STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

中性粒细胞和其他吞噬血细胞 产生高水平的活性氧物质， 各种感染性或炎症性刺激， 呼吸爆发这种活性归因于一种酶 一种叫做NADPH氧化酶的复合物，它利用分子氧和 NADPH产生超氧阴离子，杀微生物剂的前体 氧化剂这些氧化剂在宿主防御中起着关键作用， 微生物感染，并作为炎症信号的介质。 慢性肉芽肿病患者有NADPH氧化酶 导致对微生物的敏感性增强的缺陷 感染和炎症反应失调。这个项目是 探索NADPH氧化酶功能的结构基础， 呼吸爆发调节的细胞机制。 氧化酶激活是一个逐步的过程， G蛋白偶联受体的刺激和 各种激酶和磷脂酶。活性氧化酶组装体 涉及几种蛋白质成分的磷酸化， 迁移到特定的膜结构域。我们已经证明 p47phox和p67phox内的相互作用位点（SH3结构域）发挥了 在调节氧化酶组装中的重要作用，这些结构域 既能抑制又能促进活性氧化酶的形成 复杂.抑制机制包括：1）分子内SH3 p47和p67phox之间的接触，它们维持这些蛋白质在 非活性构象，2）p40phox的竞争性结合，另一个 含SH3结构域的胞质因子，和3）抑制 rac激活的激酶PAK2，其通过以下途径下调氧化酶： 在磷酸化依赖性过程中结合p47phox。的 膜结合氧化酶复合物的形成是必需的， 氧化酶激活，但不充分，因为细胞缺乏 胞质磷脂酶A2（p85）将组装氧化酶 复杂，但不产生超氧化物，除非花生四烯酸是 提供了在相关工作中，我们正在研究 磷脂酶D及其产物在吞噬细胞活化中的作用。 NADPH氧化酶的结构和功能的信息可能 为治疗策略提供了基础， 或增强呼吸爆发活动。

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 activity is attributed to an enzyme complex called NADPH oxidase, which uses molecular oxygen and NADPH to produce superoxide anion, a precursor of microbicidal oxidants. These oxidants play a key role in host defense against microbial infections and serve as mediators of inflammatory signals. Patients with chronic granulomatous disease have NADPH oxidase deficiencies which 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 that these domains can both inhibit and promote formation of the active oxidase complex. Inhibitory mechanisms include: 1) intramolecular SH3 contacts within p47 and p67phox, which maintain these proteins in inactive conformations, 2) competitive binding of p40phox, another SH3 domain-containing cytosolic factor, and 3) inhibition by a rac-activated kinase, PAK2, which down-regulates the oxidase by binding p47phox in a phosphorylation-dependent process. The formation of the membrane-bound oxidase complex is necessary for oxidase activation, but not sufficient, since cells deficient in cytosolic phospholipase A2 (p85) will assemble the oxidase complex but not produce superoxide unless arachidonic acid is provided. In related work we are examining the role of phospholipase D and its products in phagocyte activation. Information on the structure and function of NADPH oxidase may provide a basis for therapeutic strategies designed to either inhibit or enhance respiratory burst activity.