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Abstract: The superoxide-producing phagocyte NADPH oxidase gp91 phox /Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22 phox , which provides both stabilization and a docking site for organizer proteins. The p22 phox -complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47 phox or Noxo1) and a Nox activator (p67 phox or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47 phox and p67 phox or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22 phox constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22 phox -dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47 phox and either p67 phox or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47 phox , they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47 phox is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47 phox and either p67 phox or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47 phox is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67 phox as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67 phox . Since the enhancements are not observed with a mutant p67 phox defective in binding to Rac, this GTPase appear to directly recruit p67 phox to the membrane. [Copyright &y& Elsevier]

A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKCι and Par6α PB1 domains to a resolution of 1.5 Å. Both PB1 domains adopt a ubiquitin fold. PKCι PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6α PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKCι PB1 and the conserved lysine residue on the Par6α PB1. Structural comparison of the PKCι and Par6α PB1 complex with the p40phox and p67phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction. [ABSTRACT FROM AUTHOR]

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47phox, p67phox, p40phox, and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b55s (cyt b558) with different amounts of FAD. A remarkably high activity, over 100 μmol/s/μmol heme, was obtained in the oxidase with purified cyt b558, ternary complex (p47–p67-p40phox), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b558, the dissociation constant Kd of FAD in cyt b558 of reconstituted oxidase was estimated as nearly I nM. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b55s increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b558 recovered electron flow from NADPH to heme. From both Kd values of FAD and the amount of incorporated FAD in cyt b558 of reconstituted oxidase, in combination with spectra, we propose the model in which the Kd values of FAD in cyt b558 is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase. [ABSTRACT FROM AUTHOR]

Examines the role of human homologue p47 and p67 adapter proteins in the activation of superoxide-producing NAD(P)H oxidases. Structure and domain architecture of p41, a novel homologue of p47; Interaction of human p41 with other oxidase factors; Effects of p41 and p51 on activation of the phagocyte NAD(P)H oxidase.

Comments on a study that examined the multidomain protein Phox and Bem1p (PB1). Structure of PB1 domains; Function of PB1 domains; Nomenclature of homologous domains of PB1.