The phagocyte NADPH oxidase produces large quantities of superoxide in response to infection. However, superoxide and other oxidative substances contribute to inflammation seen in a number of degenerative and crippling clinical conditions such as arthritis, respiratory distress syndrome, and post- ischemic injury. The NADPH oxidase is comprised of at least five polypeptides; gp91-phox and p22-phox that make up cytochrome b558 responsible for electron transfer, and p47-phox, p67-phox, and rac which are regulatory proteins found in the cytosol of resting phagocytes. When phagocytes are activated, p47-phox, p67-phox, and rac translocate from cytosol to membrane and interact with cytochrome b558. The subject of this grant is understanding the molecular interactions between NADPH oxidase proteins during assembly and activation. Three mechanisms regulate formation of the NADPH oxidase 1) p47-phox phosphorylation, 2) GDP/GTP cycling of rac, a member of the ras superfamily of GTPases, and 3) protein-protein interactions mediated by SH3 and other peptide domains. In this grant the investigator will 1) characterize protein-protein binding between oxidase proteins in part using the two-hybrid assay, 2) characterize rac binding to p67-phox including determining amino acid sequence of the p67-phox binding site for rac, 3) examine how rac bound to p67-phox activates p67-phox participation in NADPH oxidase assembly, and 4) examine how activated phospho-p47-phox interacts with cytochrome b558 and p67-phox. The purpose of this grant is to characterize these interactions, to identify new interactions between oxidase proteins, and to determine how protein-protein binding leads to assembly of the NADPH oxidase. Novel methods, such as the yeast two-hybrid system for characterizing protein-protein binding, will be used to identify these interactions in the inactive and activated states of the oxidase. These findings may lead to new strategies for design of novel anti-inflammatory agents that will inhibit oxidase formation and production of superoxide by phagocytes.
