G PROTEIN REGULATION OF THE NEUTROPHIL NADPH OXIDASE

描述(改编自申请人的摘要)：呼吸系统 吞噬细胞的突发性氧合酶产生超氧阴离子， 作为一大群活性氧代谢物的前体 它们被这些细胞用来摧毁入侵的微生物。 NADPH氧化酶的激活和调节是由GTP- 结合蛋白(G蛋白)。申请人已提纯G- 与NADPH组分相互作用的蛋白质(Rap1A和Gox) 氧化物酶。他建议调查这样一种假设，即这些G- 蛋白质是这个重要的中性粒细胞系统的双重调节器。 Rap1A蛋白与细胞色素B558组分相互作用 氧化物酶。申请者将使用分子生物学方法进行探测 这两种蛋白质相互作用的部位。他还将评估 这种相互作用在以下方面的功能意义 多组分氧化酶系统的组装/激活。这个 Rapt A介导cAMP抑制效应的可能性- 依赖的蛋白激酶在超氧化物形成中的作用将被研究。 Gox蛋白将被鉴定和克隆。的功能 刺激氧化酶系统的蛋白质将在体外被排出 纯蛋白重组系统，以及各种突变体 采用重组DNA技术制备。抗体将被用来 评估体内和体外对氧化酶系统的影响。 野生型和突变型G和Gox(及RAP)蛋白在大肠杆菌中的表达 HL6O细胞也将用于评估G-OX在体内的作用。 与低分子量G蛋白相关的辅助分子 (GAP‘s、核苷酸交换因子等)将被确定为Gox 及其对系统调节的影响 调查过了。 拟议的研究将使申请者更好地了解 呼吸爆发氧化酶的调节，应该有 认识慢性肉芽肿的深刻意义 疾病，特别是一般的炎症。它是 预期这些研究可能导致新的治疗方法 慢性炎症性和风湿性疾病。

DESCRIPTION (adapted from the applicant's abstract): The respiratory burst oxiclase of phagocytic cells produces superoxide anion, which serves asa precursor of a large group of reactive oxygen metabolites that are used by these cells to destroy invading microorganisms. Activation and regulation of the NADPH oxidase are mediated by GTP- binding proteins (G-proteins). The applicant has purified the G- proteins (rap1A and Gox) that interact with components of the NADPH oxidase. He proposes to investigate the hypothesis that these G- proteins serve as dual regulators of this crucial neutrophil system. The rap1A protein interacts with the cytochrome B558 component of the oxidase. The applicant will use molecular biological methods to probe the site of interaction of these two proteins. Hewill also assess functional significance of this interaction in terms of assembly/activation of the multicomponent oxidase system. The possibility thatrapt A mediates the inhibitory affects of cAMP- dependent protein kinase on superoxide formation will be investigated. The Gox protein will be characterized and cloned. The function of the protein stimulating the oxidase system will be evacuated using in vitro reconstitutive systems with pure protein, as well as various mutants prepared by recombinant DNA technology. Antibodies will be used to evaluate effects upon the oxidase system in vivo and in vitro. Transfection of wild type and mutant G and Gox (and rap) protein into HL6O cells will be used to assess the role of G-ox in vivo as well. Accessory molecules associated with low molecular weight G proteins (GAP's, nucleotide exchange factors, etc.) will be identified for Gox and rap1Aand their influences upon the regulation of the system investigated. The proposed studies will enable the applicant to better understand the regulation of the respiratory burst oxidase and should have profound implications for understanding chronic granulomatous disease and in particularly for inflammation in general. It is anticipated that these studies might lead to novel therapeutic approaches to chronic inflammatory and rheumatic diseases.