Molecular mechanism for activation of the reactive-oxygen-species-producing phagocyte NADPH oxidase involved in host defense against microbial infections

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The NADPH oxidase is an enzyme that produces reactive oxygen species, which play a crucial role in host defense against microbial infections. The catalytic core of the phagocyte NADPH oxidase is membrane-integrated cytochrome b558, comprising gp91^<Phox> and p22^<Phox>. Activation of the oxidase requires stimulus-induced membrane translocation of specific adaptor proteins (p47^<Phox>, p67^<Phox>, and p40^<Phox>, each containing SH3 domain) and the small GTPase Rac. Recent studies have identified several mammalian homologues of gp91^<Phox>, presently known as Nox-family oxidases : e.g., Noxl, abundant in the colon epithelial cells, is likely involved in local host defense. In the present project, we investigated the molecular mechanism for oxidase activation, and obtained the following novel results. (1) Our biochemical analyses have revealed that, in activation of gp91^<Phox>, p47^<Phox> translocates to the membrane via both PX domain-mediated interaction with phosphoinositides and 5113 domain-mediated binding to p22^<Phox>. We have also determined the complex structure of the p47^<Phox> SH3 domains with the p22^<Phox> proline-rich region (PRR). (2) We have shown that p67^<Phox> interacts with p47^<Phox> via the C-terminal S113 domain in a novel fashion, and determined the NMR structure of a complex between the p67^<Phox> SH3 domain and the p47^<Phox> PRR. (3) We have demonstrated that p4O^<Phox> facilitates the oxidase activation by enhancing the membrane translocation of p47^<Phox> and p67^<Phox>, which effect is mediated via the PB1 domain-mediated heterodimeriztion with p67^<Phox>. (4) We have identified and cloned novel homologues of p47^<Phox> and p67^<Phox>, designated Noxol and Noxal, respectively, and shown that both proteins are required for activation of Noxl. (5) Our recent study has shown that Rac participates in Noxl activation, which is mediated via its interaction with Noxal.