Molecular Structure of the Phagocyte NADPH Oxidase

描述(申请人提供)：吞噬细胞产生02-需要组装一个称为NADPH氧化酶的膜结合复合体，它由一个完整的质膜黄色素b和四个胞浆蛋白(p40Phox、p47Phox、p67Phox和rac2)组成。虽然已经取得了进展，但关于这些蛋白质如何调控活性NADPH氧化酶复合体的形成以及类似地是什么关闭了该系统，信息有限。在之前的资助期间，我们确定了NADPH氧化酶蛋白质组分之间的一些蛋白质-蛋白质相互作用，并研究了这些相互作用与活性02生成系统的组装之间的关系。在这一更新应用中，我们建议研究NADPH氧化酶组装是如何调节的。基于前一次资助期间进行的研究、其他人对吞噬细胞NADPH氧化酶的研究以及本文描述的初步工作，我们假设中性粒细胞组装氧化酶的能力和本身在几个不同的水平上受到调节，包括Phox蛋白的转录和生物合成，活性系统的分子组装，以及质膜区的横向组织。因此，适当的NADPH氧化酶活性来自于这些多个水平的调控的整合。由于氧化酶的复杂性质，我们建议通过将这个项目主要集中在胞质中的Phox因子在氧化酶调节中的作用来解决这一假说。具体地说，这项建议描述了以下策略：1)鉴定p67Phox的启动子并确定哪些转录因子调控p67Phox的转录；2)鉴定胞液Phox蛋白的基因表达；3)分析胞液Phox蛋白的生物合成以确定这些蛋白质的相对稳定性和蛋白质周转的动力学；4)鉴定和鉴定与p67 Phox和Rac结合的黄细胞色素b结构域；以及5)建立表达GFP-Phox融合蛋白的细胞系，用于分析胞液氧化酶蛋白和体内氧化酶复合体之间的分子间相互作用。这些研究的结果将进一步加深我们对不同水平的调控输入如何整合以促进吞噬细胞NADPH氧化酶的功能组装和激活的理解。

DESCRIPTION (provided by applicant): The generation of 02- by phagocytic cells requires the assembly of a membrane-bound complex known as the NADPH oxidase, which is composed of an integral plasma membrane flavocytochrome b and four cytosolic proteins (p40 phox, p47 phox, p67 phox, and Rac2). Although progress has been made, there is limited information about how each of these proteins regulate formation of the active NADPH oxidase complex and, similarly, what turns off the system. In the previous grant period, we identified a number of protein-protein interactions among the NADPH oxidase protein components and investigated how these interactions related to assembly of the active 02- generating system. In this renewal application, we propose studies to investigate how NADPH oxidase assembly is regulated. Based on studies performed under the previous grant period, studies by others on the phagocyte NADPH oxidase, and preliminary work described here, we hypothesize that neutrophil capacity for oxidase assembly and oxidase assembly per se are regulated at several different levels, including transcription and biosynthesis of the phox proteins, molecular assembly of the active system, and lateral organization in plasma membrane domains. Consequently, appropriate NADPH oxidase activity results from integration of these multiple levels of regulation. Because of the complex nature of the oxidase, we propose to address this hypothesis by focusing this project primarily on the role of the cytosolic phox factors in oxidase regulation. Specifically, this proposal describes strategies for: 1) characterizing the promoter for p67 phox and determining what transcription factors regulate p67 phox transcription; 2) characterizing cytosolic phox protein gene expression; 3) analyzing biosynthesis of the cytosolic phox proteins to determine relative stability of these proteins and kinetics of protein turnover; 4) identifying and characterizing flavocytochrome b domains that bind p67 phox and Rac; and 5) develop cell lines expressing GFP-phox fusion proteins for use analysis of the intermolecular interactions between cytosolic oxidase proteins and the oxidase complex in vivo. The results of these studies will further our understanding of how distinct levels of regulatory input are integrated to facilitate functional assembly and activation of the phagocyte NADPH oxidase.