The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91 phox /Nox2, a membrane-integrated protein that forms a heterodimer with p22 phox to constitute flavocytochrome b 558 . The cytochrome becomes activated by interacting with the adaptor proteins p47 phox and p67 phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47 phox and p67 phox , designated p41 nox and p51 nox , respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41 nox interacts with p22 phox via the two tandem SH3 domains, as does p47 phox . The protein p51 nox as well as p67 phox can form a complex with p47 phox and with p41 nox via the C-terminal SH3 domain and binds to GTPbound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47 phox and p67 phox activate the phagocyte oxidase via the same interactions. Indeed, p41 nox and p51 nox are capable of replacing the corresponding classical homologue in activation of gp91 phox . Nox1, a homologue of gp91 phox , also can be activated in cells, when it is coexpressed with p41 nox and p51 nox , with p41 nox and p67 phox , or with p47 phox and p51 nox ; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41 nox is normally in an active state. Thus, the novel homologues p41 nox and p51 nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome bs58, composed of 91-kDa (gp91PbOx) and 22-kDa (p22PIox) subunits, and two cytosolic factors (p47PbOX and p67Pb°'), each containing two Src homology 3 (SH3) domains.Here we show that the region of the tandem SH3 domains of p47Phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22PhO and p67PhOx but fails to interact with a mutant p22Phox carrying a Pro-156 -* Gln substitution in a prolinerich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.During ingestion of microbes or upon stimulation with various soluble molecules, neutrophils and other phagocytic cells produce superoxide (O°), a precursor of microbicidal oxidants (1-4). The process involves activation ofthe phagocyte NADPH oxidase, dormant in resting cells, that catalyzes reduction of molecular oxygen to superoxide in conjunction with oxidation of NADPH. The significance of the NADPH oxidase in host defense is made evident by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease (CGD) whose phagocytes lack the superoxide-producing system (1-4).The active NADPH oxidase is found on the phagocyte membrane as an enzyme complex, the components ofwhich are identified as targets of genetic defects causing CGD. The one identified at an earlier stage is a phagocyte-specific membrane-integrated b-type cytochrome, cytochrome b558 (5-11), composed of 91-kDa and 22-kDa subunits (designated gp91Phox and p22PhOX, respectively). The cytochrome is now considered to be a flavocytochrome comprising an apparatus transporting electrons from NADPH via FAD and then heme to molecular oxygen (12)(13)(14)(15)(16) . In addition to these specialized factors, as a third cytosolic factor, the small GTPbinding protein p21rac (rac 1 and/or rac 2) is also involved in the system (22)(23)(24).Although the components of the NADPH oxidase are thus identified, little is known about the mechanism for their assembly leading to activation of the enzyme. Upon phagocyte stimulation, the cytosolic components translocate to the membrane where cytochrome b558 resides (25,26). Experiments using neutrophils from CGD patients have revealed that the t...

The small GTPase Rac functions as a molecular switch in several important cellular events including cytoskeletal reorganization and activation of the phagocyte NADPH oxidase, the latter of which leads to production of superoxide, a precursor of microbicidal oxidants. During formation of the active oxidase complex at the membrane, the GTP-bound Rac appears to interact with the N-terminal region of p67 phox , another indispensable activator that translocates from the cytosol upon phagocyte stimulation. Here we show that the p67 phox N terminus lacks the CRIB motif, a well known Rac target, but contains four tetratricopeptide repeat (TPR) motifs with highly ␣-helical structure. Disruption of any of the N-terminal three TPRs, but the last one, results in defective interaction with Rac, while all the four are required for the NADPH oxidase activation. We also find that Arg-102 in the third repeat is likely involved in binding to Rac via an ionic interaction, and that replacement of this residue with Glu completely abrogates the capability of activating the oxidase both in vivo and in vitro. Thus the TPR motifs of p67 phox are packed to function as a Rac target, thereby playing a crucial role in the active oxidase complex formation.

Chronic Granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the × chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations.

There was no major difference in AAV incidence between Japan and the U.K., but this prospective study found MPA and MPO-ANCA to be more common in Japan and granulomatosis with polyangiitis and PR3-ANCA to be more common in the U.K., in line with earlier reports.