Regulation of superoxide production by human neutrophils

说明(申请人提供)：产生超氧化物的NADPH氧化酶是一个复杂的、多组分的系统，在未受刺激的中性粒细胞中以休眠的形式存在，但在细胞暴露于广泛的刺激后被激活。超氧化物及其衍生的活性物种通常在杀死病原体方面起到有益的作用，但在炎症过程中会对正常组织造成严重损害。例如，在成人呼吸窘迫综合征中，肺损伤是由于吞噬细胞氧化剂的过度产生，而在慢性肉芽肿疾病(CGD)中，无法产生这些分子会导致危及生命的感染。这项拟议研究的长期目标是：1)阐明调节NADPH氧化酶活性的生化机制；2)开发能够调节这一活性的药物。在过度炎症的临床情况下，抑制这种活性的药物可能具有治疗作用。相反，通过提高中性粒细胞的抗菌效率来增强NADPH-氧化酶活性的药物可能对患有压倒性感染的患者有利。这个实验室已经开发了几种关键的方法来研究这个问题。一个完全可溶的无细胞系统将被用来从高度纯化和重组的氧化酶蛋白中重建氧化酶活性，以确定它们如何调节NADPH氧化酶。一组&gt；300名CGD患者具有包含四种氧化酶成分的各种突变，他们的特征已被确定，并可用于进一步研究。无细胞系统使研究这些患者表达的突变蛋白的功能缺陷成为可能。此外，还将使用在哺乳动物细胞系中表达的Phox蛋白的定点突变形式进行研究。多克隆和单抗将用于表面等离子体共振、免疫沉淀、抑制和NADPH氧化酶组分的拓扑研究。以下具体目标旨在关注目前阻碍该项目长期目标的几个主要概念和技术缺陷：1)确定胞浆因子及其辅助因子如何调节NADPH氧化酶的活性；2)研究未刺激、启动和激活的中性粒细胞中胞液因子和膜因子及其辅助因子的分子相互作用；3)鉴定和表征NADPH氧化酶组分的突变，以了解它们是如何导致CGD的。这些研究可能最终导致控制氧自由基介导的组织损伤的新的药理学方法以及对CGD的病理生理学的更好的理解。

DESCRIPTION (provided by applicant): The superoxide-generating NADPH oxidase is a complex, multicomponent system that is present in a dormant form in unstimulated neutrophils, but is activated after exposure of the cells to a wide range of stimuli. Superoxide, and the reactive species derived from it, generally serve a beneficial role in killing pathogenic microbes, but can cause serious damage to normal tissues during inflammation. For example, in adult respiratory distress syndrome, pulmonary damage results from the excessive production of phagocyte oxidants, while in chronic granulomatous disease (CGD), failure to produce these molecules leads to life-threatening infections. The long-term objectives of the proposed research are: 1) to elucidate the biochemical mechanisms that regulate NADPH oxidase activity; and 2) to develop drugs capable of modulating this activity. In clinical situations where there is excessive inflammation, agents that suppress this activity may be of therapeutic use. Conversely, drugs that augment NADPH-oxidase activity in a way that improves the antimicrobial efficiency of the neutrophil could be advantageous to patients suffering from overwhelming infections. Several key methods have been developed in this laboratory to study this problem. A fully soluble cell-free system will be used to reconstitute oxidase activity from highly purified and recombinant oxidase proteins to determine how they regulate NADPH oxidase. A group of >300 CGD patients who have a variety of mutations encompassing four of the oxidase components have been characterized and are available for further studies. The cell-free system has made it possible to study the functional defects in the mutant proteins expressed by these patients. In addition, studies will be performed using site-directed mutant forms of phox proteins expressed in mammalian cell lines. Polyclonal and monoclonal antibodies will be used in surface plasmon resonance, immunoprecipitation, inhibition and topological studies of NADPH oxidase components. The following specific aims are designed to focus on several of the major conceptual and technical deficiencies that currently impede the long-range goals of this project: 1) To determine how the cytosolic factors and their cofactors regulate the activity of NADPH oxidase; 2) To study the molecular interactions of the cytosolic and membrane factors and their cofactors in unstimulated, primed and activated neutrophils; 3) To identify and characterize mutations in NADPH oxidase components to understand how they result in CGD. These studies may ultimately lead to new pharmacological methods for controlling oxygen radical-mediated tissue damage as well as an improved understanding of the pathophysiology of CGD.