The phagocyte respiratory burst oxidase that generates the superoxide radical plays a central role in host defense and the inflammatory response. Assembly of the active oxidase complex requires the participation of both membrane and cytosolic proteins, and is regulated by small GTPases. A phagocyte-specific b-type flavocytochrome heterodimer, an integral membrane protein complex located in the plasma membrane and, in neutrophils, specific granules, is the focal point for oxidase assembly, and contains both the flavin and heme redox centers for transfer of electrons from NADPH to molecular oxygen. Genetic defects in oxidase proteins, including the two flavocytochrome subunits, result in chronic granulomatous disease (CGD), a syndrome characterized by an absent respiratory burst, recurrent infections, and chronic granulomas. The structural and functional relationships between the various oxidase subunits and the assembly of the active NADPH oxidase complex remain incompletely understood. This low-potential flavocytochrome is comprised of gp91phox, a 91-kDa glycoprotein encoded by an X-linked gene that is the site of mutations in X-linked CGD, and p22phox, a non-glycosylated peptide derived from an autosomal CGD locus. The proposed studies take a genetic approach to investigating the structure and function of the oxidase flavocytochrome b and its role as a focal point for assembly of the active NADPH oxidase, with a particular emphasis on phagocytosis-induced oxidase activation. The project has 2 specific aims, will be performed in primary phagocytes, phagocyte cell lines, and heterologous cell models we developed for expression of functional recombinant oxidase subunits and phagocytic receptors. First, studies aimed at identifying functional domains in gp91phox and p22phox that contribute to flavocytochrome b biosynthesis, trafficking during phagocytosis, and NADPH oxidase function will be pursued. p22phox, which contains 2 intracellular domains of largely unknown function, will be a particular focus. Second, oxidase activation on phagosomes will be examined, focusing on interactions between flavocytochrome b and the soluble regulatory phox subunits, including p40phox, which we recently showed to be critical for FcyR-induced oxidase activation in a model cell system, and how specific signaling cascades activate these interactions. These studies will provide further insight into the superoxide- generating system of the phagocyte, which may lead to new approaches in modulating superoxide formation in the inflammatory response and host defense.
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