NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology

Abstract

The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91^phox component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.

Keywords: electron transfer; membrane protein; modular proteins; oxidative stress; reactive oxygen species; signaling molecule.

Figure 1

Timeline of the major steps leading to the identification and mechanistic description of the NADPH oxidase family of enzymes, specialized in the deliberate production of ROS [1,2,3,4,5,9,12,14,15,23,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65].

Figure 2

Catalytic subunit of the NADPH phagocyte oxidase complex. NOX2 topology harbors 6 membrane-spanning helices inter-connected by intra and extracellular loops, as well as a cytosolic domain enclosing FAD and NADPH-binding domains. Helices 3 and 5 of NOX2 chelate two b-type heme groups. Despite various studies, the number of transmembrane segments of p22^phox is not clearly identified, and the protein has thus been represented in this review with 2 helices and a C-ter cytosolic segment presenting a PRR domain that interacts with the cytosolic factor p47^phox.

Figure 3

Activation process of the phagocytic NADPH oxidase. Detection of a pathogen triggers signaling pathways that lead to the phosphorylation of the cytosolic factors (mainly p47^phox), inducing their translocation to the membrane-bound components of NOX2 and initiating the catalysis of superoxide production. Similarly, Rac-GDP sequestered in cytosol by RhoGDI is transferred to the membrane and its GDP exchanged for GTP for final assembly with p67^phox, leading to NOX2 activation.

Figure 4

Organization of NOX2 transmembrane helices. Helices are numbered in the N to C direction. Two hemes are coordinated by conserved bis-histidyl motifs on helices III and V. (B) and (D) loops face the cytosol and contact the cytosolic DH domain. (A, C, E) loops face the extracytosolic space, which is equivalent to the interior of the phagosome, where oxygen reduction occurs.

Figure 5

Mechanism of electron transfer catalyzed by the cytochrome b558 of NADPH oxidase. (a) The NADPH substrate provides two electrons that are transferred to the FAD, reducing it to FADH2. The FADH2 transfers a first electron to the proximal heme of the cytochrome, which is rapidly transmitted to the distal heme and then to molecular oxygen, forming superoxide. The second electron carried by the FAD cofactor is transferred in the same way with the FAD as an initial donor. The order of successive steps is indicated in blue circles. (b) Redox potentials of the different couples that participate in NOX-catalyzed electron transfer.

Figure 6

Diagram of the cytosolic factors of NADPH oxidase and their interactions with partners. The p47^phox and p40^phox proteins are initially self-inhibited and require phosphorylation to reach an active conformation. The p67^phox and p40^phox factors interact through their respective PB1 domains; the SH3B domain of p67^phox binds to the PRR domain of p47^phox. After phosphorylation, the bis-SH3 domain of p47^phox is unmasked, triggering translocation of the p47^phox–p67^phox–p40^phox trimeric complex via interaction with the PRR domain of p22^phox. At the membrane, the PX domains of p47^phox and p40^phox bind to membrane lipids. Rac-GDP sequestered in cytosol by RhoGDI is transferred to the membrane and GDP is exchanged for GTP, leading to the interaction with the TPR domain of p67^phox. Domain boundaries are indi-cated by position numbers. Important phosphorylation sites in the AIR region of p47^phox are indicated as ‘S’; the major phosphorylation site of p67phox Thr233 is labeled. Dotted lines represent the inter-domain interactions. Adapted from [110].

Figure 7

Model of the trimeric cytosolic complex of NADPH oxidase in the resting state [121]. Three-dimensional model of the heterotrimer: p40^phox (cyan ribbons), p47^phox (light purple ribbons), p67^phox (gray ribbons). The N-ter extremities are shown in red and the C-ter ends in blue. In p67^phox, green spheres represent the β hairpin of the Rac interaction (residues within 115–130), yellow spheres the activation domain (residues within 199–210 [122]). The residues of the lipid interacting PX domains in p40^phox are represented by the orange spheres (R58, K92 and R105 [51]). Figure inspired by [121].

Figure 8

Atomic structure of Rac in interaction with RhoGDI or with p67^phox. The structure was solved by X-ray diffraction at a 2.7Å resolution [50]. (a) Rac is represented in blue ribbon and RhoGDI in green. The GDP molecule is depicted in stick (white) within Rac, while the geranylgeranyl tail of Rac is represented as stick (dark blue) within RhoGDI. The switch I and II regions of Rac are highlighted in yellow and red, respec-tively. (b) Binding pocket of the geranylgeranyl tail. The Rac geranylgeranyl tail, represented in blue stick, binds in a hydrophobic cavity formed by RhoGDI, represented in green ribbon and surface. The hydrophilic regions of the RhoGDI-binding pocket are colored in purple, hydrophobic regions in orange. The hydrophobic residues of RhoGDI involved in interactions with the lipidic group are labeled. (c) Atomic structure of Rac in complex with p67^phox. The structure of the Rac-p67^phox complex (1E96) [49], solved by X-ray diffraction at a 2.4Å resolution, was structurally aligned with the structure of the N-ter extremity of p67^phox (1HH8) [50]. Rac is represented in blue ribbon with switch I and II regions highlighted in yellow and red, respectively. p67^phox is represented in gray ribbon, the activation domain is represented in orange.

Figure 9

Model of the p47^phox activation mechanism showing the link between AIR and PX motif releases. (a) Model of the auto-inhibited p47^phox. This model results from the combination of SAXS and HDXMS characterization on whole p47^phox [59,61] and the insertion of the high-resolution structure of the individual domains: the PX domain (pdb: 1KQ6), autoinhibited tandem SH3, comprising the two SH3 domains locked by the AIR region (pdb: 1NG2) [55] and the polyPro of p47^phox from pdb:1K4U [54]. The PX domain is in gray, tandem SH3 in green, AIR sequence in pink and C-terminus in black, except for the polyPro motif in blue. The phosphorylation target sites involved in the activation of p47^phox (S303/S304/S328) are represented in ball and stick. (b) The insert provides a close-up showing the network of interactions maintaining PX/SH3 and AIR locked alto-gether. Polar interactions occur between residues Arg-162, Ile-164, Glu-211, and Pro-212 from SH3A (green) and residues His-309, Ser-310, Ile-311, and His-312 from AIR (pink). The docking site of the PX domain in the resting state as shown by its release upon mutation on residue 162 and 166 [61]. (c) Phosphorylation of the AIR domain leads to the release of the auto-inhibitory intramolecular interaction between the AIR domain and the bis-SH3 domain, leading to the release of the PX domain and the activation of p47^phox [61]. (d) Structure of p22^phox-p47^phox complex (pdb: 1WLP) solved by NMR [109]. The p47^phox tandem SH3s (aa 151–286) is represented in green ribbon and the p22^phox polyPro (aa 149–168) is represented in gray ribbon. This structure obtained with a truncated recombinant p47^phox bis-SH3 and p22^phox polyPro (residue 146–179) mimics the interaction between p47^phox and p22^phox following AIR release. (e) Recognition of p22^phox-(149–168) (in gray) by the SH3A and SH3B (in green) domains of p47^phox-(151–286). SH3A and SH3B (in green). The side chains of the amino acids involved in the recognition of p22^phox-(149–168) by the SH3A and SH3B domains are shown in the wire model and labelled.

Figure 10

Representation of the NADPH oxidase isoforms. Despite their similar structure and enzymatic functions, the activation mechanisms of the NOX family enzymes differ. NOX1 activity requires p22^phox, NOXO1, NOXA1 and the small GTPase Rac; NOX2 requires p22^phox, p47^phox, p67^phox and Rac. NOX3 requires p22^phox and NOXO1 and may require NOXA1 depending on the species; Rac can participate but its necessity for activity is not clearly established. NOX4 requires p22^phox in vivo and is constitutively active. NOX5, DUOX1 and DUOX2 are activated by Ca²⁺ ions; DUOX1 and DUOX2 require an association with the maturation factors DUOXA1/DUOXA2 for activation. NOX 1, 2, 3 and 5 produce mainly superoxide, Nox4 produces mainly H₂O₂, and DUOX1 and DUOX2 produce both [89].

Figure 11

Emergence of proteins of the NOX or Fre family by the fusion of two ancestral genes. (a) The transmembrane domain homologous to cytochrome b is shown in yellow, and the cytosolic domain homologous to FNR in gray. Figure adapted from [110]. (b) Topological comparison between the two-component system MsrQ/Fre, the prokaryotic homologue SpNOX and eukaryotic NOX and DUOX. The FRD domain embedded in the membrane is shown in blue. The soluble FNR domains bearing the NADPH- and FAD-binding sites are in gray; EF hands and peroxidase-like domains are also shown. The electron acceptors and products of each system are also represented in the corresponding periplasmic/extracellular compartments. Adapted from [221].

Figure 12

Structure of the DH and TM domains of CsNOX and the SANS structure of SpNOX. (a) TM domain of CsNox solved by X-ray crystallography at 2.05Å resolution [64]. The 6 transmembrane helices and the two chelated hemes are labelled. The positions of the bilipidic layer are indicated by the horizontal black lines. (b) The DH domain of CsNox was solved by X-ray crystallography at 2.2Å resolution. The FAD cofactor co-crystallized with the protein is la-belled and the unstructured EF-hand-binding loop is depicted in dotted gray (D611-T634). (c) Some of the conformations generated by Pepsi-SANS along Non-linear Normal Mode Analysis for SpNOX in a semi-transparent ribbon style [226]. The two most distant conformations have been represented in opaque ribbon; between these two configurations, the gap between the D-loop of the TM domain and the FAD-binding site varies from 34 to 43 A, highlighting the flexibility of the inter-domain linker.

Figure 13

Structure of the mouse DUOX1/DUOXA1 complex solved by cryoEM in the inactive dimer of dimer configuration (3.2 Å) and in an active heterodimer state with NADPH at, respectively, (3.3 Å). (a) The structure of DUOX1 is displayed in blue and the structure of DUOXA1 is displayed in yellow. In the dimer of dimer configuration, the cryo-EM map allowed modeling of extracellular domains and TM domains, whilst cytoplasmic domains were too flexible to be resolved. (b) The TM domain of the active heterodimer state is displayed in blue and the DH domain is displayed in gray. The FAD, NADPH and nearby lipid molecules are shown as sticks and balls and colored in yellow, green and black, respectively. (c) The insert corresponds to the frame in black present in b) and provides a close-up view showing the interactions in the region of the lipid-mediated NADPH-binding site. (d) The electron transfer path deduced from the structure of the activated DUOX with calculated distances between players in the electron transfer path. Adapted from [65].

Figure 14

Main roles of ROS produced by NOX2 during the oxidative burst. During phagocytosis, a bacterium is sequestered in the phagosome triggering the release of bactericidal content from several types of vesicles. NOX2 transmembrane electron flow is balanced by proton flow through voltage-gated proton channels. This provides protons to the phagosome interior for the conversion of O₂^●− to H₂O₂ and HOCl and relieves the cytoplasm of protons released by the oxidation of NADPH. Other transporters, including ClC-3 (a Cl/H⁺ antiporter), H⁺ -ATPase and the Na⁺/H⁺ antiporter, also contribute to pHi recovery. Adapted from [239,240].

Figure 15

Mechanism of inflammation in the absence of NOX activation. Sterile inflammation can be triggered by physical, chemical, or metabolic noxious stimuli. In these conditions, the lack of phagocytic NOX activity leads to the overproduction of IL-1α by tissue-resident macrophages, promoting the local production of G-CSF, which induces an excessive infiltration of neutrophils and monocytes at the inflammation site. This leads to increased production of cytokines and pro-inflammatory factors that prolong the inflammation, ultimately resulting in tissue damage. Inspired by [257].

Figure 16

Regulation of phosphatase, kinase and calcium channel signaling pathways by NOX. The NOX-mediated production of superoxide and resulting secondary ROS leads to the oxidation of cysteines on PTPs [295] and calcium channels, resulting in the formation of disulfide bridges. PTP activity opposes that of kinases, thus regulating a large number of proteins involved in a variety of signaling pathways; PTP activity also regulates Ca²⁺ ion flux. Secondary ROS can also activate MAP-Ks, possibly via the ERK1/2 signaling pathway through activation of epidermal growth factor (EGF) receptors, and platelet-derived growth factor (PDGF) receptors, which can stimulate Ras and the subsequent activation of the ERK pathway [296].

Figure 17

Regulation of angiogenesis steps by NOX. Hypoxia conditions activate NOX4 and NOX2, inducing the production of ROS and thereby enhancing VEGFR2 signaling and angiogenesis in ECs. Nox4-derived H₂O₂ also activates NOX2 to promote superoxide production. NOX-mediated ROS promotes lipid peroxidation activating TLR, which regulates cell proliferation. Hyperoxia conditions activate NOX1, leading to the elicitation of cell migration through the VEGF pathway. The ROS-mediated β(IKKβ)/NF-κβ and MAPK pathways can inhibit this process.

Figure 18

Activation mechanism of microglial NOX2 involved in Parkinson’s disease. a-Synuclein-induced micro-glia activation may involve different surface receptors such as P2X7, TLR2/4 and CR3 that activate kinases, leading to phosphorylation of p47^phox and NOX2 activation. Subsequent ROS production leads to migroglial chemoattraction and oxidative stress. Resulting neuronal damage then further activates the release of the protein (HMGB1) and α−synuclein. Figure is adapted from [366].

Figure 19

NOX-mediated neurodegeneration and neuroprotection. In Parkinson’s and Alzheimer’s disease models, the ROS overproduction resulting from the activity of NOX family contributes to neurodegeneration. NOX inhibition mediated by apocynin, DPI or Gp91ds-tat induces neuronal protection. Figure is inspired by [376].

Figure 20

Endogenous sources of ROS and overproduction-related consequences. The overproduction of superoxide anions and H2O2 by NOX enzymes and the subsequent increase in hydroxyl radical levels through Fenton and Haber–Weiss reactions lead to oxidation of lipids, proteins and DNA, and consequently promotes genomic instability, high mutation rate and carcinogenesis. Under these conditions, cell survival or cell death, respectively, depend on the activation of the PI3K or Ask-1 signaling pathways. High levels of ROS stimulate the Ask-1/JNK pathway, leading to cell death, while lower or transient levels of ROS may activate PI3K kinases and Ask-1/JNK inhibition, thus ensuring NF-κβ-mediated cell survival [385]. Inspired by [385].

Figure 21

Invadopodia regulation model modulated by the association of Tks5 with NOX. Initial model proposed by [406] was further adapted to include the formation of Tks-5-NOX complex assembly and subsequent activation of ROS-mediated invapodia formation. Inspired by [406].

Figure 22

Examples of signaling pathways involving NOX1, 2, 4 and 5 in the regulation of the different tumorous stages. NOX1-derived superoxide, activated by thrombin, PDGF or Ang II, can affect cell differentiation via the p38 and ERK1/2 pathways, hypertrophy via p38-mediated Akt activation, cell migration via cSrc (Proto-oncogene tyrosine-protein kinase Src) activation and cell growth via ERK1/2 activation, subsequent activation of transcription factor Ets-1 and upregulation of cyclin D. NOX1 also downregulates the expression and activity of the antiangiogenic receptor PPARα, which is known to inhibit the NF-κB transcription factor and thus angiogenesis. NOX2, activated by TNFα, thrombin or NF-κB-induced ROS, promotes migration and angiogenesis via the Akt and cSrc pathways. NOX2-mediated regulation of angiogenesis also occurs via cadherin, p38 activation or regulation of ERK1/2 activation mediated by NOX4-derived H₂O₂. NOX2 also regulates cell growth via p38 activation or inhibition of ERK1/2 differentiation, and migration via the eiF4E (Eukaryotic translation initiation factor 4E) and pRB pathway. NOX4 expression and activity are promoted by TGF-β1 or Ang II. NOX5, whose superoxide production is activated by IL4 or PDGF, promotes cell growth and inflammation, respectively, through the JAK-2 / STAT3 and NF-κB signaling pathways. Inspired by [385].

Figure 23

Role of NOX in hypertension. Angiotensin II leads to the activation and expression of NOX via the AT₂ receptor, aldosterone, inflammation and increased vascular tone. Multiple factors including prostaglandin E₂ (PGE₂), exercise, estrogen, and angiotensin, via the AT₂ receptor, contribute to limit this process. NOX located in the brain, arteries and kidneys promote the development of hypertension through various mechanisms. In the kidney, for instance, activation of NOX presumably destabilizes the tubulo-glomerular feedback loop and the glomerular filtration rate (GFR) [428].

Figure 24

Model of the crucial role of NOX2 in atherogenic EC generation. Atherogenic LDL levels activate the re-lease of intracellular ascorbic acid via phospholipase A2, leading to the translocation of p47^phox and p67^phox to NOX2. The subsequent production of O₂^●− by NOX leads to increased LDL transfer across the EC, during which the exposure of LDL to high levels of O₂^●− may cause oxidation of LDL (ox-LDL) on the abluminal side of the EC. The ox-LDL thus generated is absorbed by macrophages that are attracted by the accumulation of cell adhesion molecules, thus explaining the pro-duction of foamy cells observed in the early stages of atherosclerosis [438].