Verbatim): The superoxide(02-)-generating NADPH oxidase of phagocytes is a potent source of reactive oxygen species (ROS). Its importance to innate immunity is manifested by the inherited syndrome chronic granulomatous disease, in which oxidase activity is absent and patients are susceptible to life-threatening microbial infections. O2-and its derivatives also cause severe tissue damage, contributing to inflammatory diseases such as ischemia-reperfusion injury and acute respiratory distress syndrome, and their prolonged generation in chronic inflammation can lead to dysplasia and malignancy. ROS produced by homologous systems in non-phagocytes are implicated in abnormal cell growth. The long-term objectives of the proposed research are to understand the mechanisms that control NADPH oxidase assembly and activity, and to identify steps that can be inhibited to minimize tissue damage. These four specific aims address gaps in knowledge that impede achievement of these goals. (1) Identify the processes of molecular recognition that encode the complex of cytosolic oxidase proteins to associate with the flavocytochrome to form a isolated phagosomes and cellular fractions, will be used to study the formation of the active enzyme and determine the roles of coronin and cofilin in controlling its assembly. 2) Analyze interactions between NADPH oxidase components, and identify factors that promote their association and dissociation. A biosensor instrument will be used to quantitate interactions between oxidase-related proteins and study how they are modified by changes that mimic intracellular signals. Development of this technique will provide an assay for molecules designed to inhibit oxidase activation. 3) Identify and isolate GTPase activating proteins and guanine nucleotide exchange factors for Rac2 and study their involvement in controlling Rac and NADPH oxidase activity. The identities of the regulatory proteins that control Rae activity in the neutrophil by altering its GDP/GTP-bound state are not known. These proteins are likely to be important in the negative and positive regulation of 02- generation. 4) Identify signaling events and molecules that terminate superoxide generation. The processes that switch off the enzyme have important clinical implications for inflammatory disease, but they are not well understood. Cell-free oxidase systems and intact cells will be used to determine if a) phosphoinositides; b) changes in the phosphorylation state of phox proteins; and c) changes in the GDP/GTP-bound state of Rae are negative regulators of the enzyme.
