The phagocyte respiratory burst is an important component of host defense against bacteria and fungi. Superoxide is generated by an NADPH oxidase, a membrane-bound, multi-component electron transport chain in response to binding of a variety of stimuli to phagocyte membrane receptors. Activity of the NADPH oxidase is uniquely regulated compared to other electron transport systems. In resting phagocytes, the NADPH oxidase exists an individual membrane (cytochrome b558) and cytosol proteins (p47-phox and p67-phox). Oxidase function appears to be regulated by the GTP-binding proteins, rap-1A and rac-1/2. Stimulation of phagocytes leads to translocation of p47-phox, p67-phox, and rac from cytosol to membrane where these proteins associate with cytochrome b558 and rap1A to form the NADPH oxidase. p47-phox appears to interact directly with the carboxylterminus of the 91 kDa subunit of cytochrome b558 (gp91- phox). We hypothesize that cytochrome b558 is the membrane scaffolding on which the NADPH oxidase is formed and that phagocyte stimulation leads to binding of specific cytochrome b558 peptide domains to other oxidase components, such as p47-phox, p67-phox, rac1/2, and rap1A and possibly, to other parts of cytochrome b558 itself. The major goal of this FIRST Award proposal is to identify these cytochrome b558 binding domains and to determine how these cytochrome b558 domains facilitate oxidase assembly and maintain NADPH oxidase structure. To perform these experiments, antibodies will be generated that will recognize specifically different cytochrome b558 binding domains that interact with other oxidase proteins. These antibodies will be used to map the locations of these domains in cytochrome b558 and to determine whether p47-phox, p67-phox, or other components interact with particular binding domains. Additionally, the sequence of interactions of cytochrome b558 binding domains with other oxidase proteins at various stages of NADPH oxidase formation will be explored. We believe that these proposed studies will contribute significantly to understanding the structure of the NADPH oxidase and the mechanism of oxidase formation. These experiments will directly address the nature of changes in cytochrome b558 structure triggered by stimulation of the resting phagocyte. Determining how phagocyte stimulation leads to interactions of cytochrome b558 with other oxidase proteins will facilitate understanding the roles of these other oxidase components in oxidase function and assembly. In addition, the experiments outlined in this proposal constitute a potentially fruitful approach to future development of a new class of clinically useful agents that will inhibit specifically phagocyte superoxide production of a new class of clinically useful agents that will inhibit specifically phagocyte superoxide production in inflammatory states where the phagocyte respiratory burst contributes to pathogenesis.
NIH ExPorter
NIH ExPorter