Molecular Structure of the Phagocyte NADPH Oxidase

吞噬细胞 NADPH 氧化酶的分子结构

基本信息

批准号：
7216290
负责人：
MARK T QUINN
金额：
$ 29.49万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-09-30 至 2009-03-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：吞噬细胞产生 02- 需要组装称为 NADPH 氧化酶的膜结合复合物，该复合物由完整的质膜黄细胞色素 b 和四种胞浆蛋白（p40 phox、p47 phox）组成、p67 phox 和 Rac2）。尽管已经取得了进展，但关于这些蛋白质如何调节活性 NADPH 氧化酶复合物的形成以及类似地如何关闭该系统的信息有限。在之前的资助期间，我们确定了 NADPH 氧化酶蛋白质成分之间的许多蛋白质-蛋白质相互作用，并研究了这些相互作用如何与活性 02 生成系统的组装相关。在此更新应用中，我们提出研究来调查 NADPH 氧化酶组装是如何调节的。根据之前资助期间进行的研究、其他人对吞噬细胞 NADPH 氧化酶的研究以及此处描述的初步工作，我们假设中性粒细胞氧化酶组装的能力和氧化酶组装本身在几个不同的水平上受到调节，包括转录和生物合成phox 蛋白、活性系统的分子组装以及质膜域的横向组织。因此，适当的 NADPH 氧化酶活性是这些多级调节整合的结果。由于氧化酶的复杂性，我们建议通过将该项目主要集中在细胞质 phox 因子在氧化酶调节中的作用来解决这一假设。具体来说，该提案描述了以下策略：1）表征 p67 phox 的启动子并确定哪些转录因子调节 p67 phox 转录； 2)表征细胞质phox蛋白基因表达； 3) 分析胞质phox蛋白的生物合成以确定这些蛋白的相对稳定性和蛋白周转动力学； 4) 鉴定和表征结合 p67 phox 和 Rac 的黄素细胞色素 b 结构域； 5) 开发表达 GFP-phox 融合蛋白的细胞系，用于分析胞质氧化酶蛋白与体内氧化酶复合物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如何整合不同水平的调节输入以促进吞噬细胞 NADPH 氧化酶的功能组装和激活的理解。