This project is studying regulation of NADPH oxidase activity (respiratory burst) of phagocytes. This enzyme generates potent microbicidal oxidants; its importance is evident in chronic granulomatous disease, where oxidase defects cause enhanced susceptibility to bacterial infections. We recently proposed a model for oxidase assembly showing the importance of several SH3 domain interactions with proline-rich targets in other oxidase components. We have used gene transfection in conjunction with binding studies (with fusion proteins or the yeast two-hybrid system) to map specific contact sites between oxidase components and have explored structural details that underlie SH3 domain binding specificity. Since SH3 domains are found in a variety of intracellular proteins, insights on the oxidase may be relevant to other signal transduction systems. We have also shown inhibition of oxidase activity by disrupting SH3 interactions, both in whole cell and cell-free assays. One inhibitor is an SH3 domain-containing protein (p40-phox) that competes with the interaction between p47-phox and p67- phox. The SH3 domain of p40-phox specifically binds a proline-rich sequence in p47-phox. This domain alone down-regulate the oxidase when transfected into cells. The other inhibitor is a proline-rich natural peptide (PR-39) that avidly binds to an SH3 domain of p47-phox and inhibits its association with cytochrome b558. PR-39 is itself an antimicrobial peptide released by neutrophils that accumulates in wound fluid, thus it may directly mediate switching between oxygen-dependent and independent defense systems. Since only the porcine form of PR-39 has been described, we sought evidence for the human counterpart by isolating immunocross-reactive PR-39 clones from a human bone marrow cDNA expression library. In other work, we are screening combinatorial phage display libraries with SH3 domain probes to identify high-affinity ligands that disrupt SH3 interactions. These studies may serve as a basis for designing drugs that block production of reactive oxidants during acute or chronic inflammatory disease processes.
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