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.
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