STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

中性粒细胞和其他吞噬血细胞对各种感染或炎症刺激产生高水平的活性氧，这一过程被称为呼吸爆发。这种反应归因于NADPH氧化酶的活性，它利用分子氧和NADPH产生超氧阴离子，超氧阴离子是各种活性氧的前体，在防御微生物感染和炎症中起关键作用。慢性肉芽肿病（CGD）患者具有NADPH氧化酶缺陷，导致对微生物感染的易感性增强和炎症反应失调。本项目旨在探索NADPH氧化酶功能的结构基础和调控呼吸爆发的细胞机制。氧化酶活化是一个循序渐进的过程，从刺激g蛋白偶联受体和激活各种激酶和磷脂酶开始。活性氧化酶的组装涉及几个蛋白组分的磷酸化，这些蛋白组分迁移到特定的膜结构域。我们已经证明p47phox和p67phox中的相互作用位点（SH3结构域）在调节氧化酶组装中发挥核心作用，并通过位点定向诱变几种氧化酶成分（p22、p67、p47和p40phox）来探索蛋白质磷酸化在这一过程中的作用。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。这些研究正在探索通过共同转染几种建议的信号中间体（PI-3激酶、蛋白激酶、磷脂酶D和gtp酶、Arf和Rho）刺激趋化肽或fc - γ受体与呼吸爆发激活之间的联系。关于NADPH氧化酶的结构和功能，以及调节该酶的信号中间体的信息，可能为设计抑制或增强呼吸爆发活动的治疗策略提供基础。在其他研究中，我们正在描述各种其他组织中活性氧的来源，其中氧化剂可以作为促进程序性细胞死亡的信号，基因表达的变化或对生长因子的反应的增殖。使用p47phox缺陷小鼠模型进行的CGD研究表明，p47phox在微细胞和主动脉成纤维细胞释放活性氧化剂中起重要作用；编码p47phox的逆转录病毒的转导恢复了这些细胞中氧化剂的产生。我们还鉴定了几种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组分相互作用的能力来表征这些酶。- NADPH氧化酶，呼吸爆发，慢性肉芽肿病，信号转导，超氧化物，活性氧-人类受试者

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