STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

吞噬细胞蛋白的结构和功能

• 批准号: 3790848
• 负责人: T L LETO
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3790848
• 关键词: NAD(P)H dehydrogenase; biological signal transduction; child (0-11); cytochrome b; electron transport; enzyme reconstitution; enzyme structure; guanine nucleotide binding protein; human subject; human tissue; neutrophil; phagocytes; protein purification; protein structure function; proteins; recombinant DNA; superoxides

This project is exploring structure-function relationships in important phagocytic cell proteins. Current efforts are focused on protein structural requirements for assembly of the NADPH oxidase, a system responsible for production of superoxide and its potent microbicidal metabolites. Using purified recombinant cytosolic oxidase factors p47-phox and p67-phox, biochemical complementation studies in the last year have defined all of the protein components necessary for in vitro oxidase reconstitution. These studies suggest that a membrane associated cytochrome b558 is the sole electron transfer component of the system, while three essential cytosolic proteins (p47-phox, p67-phox, and rac) are likely to serve in oxidase activation through signal transduction mechanisms common to all eukaryotic cells. We have purified two closely related rho subfamily members of GTP binding proteins (rac-2 and CDC42Hs) from neutrophil cytosol based on complemetation assays with recombinant p47 and p67-phox. We subsequently showed that recombinant CDC42Hs was not active and are now exploring the structural basis for the functional differences between these two G-proteins. Structural variants of all three cytosolic components are being produced and characterized with recombinant DNA expression systems. The roles of src homology domains (SH3) present in p47-phox and p67-phox are also being explored through genetic deletion and reconstitution studies. Using engineered forms p67-phox from a baculovirus expression system we have identified a minimal functional domain within the amino-terminal 244 residues of p67-phox and have shown that the SH3 domains are not essential for in vitro reconstitution. Related studies using mammalian vectors are addressing the roles of SH3 domains within intact cells. In separate studies, src homology domains were fused to tag sequences to identify the targets of SH3 domains in peptide and expression libraries and to study their role in subcellular protein translocation.

该项目正在探索重要的结构功能关系 吞噬细胞蛋白。 当前的努力集中在蛋白质上 组装NADPH氧化酶的结构要求，系统 负责产生超氧化物及其有效的杀生性 代谢物。 使用纯化的重组胞质氧化酶因子 p47-phox和p67-phox，最后一次的生化互补研究 一年定义了体外所需的所有蛋白质成分 氧化酶重构。 这些研究表明膜相关 细胞色素B558是系统的唯一电子传递组件， 而三种必需的胞质蛋白（P47-PHOX，P67-PHOX和RAC） 通过信号转导可能在氧化酶激活中使用 所有真核细胞共有的机制。 我们密切净化了两个 GTP结合蛋白（RAC-2和CDC42HS）的相关RHO亚家族成员 基于重组的补充测定法，来自中性粒细胞胞质溶胶 P47和P67-Phox。 随后我们表明重组CDC42HS是 不活跃，现在正在探索功能的结构基础 这两个G蛋白之间的差异。 结构变体 正在生产和表征三个胞质成分 重组DNA表达系统。 SRC同源域的作用 （SH3）在P47-Phox和P67-Phox中的存在也正在通过 遗传缺失和重建研究。 使用工程表格 来自杆状病毒表达系统的p67-phox我们已经确定了 氨基末端244残基中的最小功能结构域 p67-phox并表明SH3域对于IN并不必不可少 体外重构。 使用哺乳动物载体的相关研究是 解决完整细胞中SH3域的作用。 在单独 研究，SRC同源域被融合到标记序列以识别 肽和表达文库中SH3域的目标，并研究 它们在亚细胞蛋白易位中的作用。