G PROTEIN REGULATION OF THE NEUTROPHIL NADPH OXIDASE

吞噬细胞的呼吸爆发氧化酶产生超氧化物 作为一大群活性氧的前体的阴离子 这些细胞用来破坏入侵的代谢物 微生物。NADPH氧化酶的激活和调节是 由GTP结合蛋白(G蛋白)介导。我们已经提纯了G 与NADPH组分相互作用的蛋白质(Rap1A和Gox) 氧化物酶。我们建议研究一种假设，即这些G蛋白 作为这一关键的中性粒细胞系统的双重调节者。The Rap1a 蛋白质与氧化酶的细胞色素b558成分相互作用。我们 将使用分子生物学方法来探测相互作用的位置 这两种蛋白质。我们还将评估这一功能的意义 多组分氧化酶组装/激活中的相互作用 系统。RAP1a介导抑制血管紧张素转换酶 CAMP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进行评估 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Gox(和RAP)蛋白转染HL-60细胞 也将被用来评估Gox在体内的作用。监管 与低分子量G蛋白(GAP‘s， 核苷酸交换因子等。)将被确定为Gox和Rap1A以及 它们对所调查的系统的监管的影响。建议数 研究将使我们能够更好地了解呼吸的调节 猝发氧化酶，应该对理解 尤其是慢性肉芽肿性疾病，以及 将军。预计这些研究将导致小说的产生 慢性炎症性和风湿性疾病的治疗方法。

The respiratory burst oxidase of phagocytic cells produces superoxide anion, which serves as 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). We have purified the G proteins (rap1A and Gox) that interact with components of the NADPH oxidase. We propose to investigate the hypothesis that these G proteins serve as dual regulators of this crucial neutrophil system. The rap1A protein interacts with the cytochrome b 558 component of the oxidase. We will use molecular biological methods to probe the site of interaction of these two proteins. We will also assess functional significance of this interaction in terms of assembly/activation of the multicomponent oxidase system. The possibility that rap1A mediates the inhibitory effects of cAMP-dependent protein kinase on superoxide formation will be investigated. The Gox protein will be characterized and cloned. The function of the protein in stimulating the oxidase system will be evaluated 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 Gox (and rap) protein into HL-60 cells will be used to assess the role of Gox in vivo as well. Regulatory molecules associated with low molecular weight G proteins (GAP's, nucleotide exchange factors, etc.) will be identified for Gox and rap1A and their influences upon regulation of the system investigated. The proposed studies will enable us to better understand the regulation of respiratory burst oxidase, and should have profound implications for understanding chronic granulomatous disease in particular, and for inflammation in general. It is anticipated that these studies should lead to novel therapeutic approaches to chronic inflammatory and rheumatic diseases.