MOLECULAR STRUCTURE OF THE PHAGOCYTE NADPH OXIDASE

吞噬细胞 NADPH 氧化酶的分子结构

• 批准号: 2769599
• 负责人: MARK T QUINN
• 金额: $ 16.53万
• 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 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2769599
• 关键词: NAD(P)H dehydrogenase; X ray crystallography; affinity labeling; binding proteins; cell free system; circular dichroism; cytochrome b; cytoplasm; enzyme activity; enzyme biosynthesis; enzyme inhibitors; enzyme structure; epitope mapping; guanine nucleotide exchange factors; guanosine triphosphate; human tissue; hybrid cells; intermolecular interaction; molecular cloning; molecular site; neutrophil; nuclear magnetic resonance spectroscopy; protein sequence; site directed mutagenesis; synthetic peptide

DESCRIPTION: (Adapted from investigator's abstract) This project examines the molecular basis for assembly of NADPH oxidase, a system responsible for generation of reactive microbicidal oxidants which can cause tissue damage at sites of inflammation. This enzyme is comprised of both membrane-bound and cytosolic components that assemble into a membrane-bound complex during oxidase activation. The applicant describes a variety of strategies for exploring the central hypothesis proposing that a series of specific protein-protein interactions occurs between oxidase components and that flavocytochrome b558 acts as a final docking site where the active enzyme assembles. Proposed experimental approaches include: 1) determination of primary sequences in cytosolic oxidase components that bind to cytochrome b558 by phage display and two-hybrid screening protocols, photoaffinity labelling and site-directed mutagenesis; 2) mapping of complementary interacting sites on the cytochrome by similar approaches; 3) studies on interactions of the GTP-binding oxidase component, Rac, with other cytosolic components and the role of the guanine nucleotide exchange inhibitor, GDI; 4) mapping of sites of interactions between the cytosolic components; 5) structural studies on oxidase proteins bound to synthetic peptides, as models of the complexes formed with other oxidase components; 6) attempts to crystallize the cytosolic components p47- and p67-phox, which would be followed by x-ray diffraction studies. These studies would provide the basis for a better understanding of the structure of the active enzyme and regulation of its assembly and may eventually lead to development of therapeutic strategies aimed at preventing tissue damage associated with overproduction of oxidants in inflammatory diseases.

描述：（改编自研究者摘要）本项目研究 NADPH氧化酶组装的分子基础，NADPH氧化酶是一种负责 产生可导致组织损伤的活性杀微生物氧化剂 在炎症部位。 这种酶由两种膜结合的 和细胞溶质成分，组装成一个膜结合的复合物， 氧化酶活化 申请人描述了多种策略， 探索中心假设，提出了一系列具体的 蛋白质-蛋白质相互作用发生在氧化酶组分之间， 黄细胞色素B558作为活性酶 集合。 建议的实验方法包括：1）测定 与细胞色素结合的细胞溶质氧化酶组分中的一级序列 b558噬菌体展示和双杂交筛选方案， 标记和定点诱变; 2）互补基因定位 通过类似的方法研究细胞色素上的相互作用位点; 3）研究 GTP结合氧化酶组分Rac与其他细胞溶质的相互作用 鸟苷酸交换抑制剂（GDI）的成分和作用; 4)胞质组分之间相互作用位点的作图; 5） 结合合成肽的氧化酶蛋白的结构研究，如 与其他氧化酶组分形成的复合物的模型; 6）试图 使胞质组分p47-和p67-phox结晶， 随后进行X射线衍射研究。 这些研究将提供 更好地了解活性酶结构的基础， 其装配的监管，并可能最终导致发展， 治疗策略旨在预防与 炎症性疾病中氧化剂的过量产生。