This project examines the molecular basis for assembly of NADPH oxidase, a system responsible for generation of reactive microbicidal oxidants which can cause tissue damage at sites of inflammation. This enzyme is comprised of both membrane-bound and cytosolic components that assemble into a membrane-bound complex during oxidase activation. The applicant describes a variety of strategies for exploring the central hypothesis proposing that a series of specific protein-protein interactions occurs between oxidase components and that flavocytochrome b558 acts as a final docking site where the active enzyme assembles. Proposed experimental approaches include: 1) determination of primary sequences in cytosolic oxidase components that bind to cytochrome b558 by phage display and two-hybrid screening protocols, photoaffinity labelling and site-directed mutagenesis; 2) mapping of complementary interacting sites on the cytochrome by similar approaches; 3) studies on interactions of the GTP-binding oxidase component, Rac, with other cytosolic components and the role of the guanine nucleotide exchange inhibitor, GDI; 4) mapping of sites of interactions between the cytosolic components; 5) structural studies on oxidase proteins bound to synthetic peptides, as models of the complexes formed with other oxidase components; 6) attempts to crystallize the cytosolic components p47- and p67-phox, which would be followed by x-ray diffraction studies. These studies would provide the basis for a better understanding of the structure of the active enzyme and regulation of its assembly and may eventually lead to development of therapeutic strategies aimed at preventing tissue damage associated with overproduction of oxidants in inflammatory diseases.
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