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.
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