Review
Molecular composition and regulation of the Nox family NAD(P)H oxidases

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Abstract

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91phox, tightly complexed with p22phox, and its activation requires the association with p47phox, p67phox, and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91phox-related enzymes of the NAD(P)H oxidase (Nox) family—seven homologues identified in humans—deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.

Section snippets

Regulation of the phagocyte NADPH oxidase gp91phox/Nox2

The catalytic subunit of the phagocyte oxidase, gp91phox/Nox2, is a highly glycosylated membrane-spanning protein. The N-terminal portion of gp91phox contains six transmembrane α-helices: the third and fifth helices each contain two invariant histidine residues that are positioned to coordinate two hemes, thereby placing one heme toward the inner face and the other toward the outer face (Fig. 2A). On the other hand, the C-terminal half of gp91phox folds into a cytoplasmic domain containing the

Regulation of Nox1, the first identified mammalian homologue of gp91phox

It was previously reported that a superoxide-producing enzyme similar to the phagocyte NADPH oxidase exists in vascular smooth muscle cells [41] and gastric pit cells in guinea pigs [42], and the responsible oxidase is currently recognized as Nox1, the first gp91phox homologue to be identified in mammals [43], [44]. Nox1 as well as gp91phox contains the N-terminal transmembrane segments and the C-terminal ferredoxin reductase domain, sharing 56% amino acid identity with gp91phox (Fig. 4A).

Regulation of Nox3, the oxidase responsible for otoconia formation in mice

The third mammalian NADPH oxidase Nox3, originally described as an enzyme expressed in several fetal tissues such as kidneys and livers [57], [58], shows the closest similarity to gp91phox (58% identity) among the Nox-family oxidases (Fig. 4). It is presently known that Nox3 exists also in the inner ear of mice and plays a crucial role in formation of otoconia, tiny mineralized structures that are required for perception of balance and gravity [59]; mice with Nox3 mutations exhibit the head tilt

Regulation of Nox4, an oxidase abundantly expressed in the kidney

Compared with Nox1 and Nox3, Nox4 is distantly related to gp91phox/Nox2, exhibiting only 39% identity in amino acid sequence (Fig. 4). Nox4 was initially identified as an NAD(P)H oxidase highly expressed in the adult and fetal kidney [62], [63]; immunohistochemical study on human renal cortex has detected Nox4 expression in epithelial cells of distal tubules. It is presently known that Nox4 is also expressed in a variety of cells including those in the cardiovasculature, especially endothelial

Regulation of Nox5, the oxidase containing four EF hands

Human Nox5 is abundantly expressed in T- and B-lymphocytes of spleen and lymph nodes, and in the sperm precursors of testis [69]; however, its biological role is presently unknown. Intriguingly, no orthologue for Nox5 is found in the mouse and rat genomes. Nox5 builds on the basic structure of gp91phox, adding an N-terminal extension that contains four Ca2+-binding sites: three canonical and one non-canonical EF-hands (Fig. 4). The gp91phox-like domain of Nox5 exhibits only 22–27% amino acid

Regulation of the dual oxidases Duox1 and Duox2

Besides a C-terminal NADPH oxidase domain, Duox enzymes feature an N-terminal peroxidase-like ectodomain that is separated from two EF hands by an additional transmembrane segment (Fig. 4), and thus called “dual” oxidases [5]. However, it is not clear whether the N-terminal domains of Duox have an appreciable peroxidase activity. Although the ectodomains are thought to belong to the MPO (myelopreoxidase)-related preoxidase family because of considerable homology in the entire regions, they lack

Cooperation between Nox oxidases and MPO-like peroxidases

Nox family oxidases often function in cooperation with MPO-like peroxidases. In the phagosome, superoxide released by the phagocyte oxidase gp91phox is dismuted into H2O2, which is further converted to HOCl, a powerful fungicidal agent, by MPO—a peroxidase that is specifically expressed in granules of neutrophils and monocytes, and released to the intraphagosomal space [1], [83]. MPO thus contributes to host defense in a manner dependent on the phagocyte NAPDH oxidase activity [84]. In thyroid

Another look

Among Nox-family oxidases, members of the Duox subfamily are found in a variety of animals. On the other hand, the existence of the closest Nox members to gp91phox (Nox1–4) is confined to the phylum Chordata. These Nox enzymes form a functional complex with p22phox, in contrast to Nox5 and Duox1/2. The requirement for p22phox appears to arise from its functional role, especially regulation by the organizers and activators (p47phox, p67phox, and their homologues), since the p22phox gene and the

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by CREST of JST (Japan Science and Technology Agency) and BIRD of JST.

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    Abbreviations: ROS, reactive oxygen species; Nox, NAD(P)H oxidase; CGD, chronic granulomatous disease; PRR, proline-rich region; AIR, autoinhibitory region; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; TPR, tetratricopeptide repeat; Noxo1, Nox organizer 1; Noxa1, Nox activator 1; RNAi, RNA interference; MPO, myeloperoixdase; TPO, thyroid peroxidase.

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