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G Protein Regulation of the Neutrophil NADPH Oxidase

G 蛋白对中性粒细胞 NADPH 氧化酶的调节

基本信息

批准号：
7851364
负责人：
Celine DerMardirossian
金额：
$ 72.67万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-09-09 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7851364
关键词：
4-ethoxymethylene-2-phenyl-2-oxazoline-5-one Acute Apoptosis Binding Binding Sites Biochemical Biochemical Genetics Biological Blood Vessels Cells Chronic Data Disease Electron Transport Enzymes Family Fluorescence GTP-Binding Proteins Genetic Goals Guanosine Triphosphate Phosphohydrolases Host Defense Human Activities Image Immune Inflammatory Response Knowledge Leukocytes Mediating Methods Molecular Monomeric GTP-Binding Proteins NADP NADPH Oxidase Natural Immunity Oxygen Pathway interactions Phagocytes Phosphorylation Phosphotransferases Physiological Play Process Proteins Reaction Reactive Oxygen Species Regulation Role Signal Transduction Site Stimulus System Work base colon cancer cell line gastrointestinal epithelium genetic analysis genetic regulatory protein human AKAP13 protein member neutrophil novel public health relevance src-Family Kinases

项目摘要

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.

描述（由申请人提供）：吞噬性白细胞形成的活性氧（ROS）在宿主防御中发挥主要作用，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，人中性粒细胞产生 ROS 的 NADPH 氧化酶 (Nox2) 的活性受到小 GTP 酶 Rac2 的调节，并且 Rac2 充当粘附中性粒细胞中 ROS 形成的关键“分子开关”。我们之前的工作提出了一种两步机制，通过该机制，Rac2 调节该多组分系统中从 NADPH 到分子氧的电子转移过程。更详细地了解 Rac GTPase 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至关重要。非吞噬细胞 Nox 酶调节 ROS 形成的分子机制仍不清楚。我们最近的数据表明，Nox1 是血管和胃肠道上皮细胞中丰富的 Nox，受到激酶依赖性途径的调节。我们已经证明，在各种结肠癌细胞系中，活性 Src 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 p40、p47 和 Nox1（已知的 Nox 调节蛋白）相关。我们建议研究 Src 激酶用于调节 Nox1 活性的分子机制，更广泛地说，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 GTPase 在调节 Nox 活性中的作用。根据我们对 Nox 蛋白上 Rac 结合位点的初步表征，我们将定义 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。对 Nox 蛋白上的 Rac 调节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 GTPase 对 Nox2 的调节。 2.0 我们将剖析 Src 酪氨酸激酶调节 Nox1 的分子基础。我们将利用生化、细胞和遗传学手段研究Src介导的NoxA1磷酸化在Nox1调节中的作用。新型 p47 相关“组织者”在调节 Nox 活性、定位和刺激反应中的作用将通过生化、细胞、遗传和成像方法来确定。公共卫生相关性：NADPH 氧化酶形成的活性氧发挥着重要的生物学作用。我们将研究 Rac GTPase 与 NADPH 氧化酶 (Nox) 的结合如何控制其活性。 Nox1 在肠道和脉管系统中含量丰富，也受到 Src 激酶的调节。我们将研究一组新鉴定的 Src 靶蛋白如何组织 Nox 组装和局部活性。了解 Nox 调节将对疾病治疗产生重要影响。