Reactive oxygen species (ROS) formed by the NADPH oxidase of phagocytic leukocytes play a primary role in host defense and inflammation, perpetuating both acute and chronic inflammatory responses. The PI has established that human neutrophil NADPH oxidase activity is regulated by the small GTPase Rac2. However, the role of Rac2 in controlling the electron transfer reactions between NADPH and molecular oxygen in this multi-protein system remains a critical undefined question. GDP Dissociation Inhibitors (GDIs) serve as key regulators of leukocyte Rac2 activity and, consequently, NADPH oxidase activity as well. The signaling mechanisms and protein components that modulate the basic Rac-GDI interaction cycle have not yet been defined.
In Aim 1, NADPH oxidase regulation by Rac GTPase will be analyzed using recombinant protein components in cell-free systems to investigate partial electron transfer reactions in the assembled NADPH oxidase. The appropriate reagents have been developed and significant technical problems overcome. The PI's is now poised to determine the molecular role of Rac in controlling the NADPH oxidase. Regulatory activities of Rac will be related to specific protein-protein interactions with other oxidase components. The role of phosphorylation in regulating the GTPase/GDI cycle will be analyzed after purification and characterization of GDI-kinase(s) present in neutrophil and brain lysates (Aim 2). Radixin inhibits the ability to displace Rho GTPases from GDI complexes (GDI-displacement factor or GDF activity). Structure-function studies on radixin and related ERM proteins to localize the putative """"""""GDF domain"""""""" (Aim 3.1) will be performed. The molecular basis for GDF activity of this core domain will be studied through structural and biochemical approaches, and conserved features of the motif will be used to identify other possible GDF's. Human neutrophil GDF activities have been identified at the biochemical level (Aim 3.2). Specific GDI-interacting proteins that represent potential GDF's or other GDI regulatory proteins have been detected. Neutrophil GDF's will be purified and their role in the Rac GTPase-GDI regulatory cycle will be investigated. These studies will yield novel insights into the overall control of oxidant formation by phagocytic leukocytes and other cells.
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