We previously demonstrated that some fish have unique response in the form of reactive oxygen species (ROS) production. In the present study, we cloned and sequenced the full-length cDNAs for carp (Cyprinus carpio) phagocyte NADPH oxidase components: gp91phox, p22phox, p47phox, p67phox and p40phox. These amino acid sequences were compared with other teleost and mammalian homologues, to elucidate the features of ROS production of fish neutrophils. The phylogeny analysis clearly demonstrates that the radiation of phagocyte oxidase components took place in the common ancestor of teleosts and mammals. Thereafter, the overall structure and expression pattern of phagocyte oxidase have been highly conserved in two different strains. However the amino acid identity of p67phox and p47phox was relatively lower than the amino acid identities of other components. Moreover, a synteny analysis supports the hypothesis that there was strong selective pressure in the p67phox and p47phox genes. Thus, it is likely that the higher divergence of p67phox and p47phox are responsible for the difference of ROS responses between different species of teleosts.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-mediated reactive oxygen species contribute to various liver diseases, including hepatocellular carcinoma (HCC). Uncertainties remain regarding the prognostic relevance of NOX1 and NOX4 protein expression in HCC.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive loss of memory. The neurodegeneration induced by AD has been linked to oxidative damage. However, little is known about the involvement of NADPH oxidase 2 (Nox2), a multisubunit enzyme that catalyzes the reduction of oxygen to produce reactive oxygen species, in the pathogenesis of AD. The main purpose of this study was to investigate the involvement of Nox2 in memory, in AD-related brain abnormalities, oxidative damage, inflammation and neuronal death in the hippocampus in the streptozotocin (STZ)-induced AD-like state by comparing the effects of that drug on mice lacking gp91phox-/- and wild-type (Wt) mice. Nox2 gene expression was found increased in Wt mice after STZ injection. In object recognition test, Wt mice injected with STZ presented impairment in short- and long-term memory, which was not observed following Nox2 deletion. STZ treatment induced increased phosphorylation of Tau and increased amyloid-β, apoptosis-inducing factor (AIF) and astrocyte and microglial markers expression in Wt mice but not in gp91phox-/-. STZ treatment increased oxidative damage and pro-inflammatory cytokines' release in Wt mice, which was not observed in gp91phox-/- mice. Nox2 deletion had a positive effect on the IL-10 baseline production, suggesting that this cytokine might contribute to the neuroprotection mechanism against STZ-induced neurodegeneration. In summary, our data suggest that the Nox2-dependent reactive oxygen species (ROS) generation contributes to the STZ-induced AD-like state.

Mitochondrial dysfunction and oxidative stress are strongly implicated in Parkinson's disease (PD) pathogenesis and there is evidence that mitochondrially-generated superoxide can activate NADPH oxidase 2 (NOX2). Although NOX2 has been examined in the context of PD, most attention has focused on glial NOX2, and the role of neuronal NOX2 in PD remains to be defined. Additionally, pharmacological NOX2 inhibitors have typically lacked specificity. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and two animal models thereof, both neuronal and microglial NOX2 are highly active in substantia nigra under chronic conditions. However, in acute and sub-acute PD models, we observed neuronal, but not microglial NOX2 activation, suggesting that neuronal NOX2 may play a primary role in the early stages of the disease. Aberrant NOX2 activity is responsible for the formation of oxidative stress-related post-translational modifications of α-synuclein, and impaired mitochondrial protein import in vitro in primary ventral midbrain neuronal cultures and in vivo in nigrostriatal neurons in rats. In a rat model, administration of a brain-penetrant, highly specific NOX2 inhibitor prevented NOX2 activation in nigrostriatal neurons and its downstream effects in vivo, such as activation of leucine-rich repeat kinase 2 (LRRK2). We conclude that NOX2 is an important enzyme that contributes to progressive oxidative damage which in turn can lead to α-synuclein accumulation, mitochondrial protein import impairment, and LRRK2 activation. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD.

NOX1 (NADPH oxidase) similar to phagocyte NADPH oxidase, is expressed mainly in the colon epithelium and it is responsible for host defense against microbial infections by generating ROS (reactive oxygen species). NOX1 is activated by two regulatory cytosolic proteins that form a hetero-dimer, Noxo1 (NOX organizer 1) and Noxa1 (NOX activator 1). The interaction between Noxa1 and Noxo1 is critical for activating NOX1. However no structural studies for interaction between Noxa1 and Noxo1 has not been reported till date. Here, we studied the inter-molecular interaction between the SH3 domain of Noxa1 and Noxo1 using pull-down assay and NMR spectroscopy. 15N/13C-labeled SH3 domain of Noxa1 has been purified for hetero-nuclear NMR experiments (HNCACB, CBCACONH, HNCA, HNCO, and HSQC). TALOS analysis using backbone assignment data of the Noxa1 SH3 domain showed that the structure primarily consists of β-sheets. Data from pull-down assay between the Noxo1 and Noxa1 showed that the SH3 domains (Noxa1) is responsible for interaction with Noxo1 C-terminal tail harboring proline rich region (PRR). The concentration-dependent titration of the Noxo1 C-terminal tail to Noxa1 shows that Noxo1 particularly in the RT loop: Q407*, H408, S409, A412*, G414*, E416, D417, L418, and F420; n-Src loop: C430, E431*, V432*, A435, W436, and L437; and terminal region: I447; F448*, F452* and V454 interact with Noxa1. Our results will provide a detailed understanding for interaction between Noxa1 and Noxo1 at the molecular level, providing insights into their cytoplasmic activity-mediated functioning as well as regulatory role of C-terminal tail of Noxo1 in the NOX1 complex.

Oxidative stress [i.e. increased levels of reactive oxygen species (ROS)] has been suggested as a pathomechanism of different diseases, although the disease-relevant sources of ROS remain to be identified. One of these sources may be NADPH oxidases. However, due to increasing concerns about the specificity of the compounds commonly used as NADPH oxidase inhibitors, data obtained with these compounds may have to be re-interpreted.

The NADPH oxidase (NOX) family of enzymes produces ROS as their sole function and is becoming recognized as key modulators of signal transduction pathways with a physiological role under acute stress and a pathological role after excessive activation under chronic stress. The seven isoforms differ in their regulation, tissue and subcellular localization and ROS products. The most studied are NOX1, 2 and 4. Genetic deletion of NOX1 and 4, in contrast to NOX2, has revealed no significant spontaneous pathologies and a pathogenic relevance of both NOX1 and 4 across multiple organs in a wide range of diseases and in particular inflammatory and fibrotic diseases. This has stimulated interest in NOX inhibitors for therapeutic application. GKT136901 and GKT137831 are two structurally related compounds demonstrating a preferential inhibition of NOX1 and 4 that have suitable properties for in vivo studies and have consequently been evaluated across a range of disease models and compared with gene deletion. In contrast to gene deletion, these inhibitors do not completely suppress ROS production, maintaining some basal level of ROS. Despite this and consistent with most gene deletion studies, these inhibitors are well tolerated and slow or prevent disease progression in a range of models of chronic inflammatory and fibrotic diseases by modulating common signal transduction pathways. Clinical trials in patients with GKT137831 have demonstrated excellent tolerability and reduction of various markers of chronic inflammation. NOX1/4 inhibition may provide a safe and effective therapeutic strategy for a range of inflammatory and fibrotic diseases.

We used apocynin to test the hypothesis that superoxide anion (O(-) (2)) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase underlies the development of diabetic nephropathy in the rat.

NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O2•-), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that lead to deficiency in NOX2 activity correlate with increased susceptibility to bacterial and fungal infections, resulting in chronic granulomatous disease. The core of NOX2 is formed by a heterodimeric transmembrane complex composed of NOX2 (formerly gp91) and p22, but a detailed description of its structural architecture is lacking. Here, we present the structure of the human NOX2 core complex bound to a selective anti-NOX2 antibody fragment. The core complex reveals an intricate extracellular topology of NOX2, a four-transmembrane fold of the p22 subunit, and an extensive transmembrane interface which provides insights into NOX2 assembly and activation. Functional assays uncover an inhibitory activity of the 7G5 antibody mediated by internalization-dependent and internalization-independent mechanisms. Overall, our results provide insights into the NOX2 core complex architecture, disease-causing mutations, and potential avenues for selective NOX2 pharmacological modulation.

Oxidative stress (OS) is a common characteristic of several neurodegenerative disorders, including Parkinson disease (PD). PD is more prevalent in men than in women, indicating the possible involvement of androgens. Androgens can have either neuroprotective or neurodamaging effects, depending on the presence of OS. Specifically, in an OS environment, androgens via a membrane-associated androgen receptor (mAR) exacerbate OS-induced damage. To investigate the role of androgens on OS signaling and neurodegeneration, the effects of testosterone and androgen receptor activation on the major OS signaling cascades, the reduced form of NAD phosphate (NADPH) oxidase (NOX)1 and NOX2 and the Gαq/inositol trisphosphate receptor (InsP3R), were examined. To create an OS environment, an immortalized neuronal cell line was exposed to H2O2 prior to cell-permeable/cell-impermeable androgens. Different inhibitors were used to examine the role of G proteins, mAR, InsP3R, and NOX1/2 on OS generation and cell viability. Both testosterone and DHT/3-O-carboxymethyloxime (DHT)-BSA increased H2O2-induced OS and cell death, indicating the involvement of an mAR. Furthermore, classical AR antagonists did not block testosterone's negative effects in an OS environment. Because there are no known antagonists specific for mARs, an AR protein degrader, ASC-J9, was used to block mAR action. ASC-J9 blocked testosterone's negative effects. To determine OS-related signaling mediated by mAR, this study examined NOX1, NOX2, Gαq. NOX1, NOX2, and the Gαq complex with mAR. Only NOX inhibition blocked testosterone-induced cell loss and OS. No effects of blocking either Gαq or G protein activation were observed on testosterone's negative effects. These results indicate that androgen-induced OS is via the mAR-NOX complex and not the mAR-Gαq complex.

Different factors are involved in thyroid function and proliferation such as thyrotropin (TSH), insulin, growth factors, iodide, etc. TSH and IGF1/insulin increase proliferation rate and stimulate genes involved in thyroid differentiation. In the present study, we analyse the physiological regulation of NOX4 expression by TSH, insulin and iodine, and the role of NOX4 on thyroid genes expression. Differentiated rat thyroid cells (FRTL-5) were incubated in the presence or absence of TSH/insulin and TTF2, PAX8, TPO, NIS, NOX4, TGFβ1, FOXO1/3 mRNA levels were examined by Real Time PCR. We showed that TSH and insulin repress NOX4 expression and appears to be inversely correlated with some thyroid genes. SiRNA targeted knockdown of NOX4 increased mRNA levels of TGFβ1, TPO, PAX8, TTF2, FOXO1 and FOXO3. A PI3K inhibitor (LY294002), increases the expression of NIS, TTF2 and FOXO1/3, however PI3K/AKT pathway does not regulate NOX4 expression. We observed that iodine increased NOX4 expression and knockdown of NOX4 reduced ROS and reversed the inhibitory effect of iodine on NIS, TPO, PAX8 and TTF2 expression. Our findings provide strong evidence that NOX4 could be a novel signaling modulator of TSH/insulin pathway and would have a critical role in the autoregulatory mechanism induced by iodine.

NADPH oxidase 4 (NOX4) is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients), esophagus (12/18 patients), bladder (10/19 patients), ovary (6/17 patients), and prostate (7/19 patients), as well as malignant melanoma (7/15 patients) when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-β1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.

NADPH oxidase 5 (NOX5) is a transmembrane oxidative signaling enzyme which produces superoxide in response to intracellular calcium flux. Increasing evidence indicates that NOX5 is involved in a variety of physiological processes as well as human disease, however, details of NOX5 signaling pathways and targets of NOX5 mediated oxidative modifications remain poorly resolved. Actin dynamics have previously been shown to be modulated by oxidative modification, however, a direct connection to NOX5 expression and activity has not been fully explored. Here we show that NOX5 and actin interact in the cell, and each modulate the activity of the other. Using actin effector molecules jasplakinolide, cytochalasin D and latrunculin A, we show that changes in actin dynamics affect NOX5 superoxide production. In tandem, NOX5 oxidatively modifies actin, and shifts the ratio of filamentous to monomeric actin. Finally, we show that knockdown of NOX5 in the pancreatic cancer cell line PSN-1 impairs cell migration. Together our findings indicate an important link between actin dynamics and oxidative signaling through NOX5.

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O2 saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O2 for 30 sec) and normoxia (21% O2 for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47phox and p67phox to the plasma membrane and their interaction with gp91phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.

Telomere shortening and oxidative stress are involved in the pathogenesis of atherosclerosis. Different studies have shown that phagocytic NADPH oxidase is associated with this disease. This study aimed to investigate the association between phagocytic NADPH oxidase and telomere shortening in human atherosclerosis. To assess this potential association, telomere length and phagocytic NADPH oxidase activity were determined by PCR and chemiluminescence, respectively, in a population of asymptomatic subjects free of overt clinical atherosclerosis. We also measured serum 8-hydroxy-2-deoxyguanosine (8-OHdG) levels (an index of oxidative stress) and carotid intima-media thickness (IMT), a surrogate marker of atherosclerosis. After adjusting them for age and sex, telomere length inversely correlated (p < 0.05) with NADPH oxidase-mediated superoxide production, with 8-OHdG values, and with carotid IMT. Interestingly, the asymptomatic subjects with plaques have a lower telomere length (p < 0.05), and higher values of plasma 8-OHdG and superoxide production (p < 0.05). These data were confirmed in a second population in which patients with coronary artery disease showed lower telomere length and higher 8-OHdG and superoxide production than the asymptomatic subjects. In both studies, NADPH oxidase-dependent superoxide production in phagocytic cells was only due to the specific expression of the Nox2 isoform. In conclusion, these findings suggest that phagocytic NADPH oxidase may be involved in oxidative stress-mediated telomere shortening, and that this axis may be critically involved in human atherosclerosis.

Several experimental studies have indicated that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) exert detrimental effects on ischemic brain tissue; Nox-knockout mice generally exhibit resistance to damage due to experimental stroke following middle cerebral artery occlusion (MCAO). Furthermore, our previous MCAO study indicated that infarct size and blood-brain barrier breakdown are enhanced in mice with pericyte-specific overexpression of Nox4, relative to levels observed in controls. However, it remains unclear whether Nox affects the stroke outcome directly by increasing oxidative stress at the site of ischemia, or indirectly by modifying physiological variables such as blood pressure or cerebral blood flow (CBF). Because of technical problems in the measurement of physiological variables and CBF, it is often difficult to address this issue in mouse models due to their small body size; in our previous study, we examined the effects of Nox activity on focal ischemic injury in a novel congenic rat strain: stroke-prone spontaneously hypertensive rats with loss-of-function in Nox. In this review, we summarize the current literature regarding the role of Nox in focal ischemic injury and discuss critical issues that should be considered when investigating Nox-related pathophysiology in animal models of stroke.

Superoxide production via NADPH-oxidase (NOX) has been shown to play a role in a variety of neurological disorders, including Alzheimer disease (AD). To improve our understanding of the NOX system and cognitive impairment, we studied the various protein components of the phagocytic isoform (gp91(phox), or NOX2) in the frontal and temporal cortex of age- and postmortem-matched samples. Individuals underwent antemortem cognitive testing and postmortem histopathologic assessment to determine disease progression and assignment to one of the following groups: no cognitive impairment (NCI), preclinical AD, mild cognitive impairment (MCI), early AD, and mild-to-moderate AD. Biochemical methods were used to determine overall NOX activity as well as levels of the various subunits (gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox)). Overall enzyme activity was significantly elevated in the MCI cohort in both cortical regions compared to the NCI cohort. This activity level remained elevated in the AD groups. Only the NOX cytosolic subunit proteins (p67(phox), p47(phox), and p40(phox) ) were significantly elevated with disease progression; the membrane-bound subunits (gp91(phox) and p22(phox)) remained stable. In addition, there was a robust correlation between NOX activity and the individual's cognitive status such that as the enzyme activity increased, cognitive performance decreased. Collectively, these data show that upregulated NADPH-oxidase in frontal and temporal cortex suggests that increases in NOX-associated redox pathways might participate in early pathogenesis and contribute to AD progression.

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3- ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.

NADPH oxidase generates reactive oxygen species that are essential to innate immunity against microbes. Like most enzymes, it is sensitive to pH, although the relative importance of pH(o) and pH(i) has not been clearly distinguished. We have taken advantage of the electrogenic nature of NADPH oxidase to determine its pH dependence in patch-clamped individual human eosinophils using the electron current to indicate enzyme activity. Electron current stimulated by PMA (phorbol myristate acetate) was recorded in both perforated-patch configuration, using an NH4+ gradient to control pH(i), and in excised, inside-out patches of membrane. No electron current was detected in cells or excised patches from eosinophils from a patient with chronic granulomatous disease. When the pH was varied symmetrically (pH(o) = pH(i)) in cells in perforated-patch configuration, NADPH oxidase-generated electron current was maximal at pH 7.5, decreasing drastically at higher or lower values. Varying pH(o) and pH(i) independently revealed that this pH dependence was entirely due to effects of pH(i) and that the oxidase is insensitive to pH(o). Surprisingly, the electron current in inside-out patches of membrane was only weakly sensitive to pH(i), indicating that the enzyme turnover rate per se is not strongly pH dependent. The most likely interpretation is that assembly or deactivation of the NADPH oxidase complex has one or more pH-sensitive steps, and that pH-dependent changes in electron current in intact cells mainly reflect different numbers of active complexes at different pH.

ROS in cancer cells play a key role in pathways regulating cell death, stemness maintenance, and metabolic reprogramming, all of which have been implicated in resistance to chemo/ immunotherapy. Adjusting ROS levels to reverse the resistance of cancer cells without impairing normal cell functions is a new therapeutic avenue. In this paper, we describe new inhibitors of NADPH oxidase (NOX), a key enzyme in many cells of the tumor microenvironment. The first inhibitor, called Nanoshutter-1, NS1, decreased the level of tumor-promoting "M2" macrophages differentiated from human blood monocytes. NS1 disrupted the active NADPH oxidase-2 (NOX2) complex at the membrane and in the mitochondria of the macrophages, as shown by confocal microscopy. As one of the characteristics of tumor invasion is hypoxia, we tested whether NS1 would affect vascular reactivity by reducing ROS or NO levels in wire and pressure myograph experiments on isolated blood vessels. The results show that NS1 vasodilated blood vessels and would likely reduce hypoxia. Finally, as both NOX2 and NOX4 are key proteins in tumors and their microenvironment, we investigated whether NS1 would probe these proteins differently. Models of NOX2 and NOX4 were generated by homology modeling, showing structural differences at their C-terminal NADPH site, in particular in their last Phe. Thus, the NADPH site presents an unexploited chemical space for addressing ligand specificity, which we exploited to design a novel NOX2-specific inhibitor targeting variable NOX2 residues. With the proper smart vehicle to target specific cells of the microenvironment as TAMs, NOX2-specific inhibitors could open the way to new precision therapies.