The reduced nicotinamide dinucleotide phosphate (NADPH) oxidase complex allows phagocytes to rapidly convert O2 to superoxide anion which then generates other antimicrobial reactive oxygen intermediates, such as H2O2, hydroxyl anion, and peroxynitrite anion. Chronic granulomatous disease (CGD) results from a defect in any of the 4 subunits of the NADPH oxidase and is characterized by recurrent life-threatening bacterial and fungal infections and abnormal tissue granuloma formation. Activation of the NADPH oxidase requires translocation of the cytosolic subunits p47phox (phagocyte oxidase), p67phox, and the low molecular weight GT-Pase Rac, to the membrane-bound flavocytochrome, a heterodimer composed of the heavy chain gp91phox and the light chain p22phox. This complex transfers electrons from NADPH on the cytoplasmic side to O2 on the vacuolar or extracellular side, thereby generating superoxide anion. Activation of the NADPH oxidase requires complex rearrangements between the protein subunits, which are in part mediated by noncovalent binding between src-homology 3 domains (SH3 domains) and proline-rich motifs. Outpatient management of CGD patients relies on the use of prophylactic antibiotics and interferon-gamma. When infection is suspected, aggressive effort to obtain culture material is required. Treatment of infections involves prolonged use of systemic antibiotics, surgical debridement when feasible, and, in severe infections, use of granulocyte transfusions. Mouse knockout models of CGD have been created in which to examine aspects of pathophysiology and therapy. Gene therapy and bone marrow transplantation trials in CGD patients are ongoing and show great promise.

Oxygen sensing is essential for homeostasis in all aerobic organisms, but its mechanism is poorly understood. Data suggest that a phagocytic-like NAD(P)H oxidase producing reactive oxygen species serves as a primary sensor for oxygen. We have characterized a source of superoxide anions in the kidney that we refer to as a renal NAD(P)H oxidase or Renox. Renox is homologous to gp91 phox (91-kDa subunit of the phagocyte oxidase), the electron-transporting subunit of phagocytic NADPH oxidase, and contains all of the structural motifs considered essential for binding of heme, flavin, and nucleotide. In situ RNA hybridization revealed that renox is highly expressed at the site of erythropoietin production in the renal cortex, showing the greatest accumulation of renox mRNA in proximal convoluted tubule epithelial cells. NIH 3T3 fibroblasts overexpressing transfected Renox show increased production of superoxide and develop signs of cellular senescence. Our data suggest that Renox, as a renal source of reactive oxygen species, is a likely candidate for the oxygen sensor function regulating oxygen-dependent gene expression and may also have a role in the development of inflammatory processes in the kidney.

Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO-catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant-generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1-dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO-mediated antimicrobial defense mechanisms on mucosal surfaces.

The importance of reactive oxygen species (ROS) in innate immunity was first recognized in professional phagocytes undergoing a "respiratory burst" upon activation. This robust oxygen consumption is related to a superoxide-generating enzyme, the phagocytic NADPH oxidase (Nox2 or phox). The oxidase is essential for microbial killing, since patients lacking a functional oxidase suffer from enhanced susceptibility to microbial infections. ROS derived from superoxide attack bacteria in the isolated niche of the neutrophil phagosome. The oxidase is electrogenic, alters ion currents across membranes, induces apoptosis, regulates cytokine production, influences gene expression, and promotes formation of extracellular traps. Recently, new homologues of Nox2 were discovered establishing the Nox family of NADPH oxidases that encompasses seven members. Nox1 is highly expressed in the colon epithelium, and can be induced by LPS or IFN-γ. Nox4 was implicated in innate immunity since LPS induces Nox4-dependent ROS generation. Duox1 and Duox2 localize to the apical plasma membrane of epithelial cells in major airways, salivary glands, and the gastrointestinal tract, and provide extracellular hydrogen peroxide to lactoperoxidase to produce antimicrobial hypothiocyanite ions. Th1 and Th2 cytokines regulate expression of Dual oxidases in human airways and may thereby act in host defense or in proinflammatory responses.

No abstract

The NADPH oxidase responsible for generatio of superoxide anion and related microbicidal oxidants by phagocytes Is assembled from at least five distinct proteins. TWo are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domine and associate with a transmembrane cytochrome bss5 upon activation. We show here that the SH3 do of p47-phox bind to proine-rich sequences in p47-phx Itself and the p22-phox subunit of cytochrome b5ss. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro'S' -* Gin) noted in a distinct form of chronic granulomatous disease and was inhibited by a short prolinerich synthetic peptide corresonding to resIdues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-defclent B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.Neutrophils respond to a number of stimuli with a burst of oxygen consumption and production of superoxide. This "respiratory burst" is attributed to NADPH oxidase, an enzyme whose importance is evident in patients with chronic granulomatous disease (CGD) (1), who suffer from increased susceptibility to bacterial and fungal infections due to deficient production ofmicrobicidal oxidants by phagocytes. The NADPH oxidase is composed of five essential components, four that are affected in different genetic types of CGD (2-6) and a fifth component, the Ras-related small GTPase p21, that bestows guanine nucleotide sensitivity to the enzyme complex (7-10). The functional center of the oxidase is flavocytochrome b558 (11, 12), composed of two subunits (gp9l-phox and p22-phox) that accept electrons from cytosolic NADPH and donate them to molecular oxygen. Two cytosolic factors, p47-and p67-phox, translocate to the membrane and associate with the cytochrome upon activation (13,14), although their precise functions remain unclear. Both factors contain two Src homology 3 (SH3) domains (3,4) shown to be critical for assembly of these proteins into the active, membrane-bound oxidase complex (15, 16).SH3 domains are conserved 60-aa sequences found in a variety of intracellular signaling and cytoskeletal proteins in organisms ranging from yeast to man (17,18 (19)(20)(21)(22)(23)(24).In light of recent observations showing that SH3 domains of both p67-and p47-phox are essential for oxidase assembly (15, 16), we examined the roles of these domains in the interactions of several oxidase components. In this report we describe specific SH...

Nox family NADPH oxidases serve a variety of functions requiring reactive oxygen species (ROS) generation, including antimicrobial defense, biosynthetic processes, oxygen sensing and redox-based cellular signaling. We explored targeting, assembly, and activation of several Nox family oxidases, since ROS production appears to be regulated both spatially and temporally. Nox1 and Nox3 are similar to the phagocytic (Nox2-based) oxidase, functioning as superoxide-generating multi-component enzymes. Factors regulating their activities include cytosolic activator and organizer proteins and GTP-Rac. Their regulation varies, with the following rank order: Nox2>Nox1>Nox3. Determinants of subcellular targeting include: 1) formation of Nox-p22phox heterodimeric complexes allowing plasma membrane translocation, 2) phospholipids-binding specificities of PX domain-containing organizer proteins (p47phox or Nox organizer 1 (Noxo1)), and 3) variably splicing of Noxo1 PX domains directing them to nuclear or plasma membranes. Dual oxidases (Duox1 and Duox2) are targeted by different mechanisms. Plasma membrane targeting results in H2O2 release, not superoxide, to support extracellular peroxidases. Human Duox1 and Duox2 have no demonstrable peroxidase activity, despite their extensive homology with heme peroxidases. The dual oxidases were reconstituted by Duox activator 2 (Duoxa2) or two Duoxa1 variants, which dictate maturation, subcellular localization, and the type of ROS generated by forming stable complexes with Duox.

Superoxide production by phagocytes involves activation of a multi-component NADPH oxidase. Recently, several homologues of the catalytic component of the phagocyte oxidase, gp91 phox , were identified in various tissues. Here we describe two proteins, p41 and p51, with significant homology to two cytosolic components of the phagocytic oxidase, p47 phox and p67 phox . Like p47 phox , p41 contains an amino-terminal Phox homology domain, two SH3 domains, and a conserved carboxylterminal, proline-rich motif. Similarly, p51 is homologous to p67 phox , containing four amino-terminal tetratrico-peptide repeats, a conserved "activation domain" motif, a PB1 domain, and a carboxyl-terminal SH3 domain. The highest levels of p41 transcript are detected in the colon and in other gastrointestinal tissues that express Nox1, the predominant gp91 phox homologue in these tissues. In contrast, the p51 transcript showed a more widespread expression pattern, suggesting that it may support other tissue-specific oxidases. Mouse colon in situ hybridization detected both transcripts in the epithelial cells of colon crypts. Heterologous co-expression of p41 and p51 significantly enhances the superoxide-generating activity of Nox1-expressing cells; thus, p41 and p51 appear to be novel regulators of Nox1. These proteins also support the activity of gp91 phox , albeit at much lower levels than the cytosolic phox counterparts. Our results suggest colon epithelial cells contain a multi-component NAD(P)H oxidase with a molecular architecture similar to the phagocytic oxidase.