This project studies NADPH oxidase involved in human neutrophill and monocyte oxidative responses to inflammation and infection, including: (i) biochemistry of oxidase activation; (ii) gene regulation of NADPH oxidase activity; (iii) NADPH oxidase component gene defects causing Chronic Granulomatous Diseases of Childhood (CGD); (iv) retrovirus expression of oxidase components with in vitro model systems for development of gene therapy of CGD. Cytoplasmic oxidase components p47-phox, P67-phox, NCF-3 and membrane cytochrome b558 (CYTO b) subunits p22-phox and gp9l-phox interact to form a membrane-bound complex producing superoxide. We show that oxidase activation requires an early reaction corresponding to multiple phosphorylation of p47-phox and translocation to the membrane where it interacts with CYTO b. We identified a carboxyterminus sequence of gp9l-phox, arg-gly-val-his-phe-ile-phe, which is critical to oxidase activation. Oligopeptides containing this sequence inhibit oxidase activation. Knowledge of phosphorylation events and critical protein domains involved in oxidase activation may allow development of novel therapies to augment or inhibit inflammation oxidants. We show that regulation of oxidase activity in cultured human monocytes correlates most closely with changing levels of cytoplasmic p47-phox protein. In myeloid cells during differentiation p67-phox was the last component to appear and was therefore the limiting oxidase factor under these conditions. In other studies, delineation of the structure of the p67-pliox gene identified normal allelic restriction-length polymorphisms which could be used to mark the parental origin of p67-phox gene. More recently, we have inserted p47-phox cDNA into a retrovirus vector and used this to restore the missing protein to cultured blood cells from a CGD patient with p47- phox deficiency. In related studies CYTO b subunits have been expressed in a baculovirus expression system and cDNA encoding these subunits have also been inserted into retroviral vectors. These studies represent early steps toward the development of genetic reconstitutional therapy for CGD.
