This project explores structure-function relationships in important host defense systems of phagocytic cells. Current efforts are focused on the assembly of the NADPH oxidase, an enzyme responsible for production of superoxide anion and related microbicidal oxidants. We have recently proposed a model for NADPH oxidase assembly involving interaction of conserved Src Homology 3 (SH3) domains in two cytosolic components (p47- phox and p67-phox) with proline-rich target sequences in other oxidase components. The model is based on in vitro binding experiments, using isolated domains of oxidase components, and oxidase assembly studies that exploit recently developed gene transfer technology. We have shown that a point mutation in one of the putative proline-rich targets in the cytochrome b558 small subunit (p22-phox, P156Q) that has been associated with chronic granulomatous disease (CGD) interferes with membrane translocation of p47-phox and disrupts p47-phox SH3 binding. The model proposing direct p47-p22-phox interactions was further supported by inhibitory effects of two point mutations in the first SH3 domain of p47- phox and experiments showing that expression of p22-phox alone is sufficient to enable p47-phox translocation. Similar experiments involving mutagenesis of a second proline-rich target sequence (p47-phox, res. 358-372) confirmed the importance of yet another SH3-mediated interaction, observed between the C-terminal domains of p67-phox and p47- phox. We have also recognized another structural motif, identified by computer searches as 34 residue repeats within p67-phox. Their role is being explored with regards to oxidase reconstitution and binding to another essential oxidase component, Rac. Structural features critical to oxidase regulation by this Ras-related GTPase have been defined through analysis of chimeric Rac proteins that contain sequences from inactive Ras homologs. These studies may provide a structural basis for design of agents that can modulate this important aspect of the inflammation and may facilitate developments in the area of gene therapy for CGD. The work also provides insights on a variety of other signal transduction systems that involve structurally related proteins or protein sequence motifs.
Showing the most recent 10 out of 41 publications
