Biological Roles for the NOX Family NADPH Oxidases

Linking the phagocyte defect in patients with chronic granulomatous disease (CGD) 2The abbreviations used are: CGD, chronic granulomatous disease; ROS, reactive oxygen species; NOX, NADPH oxidase. with the biochemical basis for oxygen consumption by stimulated neutrophils (1Baehner R.L. Nathan D.G. Science. 1967; 155: 835-836Crossref PubMed Scopus (257) Google Scholar) represented a seminal advance in understanding the molecular basis for a key component of innate immunity. In subsequent decades, the catalytic and regulatory elements of the “respiratory burst oxidase” were elucidated, tacitly assuming all along that the NADPH-dependent oxidase under study represented a system uniquely expressed in phagocytic cells and dedicated to generating relatively large amounts of reactive oxygen species (ROS) destined to destroy invading microbes. Development of more sensitive analytical systems for ROS detection revealed that some physiological and pathophysiological events in non-phagocytic cells were associated with ROS generation, although the subcellular source of oxidants remained uncertain. With the identification of mox1 in 1999 (2Suh Y.-A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1278) Google Scholar) as a homolog of gp91phox, the catalytic component of the phagocyte oxidase, came the birth of the NADPH oxidase (NOX) protein family and the eventual identification of its seven members. With remarkable rapidity, many features of the structure, activity, cell biology, and physiology of the NOX proteins have been described, as summarized in several excellent and comprehensive recent reviews (3Lambeth J.D. Kawahara T. Diebold B. Free Radic. Biol. Med. 2007; 43: 319-331Crossref PubMed Scopus (409) Google Scholar, 4Geiszt M. Cardiovasc. Res. 2006; 71: 289-299Crossref PubMed Scopus (179) Google Scholar, 5Bedard K. Krause K.-H. Physiol. Rev. 2007; 87: 245-313Crossref PubMed Scopus (4958) Google Scholar). This more circumscribed minireview provides an overview of the organizing features of the protein family, a summary of the physiology and pathophysiology in which NOX proteins participate (or might participate), and identification of some of the remaining unanswered questions in the field. The patriarch of the NOX protein family is gp91phox (NOX2), the heavy subunit of flavocytochrome b558, the catalytic component of the phagocyte NADPH oxidase (6Babior B.M. Curr. Opin. Immunol. 2004; 16: 42-47Crossref PubMed Scopus (655) Google Scholar). Associated with p22phox in plasma membrane and membranes of selected intracellular compartments, gp91phox operates as an electron transferase, shuttling electrons from NADPH in the phagocyte cytoplasm, across two nonequivalent hemes (7Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) buried in the membrane, to O2, the electron acceptor, thereby generating superoxide anion. Substantial evidence suggests a stepwise flow of electrons during oxidase activity: a single electron transfers sequentially from cytosolic NADPH to NOX2-associated FAD, to the more proximal heme in the membrane (Em7 = –225 mV) (7Cross A.R. Rae J. Curnutte J.T. J. Biol. Chem. 1995; 270: 17075-17077Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), to the distal heme (Em7 = –265 mV), and finally to molecular O2. The univalent nature of the reaction with O2 produces superoxide anion as the immediate product of the oxidase, with hydrogen peroxide subsequently generated by dismutation of the superoxide. As discussed below, some of the non-phagocyte NOX proteins display unanticipated and unexplained reactivity. Although the crystal structure of flavocytochrome b558 has not been solved, mutagenesis data suggest that the NOX2 subunit contains both hemes, where they are bishistidine-ligated in parallel transmembrane helices, coordinated with His101– His115 and His209–His222, respectively (8Biberstine-Kinkade K.J. DeLeo F.R. Epstein R.I. LeRoy B.A. Nauseef W.M. Dinauer M.C. J. Biol. Chem. 2001; 276: 31105-31112Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The currently accepted model resembles that suggested for FRE1, the iron reductase of Saccharomyces cerevisiae (9Finegold A.A. Shatwell K.P. Segal A.W. Klausner R.D. Dancis A. J. Biol. Chem. 1996; 271: 31021-31024Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), with the paired hemes stacked between two α-helices, perpendicular to the plane of the membranes, and coordinated with histidines 12–13 amino acids apart in the linear sequence (10Taylor R.M. Baniulis D. Burritt J.B. Gripentrog J.M. Lord C.I. Riesselman M.H. Maaty W. Bothner B.P. Angel T.E. Dratz E.A. Linton G.F. Malech H.L. Jesaitis A.J. J. Biol. Chem. 2006; 281: 37045-37056Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although gp91phox is the catalytic component of flavocytochrome b558, its partner, p22phox, is essential for optimal activity. In myeloid cells, coexpression of p22phox with gp91phox and subsequent heterodimer formation are prerequisites for egress from the endoplasmic reticulum and proper localization in the plasma membrane. Expression of either subunit alone results in retention of the expressed protein in the endoplasmic reticulum, with subsequent degradation, in part mediated by the proteasome (11DeLeo F.R. Burritt J.B. Yu L. Jesaitis A.J. Dinauer M.C. Nauseef W.M. J. Biol. Chem. 2000; 275: 13986-13993Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Consistent with the requirement of heterodimer formation for stability of the individual subunits, neutrophils from patients with genetic defects in either gp91phox or p22phox lack both proteins (12Heyworth P.G. Cross A.R. Curnutte J.T. Curr. Opin. Immunol. 2003; 15: 578-584Crossref PubMed Scopus (337) Google Scholar). When expressed heterologously in COS-7 cells, gp91phox alone reaches the cell surface (albeit less efficiently), exhibits the same spectral properties as flavocytochrome b558, and contains hemes with mid-potentials of –264 and –233 mV, nearly the same as the native protein. However, membrane-dependent generation of superoxide in the cell-free system from the COS-7 system absolutely requires coexpression of both subunits (13Yu L. Quinn M.T. Cross A.R. Dinauer M.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7993-7998Crossref PubMed Scopus (178) Google Scholar). The phagocyte oxidase is agonist-dependent, exhibiting no detectable constitutive activity, and transfers electrons and consumes oxygen nearly instantaneously when stimulated. Characterization of NOX2 benefited not only from seminal studies of patients with CGD, but also from the development of the broken cell superoxide-generating system. First described by Bromberg and Pick (14Bromberg Y. Pick E. Cell. Immunol. 1984; 88: 213-221Crossref PubMed Scopus (249) Google Scholar) using macrophages and by several other laboratories (15McPhail L.C. Shirley P.S. Clayton C.C. Snyderman R. J. Clin. Investig. 1985; 75: 1735-1739Crossref PubMed Scopus (228) Google Scholar, 16Curnutte J.T. J. Clin. Investig. 1985; 75: 1740-1743Crossref PubMed Scopus (199) Google Scholar) using neutrophil subcellular components, the broken cell system combines membrane and cytosol from resting phagocytes, which lack oxidase activity, in the presence of anionic amphiphiles to generate superoxide anion in an NADPH-dependent fashion. Availability of the broken cell system was essential for the identification of p47phox, p67phox, and Rac as cytosolic components required for phagocyte oxidase activity (17Nunoi H. Rotrosen D. Gallin J.I. Malech H.L. Science. 1988; 242: 1298-1301Crossref PubMed Scopus (221) Google Scholar, 18Volpp B.D. Nauseef W.M. Clark R.A. Science. 1988; 242: 1295-1298Crossref PubMed Scopus (277) Google Scholar, 19Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (765) Google Scholar) and has been a powerful vehicle for applying a reductionist approach to dissecting the biochemistry of this oxidase. Regulation of the phagocyte system relies on spatial segregation of its essential components (Fig. 1); upon stimulation, regulatory components residing in the cytoplasm of resting phagocytes translocate to the target membranes and associate there with flavocytochrome b558 to assemble into a functional NADPH oxidase (20Nauseef W.M. Histochem. Cell Biol. 2004; 122: 277-291Crossref PubMed Scopus (322) Google Scholar). In the cytoplasm of resting cells are two protein complexes, one composed of p47phox, p67phox, and p40phox and the other containing Rac and RhoGDI, the GDP dissociation inhibitor for Rho. Activation-dependent phosphorylation of p47phox and RhoGDI triggers conformational changes in both targets, thereby exposing otherwise cryptic sites in p47phox that bind p22phox, membrane phospholipids, and perhaps other sites and generating Rac-GTP that subsequently translocates to the membrane. Although the multiple intermolecular interactions that mediate oxidase assembly have been extensively studied, it suffices for the purpose of this minireview to recognize that regulation of the phagocyte oxidase depends on compartmentalization of its components in the resting cell and agonist-dependent assembly at the integral membrane protein, flavocytochrome b558. Biochemical studies using the broken cell system have demonstrated convincingly that p47phox serves as an organizing adaptor protein that lacks intrinsic catalytic activity. On the other hand, p67phox represents an essential activating cofactor, possessing a domain that regulates the reduction of FAD by NADPH (21Nisimoto Y. Motalebi S. Han C.H. Lambeth J.D. J. Biol. Chem. 1999; 274: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Whereas p47phox and p67phox translocate to the membrane in a complex with p40phox, Rac2 translocates in its GTP-bound form independent of p47phox and p67phox (22Heyworth P.G. Bohl B.P. Bokoch G.M. Curnutte J.T. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar). Rac2 participates in the catalytic activity of the phagocyte oxidase directly, via its interactions with p67phox, or both (23Bokoch G.M. Diebold B.A. Blood. 2002; 100: 2692-2696Crossref PubMed Scopus (278) Google Scholar, 24Sarfstein R. Gorzalczany Y. Mizrahi A. Berdichevsky Y. Molshanski-Mor S. Weinbaum C. Hirshberg M. Dagher M.C. Pick E. J. Biol. Chem. 2004; 279: 16007-16016Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Additional factors modulate the stability and activity of the assembled complex, although the extent of their contribution to oxidase function is incompletely understood. In summary, the assembled and active phagocyte oxidase includes an electron transferase and associated organizing, activating, and stabilizing elements. The seven-member NOX protein family is defined by its shared sequence homology with gp91phox: all members have six predicted transmembrane domains, motifs for NADPH and FAD binding, and conserved paired histidines that could ligate heme groups. NOX5, Duox1, and Duox2 have additional structural features not shared by the other NOX proteins. All three have cytosolic EF-hands (four in NOX5 and two in Duox proteins) that subserve calcium-dependent regulation of their oxidase activity. In addition, Duox has a long (>550 amino acids) N-terminal extracellular region with sequence homology to the animal peroxidases (Fig. 2). Although there is ∼25% amino acid identity between this N-terminal region and myeloperoxidase, the invariant histidines that provide the proximal and distal axial bonds with heme iron in animal peroxidases (25Fiedler T.J. Davey C.A. Fenna R.E. J. Biol. Chem. 2000; 275: 11964-11971Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) are not conserved in Duox but are replaced by serine residues. This observation is not evidence against the region being a functional peroxidase domain, as alternative residues could form the heme pocket in Duox. However, the lack of homology in residues that characterize the animal peroxidases serves as a caveat to extrapolation from sequence similarity. Furthermore, were the terminal region in Duox capable of peroxidase activity, one would anticipate that it would support normal thyroid hormone synthesis in those patients in whom defective thyroid peroxidase activity causes hypothyroidism (26Park S.M. Chatterjee V.K. J. Med. Genet. 2004; 42: 379-389Crossref Scopus (298) Google Scholar). However, thyroid hormone synthesis requires functional thyroid peroxidase, suggesting that Duox cannot mediate iodination of thyroid hormone. As iodination ranks low in the hierarchy of halogenation reactions mediated by animal peroxidases, the absence of a detectable contribution to thyroid synthesis suggests that Duox lacks peroxidase activity in this particular in vivo setting (27Harper R.W. Xu C. McManus M. Heidersbach A. Eiserich J.P. FEBS Lett. 2006; 580: 5150-5154Crossref PubMed Scopus (34) Google Scholar). Interferon-γ-induced augmentation of peroxidase activity by cultured human bronchial epithelial cells is inhibited by the stable expression of short hairpin RNA and parallels the decrease in Duox2 mRNA, consistent with Duox2 exhibiting peroxidase activity in this experimental setting. In the absence of spectral data or rigorous characterization of the enzymology of the purified protein or putative peroxidase domain, the function of this structural feature in Duox remains undefined. Whereas correct cellular localization and optimal function of flavocytochrome b558 require NOX2 to associate with p22phox, this organizational feature is not shared by all NOX protein family members; only NOX1, NOX3, and NOX4 also form heterodimers with p22phox. As with NOX2, the structural bases and the functional consequences of the association of NOX with p22phoxare not completely delineated. It is noteworthy that the EF-hand-containing, calcium-activated NOX proteins, specifically NOX5, Duox1, and Duox2, are those that function without an association with p22phox, perhaps hinting of a specific functional requirement fulfilled by p22phox or simply marking concurrent modifications during the evolution of the protein family (28Kawahara T. Quinn M.T. Lambeth J.D. BMC Evol. Biol. 2007; 7: 109Crossref PubMed Scopus (243) Google Scholar). Just as the phagocyte oxidase has components in addition to the membrane electron transferase, so too do related NOX proteins have associated factors. Homologs of p47phox and p67phox, named NOX organizing (NOXO1) and activating (NOXA1) proteins to reflect their presumed function, have been identified (29Bánfi B. Clark R.A. Steger K. Krause K.-H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Lambeth J.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), of described R. H. 2006; PubMed Scopus Google Scholar, T. K. S. N. Free Radic. Biol. Med. 2007; 42: PubMed Scopus (34) Google Scholar), and in the activity of of on exhibits species requires both and for activity, optimal activity of human depends on alone D. Lambeth J.D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Y. J. R. E. B. Curr. Biol. 2006; 16: Full Text Full Text PDF PubMed Scopus Google Scholar). and are at the amino acid with at the is amino acids its homolog and includes a at the Although the basis for this species is not it questions the regulation of activity by cytosolic factors. are data the contribution of Rac to activity, and there is no evidence that Rac in the of NOX5, or Duox proteins. NOX5 and the Duox proteins, serves as the with phosphorylation of NOX5 a for its to D. B. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the NOX proteins a of on subunits from NOX2, which depends on multiple factors in the membrane and from the cytoplasm, to as an independent membrane protein. ROS are generated in a of both physiological and and in nearly all and of and K. B. Krause K.-H. 2007; PubMed Scopus Google Scholar). Although NOX proteins the of ROS, identification of the specific NOX for ROS in a particular or system has been by of for several against NOX2 are no at this for for other non-phagocyte NOX proteins are or incompletely are recognize more one NOX or properties that for their recognize protein in in which the the NOX protein is not localization studies have on detection of and not protein, or the identification of p22phox, as a for It is in this using the that NOX proteins have been identified in nearly and with more one in a activity. In some identification of multiple subcellular or dedicated a cell might have a active NOX at the plasma membrane, a of oxidants at the cell surface to a specific extracellular NOX in serves as an agonist-dependent source of oxidants as for intracellular When analytical are to expression of NOX proteins in it to and to the that results in of specific from that from the of With the of the contribution of NOX2 to activity, there are functional of NOX proteins in specific and are essential for normal formation in the and the absence of of the three components results in J. Y. J. R. E. B. Curr. Biol. 2006; 16: Full Text Full Text PDF PubMed Scopus Google Scholar, R. R.A. W. Y. A. A. J. A. 2004; PubMed Scopus Google Scholar, Y. Nauseef W.M. S.M. B. J. Clin. Investig. Google Scholar). Duox serves as the source in the thyroid that thyroid iodination of thyroid hormone C. A. R. J. D. J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). Although of hypothyroidism to reflect in thyroid peroxidase, defects in Duox are in some patients T.J. 2007; PubMed Scopus Google Scholar). Duox also is in by M. J. J. K. J. 2003; PubMed Scopus Google Scholar), oxidants to support the system R. M. J. Physiol. 2004; Scopus Google Scholar, D. K.J. J. Nauseef W.M. C. B. J. Med. 2007; PubMed Scopus Google Scholar). in optimal activity of this system and in part to the in patients with this comprehensive of from several NOX activity to a of physiological This is in A. R. S. 2006; PubMed Scopus Google Scholar), where NOX1, NOX2, and NOX4 have been in many NOX2, and and and and NOX2 = NOX to of of oxygen and of cell and The that reflect the complex between ROS from NOX proteins and reactive species by The interactions of reactive species or in and the localization of their cellular NOX activity cell and to and the of that As predicted from the identification of NOX expression in nearly all the in which NOX proteins are are of the and systems as as and a of J.D. Free Radic. Biol. Med. 2007; 43: PubMed Scopus Google Scholar, K.-H. 2007; 42: PubMed Scopus (155) Google Scholar). into the in NOX that study and the that in the it to the of factors that of the phagocyte oxidase. the phagocyte oxidase exhibits stimulated human neutrophils generate of cells, which requires the of or a of R. T.E. 2006; PubMed Scopus Google Scholar). as superoxide reduction of and oxygen consumption oxidase activity. the source of the phagocyte oxidase is and large of neutrophils of in for study relatively large amounts of NOX2 of is no evidence that phagocytes NOX other NOX2, as there is no spectral evidence for a flavocytochrome or oxidase activity of neutrophil plasma membranes from patients with CGD, and there is no in for of the NOX in neutrophils of patients with R. NADPH-dependent the activity of provides a by which to and to related to the function, and physiology of the phagocyte oxidase. In the for the non-phagocyte oxidase The non-phagocyte are expressed at low in complex composed of multiple cell and NOX with analytical provide an to in the by have many features of the NOX proteins undefined. data the enzymology of the non-phagocyte NOX no studies the or have been as is the that other oxygen the target of electron by the NOX proteins in of spectral properties is to a single of heterologously expressed Y. B. Jesaitis A.J. Dinauer M.C. Nauseef W.M. J. 2007; PubMed Scopus Google Scholar), and only one the activity of membranes in a broken cell the the of NOX4 for NADPH L. L. K. B. B. A. L. W. Krause K.-H. J. 2007; PubMed Scopus Google Scholar). of analytical the biochemical basis for the observation the proximal detectable product generated by some of the NOX family members. Whereas nearly all NOX protein members generate superoxide anion by of single electron reduction of molecular only has been of Duox. the extracellular domain to Duox serves as a superoxide the superoxide anion generated by the oxidase to the (Fig. or the activity is currently not of NOX4 the NADPH-dependent of not superoxide anion K. Dinauer M.C. Cell. 2006; PubMed Scopus Google Scholar), and NOX4 membranes as by NADPH-dependent of although NOX4 which superoxide anion generation the exhibits L. L. K. B. B. A. L. W. Krause K.-H. J. 2007; PubMed Scopus Google Scholar). NOX4 expressed in cells superoxide in to hinting that some of the reflect subcellular or both related to of the understanding of the biochemistry of the non-phagocyte NOX proteins from studies of their in Although adaptor and organizing factors under the specific in have not been defined in all In many has been identified in where the NOX protein would require for activity. with the of cells T. T. J. Y. M. H. M. Kawahara T. K. S. M. PubMed Scopus (113) Google Scholar), expression of or has not been only are the essential in in many but the physiological and pathophysiological that modulate activity, both as and are incompletely In multiple NOX proteins, of the of particular and the contribution of their subcellular compartmentalization to the hierarchy of generation The of ROS in as as cellular and provides to characterize the NOX proteins, both in and in their physiological Although the phagocyte has many features of the NOX protein family members for their of and provide to the of in their As by the at the NOX protein are in the oxidase with