最近的数据表明，Nox1是一种在血管和胃肠道上皮细胞中大量存在的Nox，受依赖于激酶的通路的调节。我们已经证明，在不同的结肠癌细胞系中，活性Src存在的水平与Nox1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”分子发挥作用，这些分子在结构上与已知的NOx调节蛋白p40、p47和Nox1相关。我们建议研究Src激酶调节NOx1活性的分子机制，更广泛地说，探索NOx调控组织者家族的新成员(S)的生物学作用。为了实现这些目标，我们将结合使用生化、遗传、生物物理和细胞方法。1.0我们将研究Rac GTP酶在调节NOx活性中的作用。基于我们对Nox蛋白上RAC结合位点的初步鉴定，我们将确定rac2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(S)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被用来评估RAC GTPase对NOX2的调控。2.0我们将剖析Src酪氨酸激酶调节Nox1的分子基础。我们将使用生化、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“组织者”在调节NOx活性、定位和对刺激的反应中的作用将通过生化、细胞、遗传和成像方法来确定。 与公众健康相关：被称为NADPH氧化酶的酶形成活性氧起着重要的生物学作用。我们将研究RAC GTP酶与NADPH氧化酶(NOx)的结合是如何控制其活性的。Nox1在肠道和血管系统中大量存在，也受Src激酶的调节。我们将研究一组新发现的Src靶蛋白如何作用于组织NOx组装和局部活性。了解NOx的调控机制将对疾病治疗具有重要意义。

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.