STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

吞噬细胞蛋白的结构和功能

• 批准号: 3803285
• 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/3803285
• 关键词: DNA virus; NAD(P)H dehydrogenase; bactericidal immunity; cell free system; child (0-11); chronic granulomatous disease; enzyme complex; gene deletion mutation; human subject; membrane proteins; molecular cloning; phagocytes; protein engineering; protein purification; protein structure function; superoxides; transposon /insertion element

The purpose of this project is to explore structure-function relationships in important phagocytic cell proteins. Current efforts are focused on the components of the NADPH oxidase, a system comprised of both membrane bound and soluble proteins. Interest in this system lies at two levels: 1) Studies on the the structure and function of this enzyme complex will address the molecular basis of a critical host defense mechanism of phagocytic white blood cells, since the NADPH oxidase is responsible for the generation of superoxide and its potent microbicidal oxygen metabolites. 2) The activation and assembly of the oxidase is thought to involve mechanisms common to other intercellular signal transduction systems relevant in all cells, since two of the oxidase components contain sequence motifs (SH3 domains) exhibiting significant homology to p60-src and other important intracellular proteins. We have recently cloned full length cDNAs encoding the four of oxidase factors p47-phox, p-67-phox, p22-phox, and gp91-phox) affected in Chronic Granulomatous Disease of Childhood (CGD) within a number of expression vectors. Two of these factors (p47-phox and p67-phox) have been purified to homogeneity from a recombinant baculovirus system, allowing detailed biochemical studies previously not feasible with the native proteins. These recombinant factors were used to restore cell-free NADPH oxidase activity of CGD patient cytosolic preparations to nearly normal levels. While the two recombinant proteins together were not sufficient to reconstitute the NADPH oxidase when mixed with membranes, a third soluble component (P28-phox) was identified and purified from crude cytosol based on its ability to complement the activities of recombinant p47-phox and p67-phox in a cell-free oxidase assay system. Finally, the roles of src-like sequence motifs in p67-phox have been explored by (deletion mutagenesis and reconstitution using the genetically modified recombinant proteins. These src-like sequences do not appear to be critical for oxidase activity, but are thought to function in cellular localization and activation processes.

该项目的目的是探索结构功能 重要的吞噬细胞蛋白中的关系。 当前的努力 专注于NADPH氧化酶的成分，该系统包括 膜结合和可溶性蛋白质的。 对该系统的兴趣 位于两个级别：1）研究的结构和功能 酶复合物将解决关键宿主的分子基础 吞噬白细胞的防御机理，因为NADPH 氧化酶负责产生超氧化物及其有效性 微生物氧代谢产物。 2）激活和组装 氧化酶被认为涉及其他细胞间常见的机制 信号转导系统在所有细胞中相关，因为两个 氧化酶成分包含序列基序（SH3结构域） 与P60-SRC和其他重要细胞内的重要同源性 蛋白质。 我们最近将全长cdnas克隆了编码四个 氧化酶因子p47-phox，p-67-phox，p22-phox和gp91-phox） 受到慢性肉芽肿性儿童期（CGD）的影响 表达向量的数量。 其中两个因素（p47-phox和 p67-phox）已从重组纯化为均匀性 杆状病毒系统，以前允许详细的生化研究 与天然蛋白质可行。 使用这些重组因素 恢复CGD患者的无细胞NADPH氧化酶活性 准备水平几乎正常。 而两个重组 蛋白质不足以重建NADPH氧化酶 当与膜混合时，第三个可溶成分（p28-phox）为 根据其根据其的能力来识别和纯化。 补充重组P47-Phox和P67-Phox的活性 无细胞氧化酶测定系统。 最后，SRC样序列的作用 P67-Phox中的基序已通过（缺失诱变和 使用转基因重组蛋白重建。 这些类似SRC的序列对于氧化酶似乎并不重要 活动，但被认为在细胞定位和 激活过程。