Molecular Structure of the Phagocyte NADPH Oxidase

吞噬细胞 NADPH 氧化酶的分子结构

• 批准号: 6614243
• 负责人: MARK T QUINN
• 金额: $ 33.71万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6614243
• 关键词: NAD(P)H dehydrogenase; X ray crystallography; affinity labeling; binding proteins; circular dichroism; clinical research; cytochrome b; cytoplasm; enzyme activity; enzyme biosynthesis; enzyme inhibitors; enzyme structure; guanine nucleotide exchange factors; guanosine triphosphate; human tissue; intermolecular interaction; laboratory rabbit; molecular cloning; molecular site; neutrophil; nuclear magnetic resonance spectroscopy; phagocytes; protein protein interaction; protein sequence; synthetic peptide; transcription factor

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O2-需要组装称为NADPH氧化酶的膜结合复合物，其由完整的质膜黄细胞色素B和四种胞质蛋白（p40 phox、p47 phox、p67 phox和Rac 2）组成。虽然已经取得了进展，但关于这些蛋白质中的每一种如何调节活性NADPH氧化酶复合物的形成以及类似地关闭系统的信息有限。在前一个资助期，我们鉴定了NADPH氧化酶蛋白质组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O2生成系统的组装相关。在这个更新的应用程序中，我们提出的研究，调查NADPH氧化酶组装是如何调节。根据研究进行的前一个补助期，其他人的吞噬细胞NADPH氧化酶的研究，和这里描述的初步工作，我们假设，中性粒细胞的氧化酶组装和氧化酶组装本身的能力在几个不同的水平，包括转录和生物合成的phox蛋白，分子组装的活性系统，并在质膜结构域的横向组织进行调节。因此，适当的NADPH氧化酶活性来自于这些多水平调节的整合。由于氧化酶的复杂性，我们建议解决这一假设，主要集中在这个项目的作用，细胞质phox因子在氧化酶的调节。具体地说，该建议描述了以下策略：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质周转的动力学; 4）鉴定和表征结合p67 phox和Rac的黄细胞色素B结构域;和5）开发表达GFP-phox融合蛋白的细胞系，用于分析体内细胞溶质氧化酶蛋白和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我们的理解不同水平的监管输入是如何整合，以促进功能组装和激活的吞噬细胞NADPH氧化酶。