NADPH oxidases: redox regulation of cell homeostasis and disease

Abstract

The redox signaling network in mammals has garnered enormous interest and taken on major biological significance in recent years as the scope of NADPH oxidases (NOXs) as regulators of physiological signaling and cellular degeneration has grown exponentially. All NOX isoforms have in common the capacity to generate reactive oxygen species (ROS) superoxide anion (O₂^•-) and/or hydrogen peroxide (H₂O₂). A baseline, normal level of ROS formation supports a wide range of processes under physiological conditions. A disruption in redox balance caused by either the suppression or "super" induction of NOX off balance with antioxidant systems is associated with myriad diseases and cell/tissue damage. Over the past two to three decades, our understanding of NOXs has progressed from almost entirely a phagocyte-, antimicrobial-centered perspective to that of a family of enzymes that is vital to broad cellular function and organismal homeostasis. It is becoming increasingly evident that highly regulated, targeted oxidative protein modifications are elicited in a spatiotemporal manner and initiated at cell membranes in humans by seven NOX isoforms [NOXs 1, 2, 3, 4, 5 and dual oxidases (DUOXs) 1 and 2]. In a sense, this renders NOX-ROS signaling akin to that of other second messenger systems involving localized Ca²⁺ dynamics and tyrosine kinase transactivation. Accordingly, the study of ROS compartmentalization in subcellular organelles has been shown to be crucial to elucidating their role in cell phenotype modulation under physiological and pathophysiological conditions. The NOXs are as distinct in their distribution and activation as they are in their cellular functions, ranging from host defense, second messenger posttranslational modifications (PTMs) to transcriptional, epigenetic, and (de)differentiating effects. This review integrates past knowledge in the field with new focus areas on the leading edge of NOX-centered ROS signaling, including how a new wave of structural information provides insights for NOX biology and targeted therapies.

Keywords: NADPH oxidase; NOX; disease; physiology; redox signaling.

Figure 1.. NADPH oxidase (NOX) family members.

NOXs comprise a family of transmembrane proteins with 6 or 7 transmembrane (TM) domains. NOX1–3 require assembly of cytosolic regulatory subunits (NOXA1, NOXO1, p47^phox, p67^phox and Rac1/2) for activation. NOX4 is constitutively active though its catalytic activity is increased by Poldip2 and tyrosine kinase substrates with 4 or 5 Src (SH3) homology domains (Tks 4/5). Tks 4/5 can also activate NOX1. NOXs 1–4 catalytic subunits are associated with p22^phox which stabilizes the complex. NOX5 and DUOX1 & 2 do not bind to p22^phox but have 4 or 2 Ca²⁺ binding sites, EF-hands, respectively. NOX5 possesses a calmodulin binding-domain, and its stability is regulated by heat shock protein 90 (HSP90). DUOX1 & 2 have an extra TM from which a peroxidase-like domain extends extracellularly at the N-terminus of the isoform.

Figure 2.. Identifying domain maps of NOX membrane and cytosolic regulatory subunits.

Structural domains within membrane and cytosolic subunits of the NOX isoforms. NOXs 1 – 5 contain requisite 6 transmembrane domains (TM) and cytosolic NADPH and FAD binding domains. NOX5 and DUOXs 1 & 2 possess Ca²⁺ binding EF hand domains toward their N-terminus; wherein the DUOXs 1 & 2 also contain an extra TM domain with a tethered peroxidase-like domain. A hallmark proline rich domain (PRR) in p22^phox, p47^phox and NOXO1 is key for its interaction with Src homology domains (SH3) within p47^phox, p67^phox and NOXA1, respectively. Tetratricopeptide repeats (TPRs) in p67^phox permits its binding to Rac1/2 and a Phox and Bem1 (PB1) domain is required for its interaction with p40^phox. PX domains in p47^phox, p40^phox and NOXO1 facilitate binding to phosphoinositides on the plasma membrane. Different domains in Rac1 include the nucleotide binding sites (NBS), switch 1, switch 2, polybasic region (PRB), insert region (IR) and the CAAX box.

Figure 3.. Activation of NOX2 is driven by p47^phox S-379 phosphorylation leading to the interaction between regulatory and membrane components.

In the resting state, NOX2 oxidase is disassociated into its subunits and cofactors, while NOX2 and p22^phox are in the membrane, p47^phox–p67^phox – p40^phox are in an associated, yet dephosphorylated, state in a soluble trimeric complex, in which the p67^phox and p40^phox PB1 domains bind and a SH3 domain at the C-terminus of p67^phox binds to the PRR of p47^phox. At this time, the SH3 super-groove and PX domain of p47^phox are masked by the polybasic auto-inhibitory region (AIR) which restricts p47^phox to its folded and inactive state. Upon activation by diverse stimuli, protein kinase C (PKC) gets activated and phosphorylates p47^phox on critical serines, this in turn disrupts the hydrogen bonds linking the C-terminal AIR and the tandem SH3 domains and exposes the supergroove pocket. This allows for the secondary phosphorylation of serine residues (S379) (see insert) permitting translocation and binding of p47^phox to p22^phox’s PRR domain. p47^phox and p40^phox acting through hallmark binding domains outlined in Figure 2 function to engage p67^phox and Rac1 and scaffold the entire complex in place for NOX2 activation.

Figure 4.. Tissue distribution of the NOX in human tissues.

Graphs correspond to the consensus dataset of expression levels/tissue expressed as RPKM (reads per kilobase of transcript per million mapped reads) created by HPA and GTEx transcriptomics datasets. Y axis in each graph is adjusted to the maximum expression for the given NOX. Anatomical display of the expression levels shown in males. Adapted from https://www.proteinatlas.org/. More details and information can be found under HELP: Assays & Annotations on the Protein atlas website. Note that the website is constantly been updated with new information, new tissues, etc.

Figure 5.. Model of compartmentalized NOX-ROS downstream signaling. Constellation of intracellular mechanisms channeling NOX-derived H₂O₂ to kinase targets and various phenotype shifts.

Diagrammatic representation of how oxidative and phosphorylative mechanisms in the vicinity of the NOX2 source in lipid rafts can theoretically channel NOX-derived H₂O₂ for redox signaling. In this scenario, growth factor receptor (GFR) transactivates and stimulates NOX2 assembly and H₂O₂ production which, in turn, activates a target kinase. GFR’s phosphorylation and inhibition of peroxiredoxin I (Prx I) prevent its degradation of NOX2-derived H₂O₂. Simultaneously, NOX2 H₂O₂ oxidatively inactivates Prx II further augmenting steady-state H₂O₂ in the vicinity of NOX2 and its target kinase. Additionally, H₂O₂ via cysteine oxidation of phosphatases inhibits phosphatase activity which is permissive of target kinase activity and feedforward signaling. Away from NOX2, Prx I and II remain active and scavenge H₂O₂ from other potentially interfering sources. Collectively, these effects protect and allow localized H₂O₂ to rise close to target kinase(s) and propagate downstream signaling leading to an array of phenotypic shifts. In sum, these reactions restrict and direct NOX- H₂O₂ while, in other areas of the cell, active Prxs degrade H₂O₂ preventing interference with signaling (see section 2.4).

Figure 6.. NOX2 regulation of NFκB is at the intersection between inflammatory and autoimmune disease.

In macrophages, NOX2-derived oxidants prevent thioredoxin-1 (TRX1) reduction by thioredoxin reductase (TR-1) keeping TRX1 from entering the nucleus, thereby preventing NFκB-mediated pro-inflammatory cytokine production (TNFα, IL1 and IL6) and inflammatory disease (A). In contrast, regulatory T-cells (Tregs) play an essential role in protection against autoimmunity by suppressing T-cell responses. In NOX2-deficient Tregs, TRX1 levels accumulate in the nucleus and augment NFκB-mediated transcription of immunosuppressive cytokines (CD25, CD39 and CD73), which in turn, cause an increase in Treg-suppressive activity and the infiltration/proliferation of effector T cells, thus, inhibiting a hyperimmune/autoimmune response, and transplant rejection (B).

Figure 7.. NOX signaling in vascular tone maintenance and diseases of the cardiovascular system.

Stimuli such as hypoxia, AngII or a high fat diet increase expression and enzymatic activity of NOX1, NOX2 and NOX4. O₂^·- production from NOX1 and NOX2 decreases nitric oxide (NO) availability causing an increase in vascular tone. Furthermore, increased oxidative stress leads to activation of HIF1 and its target genes such as VEGF, a master regulator of proliferation that underpins vascular remodeling seen in many vascular pathologies. NOX1 and NOX2 and NOX5 also acting via the activation of NFκB and decreased NO promote pro-inflammatory effects on vascular tissue leading to vascular remodeling, neointimal growth and atherosclerosis. Distinctly, NOX4 is generally deemed vasculoprotective in part the consequence of H₂O₂’s vasorelaxant properties and, in part, via activation of Nrf2. NOX5, activated through calcium-binding and acting via redox activation of kinases such as c-Src is involved in vascular hypercontractility and vascular dysfunction in hypertension and atherogenesis. A combination of more than one NOX is observed in cardiovascular diseases. MMP2/9: Matrix Metalloproteinase 2/9; NFκB: Nuclear Factor Kappa B; VEGF: Vascular Endothelial Growth Factor; HIF1: Hypoxia inducible factor 1. c-Src: Proto-oncogene, non-receptor tyrosine kinase Src.

Figure 8.. NOX activation, signaling and lung disease phenotypes.

Damaging stimuli, such as microbes, cigarette smoking and pollution activate distinct NOXs beyond their physiological signaling and stimulate their participation in the development of various lung diseases including lung injury, fibrosis, COPD and asthma. NOX2 is reportedly fundamental to the activation of ENaC channels and alveolar fluid clearance by epithelial cells and thus is deemed homeostatic in the lung. On the other hand, NOX2 can effect lung injury via NFκB-mediated pathways. Lung fibrosis is under the effect of NOX4 expression. COPD involves the participation of NOX1, NOX2 and NOX4 and part of that pathology is mediated via the suppression of SIRT1 and disinhibition of MMP9. Asthma is brought about by NOX2, NOX4 and DUOX1 through a variety of factors including CK2α-mediated NFκB as well as, in the case of DUOX, EGFR and activation of pro-inflammatory cytokines. While NOX5 has been correlated with a number of these disorders, no studies to date appear to have demonstrated causality in in vitro or in vivo models. COPD: Chronic obstructive pulmonary disease; CK2 α: casein kinase 2α; SIRT1: member of the sirtuin family; MMP9: Matrix Metalloproteinase 9; EGFR: epidermal growth factor.

Figure 9.. NOXs in human cancer.

Differential expression between tumor and adjacent normal tissues for NOX enzymes across tumor types in The Cancer Genome Atlas (TCGA). Distributions of gene expression levels are displayed using box plots. Genes that are upregulated or downregulated in the tumors are compared to normal tissues for each cancer type. mRNA expression of the indicated NOX isoform in normal (blue) and tumor (red) samples, each dot represents a different patient obtained from the TCGA database (https://portal.gdc.cancer.gov). BLCA=Bladder Urothelial Carcinoma, BRCA=Breast invasive carcinoma, CESC=Cervical squamous cell carcinoma and endocervical adenocarcinoma, CHOL=Cholangiocarcinoma, COAD=Colon adenocarcinoma, ESCA=Esophageal carcinoma, GBM=Glioblastoma multiforme, HNSC=Head and Neck squamous cell carcinoma, KICH=Kidney Chromophobe, KIRC=Kidney renal clear cell carcinoma, KIRP=Kidney renal papillary cell carcinoma, LIHC=Liver hepatocellular carcinoma, LUAD=Lung adenocarcinoma, LUSC=Lung squamous cell carcinoma, PAAD=Pancreatic adenocarcinoma, PCPG=Pheochromocytoma and Paraganglioma, PRAD=Prostate adenocarcinoma, READ=Rectum adenocarcinoma, SKCM=Skin Cutaneous Melanoma, STAD=Stomach adenocarcinoma, THCA=Thyroid carcinoma, UCEC=Uterine Corpus Endometrial Carcinoma. Wilcoxon test showing significance between tumor and adjacent tissue at * p<0.05, **p<0.01, ***p<0.001.

Figure 9.. NOXs in human cancer.

Differential expression between tumor and adjacent normal tissues for NOX enzymes across tumor types in The Cancer Genome Atlas (TCGA). Distributions of gene expression levels are displayed using box plots. Genes that are upregulated or downregulated in the tumors are compared to normal tissues for each cancer type. mRNA expression of the indicated NOX isoform in normal (blue) and tumor (red) samples, each dot represents a different patient obtained from the TCGA database (https://portal.gdc.cancer.gov). BLCA=Bladder Urothelial Carcinoma, BRCA=Breast invasive carcinoma, CESC=Cervical squamous cell carcinoma and endocervical adenocarcinoma, CHOL=Cholangiocarcinoma, COAD=Colon adenocarcinoma, ESCA=Esophageal carcinoma, GBM=Glioblastoma multiforme, HNSC=Head and Neck squamous cell carcinoma, KICH=Kidney Chromophobe, KIRC=Kidney renal clear cell carcinoma, KIRP=Kidney renal papillary cell carcinoma, LIHC=Liver hepatocellular carcinoma, LUAD=Lung adenocarcinoma, LUSC=Lung squamous cell carcinoma, PAAD=Pancreatic adenocarcinoma, PCPG=Pheochromocytoma and Paraganglioma, PRAD=Prostate adenocarcinoma, READ=Rectum adenocarcinoma, SKCM=Skin Cutaneous Melanoma, STAD=Stomach adenocarcinoma, THCA=Thyroid carcinoma, UCEC=Uterine Corpus Endometrial Carcinoma. Wilcoxon test showing significance between tumor and adjacent tissue at * p<0.05, **p<0.01, ***p<0.001.

Figure 10.. c-Met/NOX4 axis contributes to metastasis and drug resistance in BRAF mutated melanoma.

Proposed role of the c-MET axis and NOX4 in melanoma resistance and progression. Putative NOX4-mediated oxidation of BRAF at CR1 heightens NRAS leads to epithelial to mesenchymal transition and resistance to BRAF inhibitors in BRAF-mutated melanoma. BRAF proteins have 3 conserved (CR1, CR2, CR3), CR1 contains Ras binding domain where NRAS can bind, and a cysteine-rich subdomain that can be oxidized by NOX4-derived H2O2 leading to exaggerated resistance to BRAF inhibitors and melanoma progression. CR, Conserved region; RBD, Ras binding domain; CRB, cysteine-rich subdomain; AS, activation segment; NRAS: neuroblastoma Ras viral oncogene homolog.

Figure 11.. Salient non-myeloid NOX signaling pathways in cancer development and progression.

Non-myeloid NOX2-derived ROS may facilitate cancer expansion by promotion of cell proliferation and a phenomenon described as “apoptotic escape”; ROS can evoke the capacity to redirect osteosarcoma from programmed death to survival. Adaptor Hic-5, which reportedly suppresses NOX4, is downregulated in metastatic cancer cells and unleashes NOX4-mediated invasiveness. NOX4-derived H2O2 causes oxidation of CR1 in BRAF propagating exaggerated NRAS-mediated MEK and ERK activation, and melanoma progression. NOX4 overexpression is linked to EMT and invasion via JAK2/STAT3. DUOX2 is implicated in PKC-induced Akt/MAPK activation and proliferation, migration, and invasion in colon cancer. NOX1 via MAPK induces cyclin D1 and proliferation as well as activates ADAM17-EGFR-PI3K-Akt signaling and increases the expansion of colon cancer cells.

Figure 12.. Location of NOX single nucleotide variations (SNVs) and single nucleotide polymorphisms (SNPs).

Diagrammatic representation of the specific locations of SNVs (red dots) and SNPs (black dots) in NOX catalytic subunits discussed in the text. EFh, EF-hand domain (calcium-binding domain); FAD, flavin adenine dinucleotide domain; FRD, ferric reductase domain; NADPH, nicotinamide-adenine dinucleotide phosphate domain; Peroxidase, peroxidase domain; TM, transmembrane domain. Note: For visualization purposes, only NOXs that contain pertinent coding non-synonymous mutations discussed in the manuscript are shown.

Figure 13.. Localization of single nucleotide variations (SNVs, black font) and single nucleotide polymorphisms (SNPs, gray font) for NOX2.

Virtual 3D-image was obtained from the protein data bank (PDB) with the ID# 8GZ3, and edited using the software UCSF Chimera, version 1.17.2 (May 2024). Briefly, chain B (cytochrome b-245 heavy chain, NOX2) of PDB 8GZ3 was isolated and colorized according to domain classifications: 6 transmembrane domains (TMDs) are depicted in orange, extracellular regions are depicted in gray, and intracellular regions are in magenta. Location of five mutations discussed in the manuscript are highlighted in cyan and magnified to show the amino acid side chain. These include rs137854588 (R73*, intracellular), rs137854591 (R91*, intracellular), rs139670417 (R229H, extracellular), rs137854585 (P415H, intracellular), and rs13306300 (G472S, intracellular) (Table 2). Bold text indicates an SNP. If the mutations result in the substitution of an amino acid, the residue represented in the structure is the changed amino acid. If the mutations result in a termination codon it is denoted by * and no change in amino acid is represented. Note that rs137854593 and rs151344490 (at amino acids 500 and 505) could not be represented because structural data at the C-terminal region was disordered and remains undetermined. Eighty-five mutations spanning extracellular, TMD and intracellular domains causing chronic granulomatous disease are shown in green (from Magnani, et al. (721), Table S5) are highlighted in green. Note that mutations Ala488Asp, His495Pro, Asp500Tyr/Phe/His/Asn/Gly, and Leu505Arg could not be represented because structural data was disordered and remains undetermined.