Neutrophils and other phagocytic blood cells generate high levels of reactive oxygen species in response to a variety of infectious or inflammatory stimuli in a process known as the respiratory burst. This response is attributed to activity of NADPH oxidase, which uses molecular oxygen and NADPH to produce superoxide anion, a precursor of various reactive oxygen species that have key roles in defense against microbial infections and inflammation. Patients with chronic granulomatous disease (CGD) have NADPH oxidase deficiencies that result in enhanced susceptibility to microbial infections and dysregulated inflammatory responses. This project is exploring the structural basis for NADPH oxidase function and the cellular mechanisms underlying regulation of the respiratory burst. Oxidase activation is a stepwise process that begins with stimulation of G-protein-coupled receptors and activation of various kinases and phospholipases. Active oxidase assembly involves phosphorylation of several protein components that transmigrate to specific membrane domains. We have shown that interacting sites (SH3 domains) within p47phox and p67phox play a central role in regulating oxidase assembly and are exploring roles of protein phosphorylation in this process through site-directed mutagenesis of several oxidase components (p22, p67, p47, and p40phox). In work aimed at defining cellular signals that trigger oxidase activation, we have reconstituted receptor-mediated activation of the respiratory burst in transfected K562 cells. These studies are exploring links between stimulation of chemotactic peptide or Fc-gamma receptors and activation of the respiratory burst by co-transfection of several proposed signaling intermediates (PI-3 kinase, protein kinases, phospholipase D, and the GTPases, Arf and Rho). Information on the structure and function of NADPH oxidase, as well as the signaling intermediates that modulate this enzyme, may provide a basis for therapeutic strategies designed to either inhibit or enhance respiratory burst activity. In other studies we are characterizing sources of reactive oxygen species in a variety of other tissues where oxidants can serve as signals promoting programmed cell death, changes in gene expression, or proliferation in response to growth factors. Studies using a p47phox-deficient mouse model for CGD indicate an essential role for p47phox in the release of reactive oxidants by microgial cells and aortic fibroblasts; transduction by p47phox- encoding retrovirus restores oxidant production in these cells. We have also identified three distinct gp91phox homologues produced in several tissues and will characterize these enzymes by assessing their abilities to interact with other oxidase components. - NADPH oxidase, respiratory burst, chronic granulomatous disease, signal transduction, superoxide, reactive oxygen species - Human Subjects
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