Structure/Function Analysis of Phagocyte Proteins

描述（由申请人提供）：产生超氧自由基的吞噬细胞呼吸爆发氧化酶在宿主防御和炎症反应中起核心作用。活性氧化酶复合物的组装需要膜蛋白和细胞质蛋白的参与，并由小gtp酶调节。吞噬细胞特异性b型黄细胞色素异二聚体是一种完整的膜蛋白复合物，位于质膜和中性粒细胞特异性颗粒中，是氧化酶组装的焦点，并且包含黄素和血红素氧化还原中心，用于将电子从NADPH转移到分子氧。氧化酶蛋白的遗传缺陷，包括两种黄细胞色素亚基，导致慢性肉芽肿病（CGD），一种以无呼吸爆发、反复感染和慢性肉芽肿为特征的综合征。各种氧化酶亚基与活性NADPH氧化酶复合物组装之间的结构和功能关系尚不完全清楚。这种低电位黄细胞色素由gp91phox和p22phox组成，gp91phox是一种91 kda的糖蛋白，由x连锁CGD的突变位点x连锁基因编码，p22phox是一种来自常染色体CGD位点的非糖基化肽。建议的研究采用遗传学方法来研究氧化酶黄细胞色素b的结构和功能及其作为活性NADPH氧化酶组装的焦点的作用，特别强调吞噬诱导的氧化酶活化。该项目有两个具体目标，将在原代吞噬细胞、吞噬细胞系和我们开发的表达功能性重组氧化酶亚基和吞噬受体的异源细胞模型中进行。首先，研究旨在确定gp91phox和p22phox中参与黄细胞色素b生物合成、吞噬过程中运输和NADPH氧化酶功能的功能域。P22phox包含2个功能未知的胞内结构域，将成为特别关注的焦点。其次，将研究吞噬体上的氧化酶激活，重点关注黄细胞色素b与可溶性调节phox亚基（包括p40phox）之间的相互作用，我们最近发现p40phox对模型细胞系统中fcyr诱导的氧化酶激活至关重要，以及特定的信号级联如何激活这些相互作用。这些研究将进一步深入了解吞噬细胞的超氧化物生成系统，这可能会导致在炎症反应和宿主防御中调节超氧化物形成的新方法。

DESCRIPTION (provided by applicant): The phagocyte respiratory burst oxidase that generates the superoxide radical plays a central role in host defense and the inflammatory response. Assembly of the active oxidase complex requires the participation of both membrane and cytosolic proteins, and is regulated by small GTPases. A phagocyte-specific b-type flavocytochrome heterodimer, an integral membrane protein complex located in the plasma membrane and, in neutrophils, specific granules, is the focal point for oxidase assembly, and contains both the flavin and heme redox centers for transfer of electrons from NADPH to molecular oxygen. Genetic defects in oxidase proteins, including the two flavocytochrome subunits, result in chronic granulomatous disease (CGD), a syndrome characterized by an absent respiratory burst, recurrent infections, and chronic granulomas. The structural and functional relationships between the various oxidase subunits and the assembly of the active NADPH oxidase complex remain incompletely understood. This low-potential flavocytochrome is comprised of gp91phox, a 91-kDa glycoprotein encoded by an X-linked gene that is the site of mutations in X-linked CGD, and p22phox, a non-glycosylated peptide derived from an autosomal CGD locus. The proposed studies take a genetic approach to investigating the structure and function of the oxidase flavocytochrome b and its role as a focal point for assembly of the active NADPH oxidase, with a particular emphasis on phagocytosis-induced oxidase activation. The project has 2 specific aims, will be performed in primary phagocytes, phagocyte cell lines, and heterologous cell models we developed for expression of functional recombinant oxidase subunits and phagocytic receptors. First, studies aimed at identifying functional domains in gp91phox and p22phox that contribute to flavocytochrome b biosynthesis, trafficking during phagocytosis, and NADPH oxidase function will be pursued. p22phox, which contains 2 intracellular domains of largely unknown function, will be a particular focus. Second, oxidase activation on phagosomes will be examined, focusing on interactions between flavocytochrome b and the soluble regulatory phox subunits, including p40phox, which we recently showed to be critical for FcyR-induced oxidase activation in a model cell system, and how specific signaling cascades activate these interactions. These studies will provide further insight into the superoxide- generating system of the phagocyte, which may lead to new approaches in modulating superoxide formation in the inflammatory response and host defense.