This project is exploring structure-function relationships in important phagocytic cell proteins. Current efforts are focused on protein structural requirements for assembly of the NADPH oxidase, a system responsible for production of superoxide and its potent microbicidal metabolites. Using purified recombinant cytosolic oxidase factors p47-phox and p67-phox, biochemical complementation studies in the last year have defined all of the protein components necessary for in vitro oxidase reconstitution. These studies suggest that a membrane associated cytochrome b558 is the sole electron transfer component of the system, while three essential cytosolic proteins (p47-phox, p67-phox, and rac) are likely to serve in oxidase activation through signal transduction mechanisms common to all eukaryotic cells. We have purified two closely related rho subfamily members of GTP binding proteins (rac-2 and CDC42Hs) from neutrophil cytosol based on complemetation assays with recombinant p47 and p67-phox. We subsequently showed that recombinant CDC42Hs was not active and are now exploring the structural basis for the functional differences between these two G-proteins. Structural variants of all three cytosolic components are being produced and characterized with recombinant DNA expression systems. The roles of src homology domains (SH3) present in p47-phox and p67-phox are also being explored through genetic deletion and reconstitution studies. Using engineered forms p67-phox from a baculovirus expression system we have identified a minimal functional domain within the amino-terminal 244 residues of p67-phox and have shown that the SH3 domains are not essential for in vitro reconstitution. Related studies using mammalian vectors are addressing the roles of SH3 domains within intact cells. In separate studies, src homology domains were fused to tag sequences to identify the targets of SH3 domains in peptide and expression libraries and to study their role in subcellular protein translocation.
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