STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

吞噬细胞蛋白的结构和功能

• 批准号: 6288893
• 负责人: THOMAS 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/6288893
• 关键词: NAD(P)H dehydrogenase; bacterial disease; binding proteins; child (0-11); chimeric proteins; chronic granulomatous disease; disease /disorder proneness /risk; enzyme activity; enzyme inhibitors; enzyme structure; gene mutation; human genetic material tag; human subject; human tissue; inflammation; leukocyte oxidative burst; molecular cloning; phagocytes; protein structure function; tissue /cell culture; transfection

Neutrophils and other phagocytic blood cells generate high levels of reactive oxygen species in response to a variety of infectious or inflammatory stimuli in a process known as the respiratory burst. This response is attributed to activity of NADPH oxidase, which uses molecular oxygen and NADPH to produce superoxide anion, a precursor of various reactive oxygen species that have key roles in defense against microbial infections and inflammation. Patients with chronic granulomatous disease (CGD) have NADPH oxidase deficiencies that result in enhanced susceptibility to microbial infections and dysregulated inflammatory responses. This project is exploring the structural basis for NADPH oxidase function and the cellular mechanisms underlying regulation of the respiratory burst. Oxidase activation is a stepwise process that begins with stimulation of G-protein-coupled receptors and activation of various kinases and phospholipases. Active oxidase assembly involves phosphorylation of several protein components that transmigrate to specific membrane domains. We have shown that interacting sites (SH3 domains) within p47phox and p67phox play a central role in regulating oxidase assembly and are exploring roles of protein phosphorylation in this process through site-directed mutagenesis of several oxidase components (p22, p67, p47, and p40phox). In work aimed at defining cellular signals that trigger oxidase activation, we have reconstituted receptor-mediated activation of the respiratory burst in transfected K562 cells. These studies are exploring links between stimulation of chemotactic peptide or Fc-gamma receptors and activation of the respiratory burst by co-transfection of several proposed signaling intermediates (PI-3 kinase, protein kinases, phospholipase D, and the GTPases, Arf and Rho). Information on the structure and function of NADPH oxidase, as well as the signaling intermediates that modulate this enzyme, may provide a basis for therapeutic strategies designed to either inhibit or enhance respiratory burst activity. In other studies we are characterizing sources of reactive oxygen species in a variety of other tissues where oxidants can serve as signals promoting programmed cell death, changes in gene expression, or proliferation in response to growth factors. Studies using a p47phox-deficient mouse model for CGD indicate an essential role for p47phox in the release of reactive oxidants by microgial cells and aortic fibroblasts; transduction by p47phox- encoding retrovirus restores oxidant production in these cells. We have also identified three distinct gp91phox homologues produced in several tissues and will characterize these enzymes by assessing their abilities to interact with other oxidase components. - NADPH oxidase, respiratory burst, chronic granulomatous disease, signal transduction, superoxide, reactive oxygen species - Human Subjects

在称为呼吸爆发的过程中，中性粒细胞和其他吞噬血细胞对各种感染性或炎症刺激产生高水平的活性氧。该反应归因于NADPH氧化酶的活性，该酶的活性使用分子氧和NADPH产生超氧化阴离子，这是各种活性氧的前体，在防御微生物感染和炎症中具有关键作用。患有慢性肉芽肿性疾病（CGD）的患者患有NADPH氧化酶缺陷，从而增强对微生物感染的易感性和失调的炎症反应。该项目正在探索NADPH氧化酶功能的结构基础和呼吸爆发调节的细胞机制。氧化酶活性是一个逐步过程，始于刺激G蛋白偶联受体和各种激酶和磷脂酶的激活。活性氧化酶的组装涉及几种将迁移到特定膜结构域的蛋白质成分的磷酸化。我们已经表明，p47phox和p67phox内的相互作用位点（SH3结构域）在调节氧化酶的组装中起着核心作用，并正在通过几种氧化酶组分的位置定向诱变在此过程中探索蛋白质磷酸化的作用（p22，p67，p47，p47和p40phox）。在旨在定义触发氧化酶激活的细胞信号的工作中，我们在转染的K562细胞中重构了受体介导的呼吸爆发激活。这些研究是探索趋化肽或FC-γ受体的刺激之间的联系，以及通过共转染几种建议的信号中间体（PI-3激酶，蛋白激酶，磷脂酶D，磷脂酶D和GTPases，ARF和RHO），通过共转染呼吸道破裂。 NADPH氧化酶的结构和功能以及调节该酶的信号传导中间体的信息可能为抑制或增强呼吸爆发活性的治疗策略提供基础。在其他研究中，我们正在表征许多其他组织中活性氧的来源，在这些组织中，氧化剂可以用作促进程序性细胞死亡的信号，基因表达的变化或响应生长因子的响应。使用P47Phox缺乏小鼠模型进行CGD的研究表明，P47Phox在微型细胞和主动脉成纤维细胞释放反应性氧化剂中的重要作用。 P47Phox-编码逆转录病毒的转导可恢复这些细胞中的氧化剂产生。我们还确定了在几个组织中产生的三种不同的GP91Phox同源物，并将通过评估它们与其他氧化酶成分相互作用的能力来表征这些酶。 - NADPH氧化酶，呼吸爆发，慢性肉芽肿性疾病，信号转导，超氧化物，活性氧 - 人类受试者