Dual oxidases (DUOX) are enzymes that contain an NADPH oxidase domain that produces hydrogen peroxide (H2O2) and a peroxidase domain that can utilize H2O2 to carry out a variety of reactions. The model organism Caenorhabditis elegans produces the DUOX, BLI-3, which has roles in both cuticle development and in protection against infection. In previous work, we demonstrated that while certain peroxidases were protective against the human bacterial pathogen Enterococcus faecalis, the peroxidase domain of BLI-3 was not, leading to the postulate that the NADPH oxidase domain is the basis for BLI-3's protective effects. In this work, we show that a strain carrying a mutation in the NADPH oxidase domain of BLI-3, bli-3(im10), is more susceptible to E. faecalis and the human fungal pathogen Candida albicans. Additionally, less H2O2 is produced in response to pathogen using both an established Amplex Red assay and a strain of C. albicans, WT-OXYellow, which acts as a biosensor of reactive oxygen species (ROS). Finally, a C. elegans line containing a BLI-3::mCherry transgene was generated. Previous work suggested that BLI-3 is produced in the hypodermis and the intestine. Expression of the transgene was observed in both these tissues, and additionally in the pharynx. The amount and pattern of localization of BLI-3 did not change in response to pathogen exposure.

A small series of nitro group-bearing enamides was designed, synthesized (NEA1-NEA5), and evaluated for their inhibitory profiles of monoamine oxidases (MAOs) and β-site amyloid precursor protein cleaving enzyme 1 (β-secretase, BACE1). Compounds NEA3 and NEA1 exhibited a more potent MAO-B inhibition (IC50 value = 0.0092 and 0.016 µM, respectively) than the standards (IC50 value = 0.11 and 0.14 µM, respectively, for lazabemide and pargyline). Moreover, NEA3 and NEA1 showed greater selectivity index (SI) values toward MAO-B over MAO-A (SI of >1652.2 and >2500.0, respectively). The inhibition and kinetics studies suggested that NEA3 and NEA1 are reversible and competitive inhibitors with Ki values of 0.013 ± 0.005 and 0.0049 ± 0.0002 µM, respectively, for MAO-B. In addition, both NEA3 and NEA1 showed efficient BACE1 inhibitions with IC50 values of 8.02 ± 0.13 and 8.21 ± 0.03 µM better than the standard quercetin value (13.40 ± 0.04 µM). The parallel artificial membrane permeability assay (PAMPA) method demonstrated that all the synthesized derivatives can cross the blood-brain barrier (BBB) successfully. Docking analyses were performed by employing an induced-fit docking approach in the GLIDE module of Schrodinger, and the results were in agreement with their in vitro inhibitory activities. The present study resulted in the discovery of potent dual inhibitors toward MAO-B and BACE1, and these lead compounds can be fruitfully explored for the generation of newer, clinically active agents for the treatment of neurodegenerative disorders.

The gut is densely inhabited by commensal bacteria, which metabolize dietary fibers/undigested carbohydrates and produce short-chain fatty acids such as acetate. GPR43 is one of the receptors to sense short-chain fatty acids, and expressed in various immune and non-immune cells. Acetate/GPR43 signaling has been shown to affect various inflammatory diseases through Th17 responses and NADPH oxidase (NOX)-derived reactive oxygen species (ROS) generation. However, no study has previously explored the effects of GPR43 activation during psoriasis-like inflammation. Therefore, this study investigated the effect of acetate/phenylacetamide (GPR43 agonists) on imiquimod induced skin inflammation in mice. Mice were administered phenylacetamide/acetate followed by assessment of skin inflammation, NOXs (NOX-2, NOX-4, dual oxidases), and Th17 related signaling. Our study showed induction of epidermal GPR43 after imiquimod treatment, i.e. psoriasis-like inflammation. Acetate administration in psoriatic mice led to further increase in skin inflammation (ear thickness/myeloperoxidase activity) with concurrent increase in Th17 immune responses and epidermal dual oxidase-2 signaling. Further, topical application of GPR43 agonist, phenylacetamide led to enhanced ear thickness with concomitant epidermal IL-6 signaling as well as dual oxidase-2 upregulation which may be responsible for increased psoriasis-like inflammation. Taken together, dual oxidase-2 and IL-6 play important roles in GPR43-mediated skin inflammation. The current study suggests that GPR43 activation in psoriatic patients may lead to aggravation of psoriatic inflammation.

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase-activated receptor 2 (PAR-2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX-2, have been shown to be involved in airway inflammation in response to Toll-like receptor activation. However, the association between PAR-2 and DUOX-2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX-2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR-2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX-2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl-2, caspase-3). Challenge with CE led to up-regulation of DUOX-2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD-1068, a small molecule antagonist of PAR-2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX-2/ROS pathway; however, these effects were less pronounced than PAR-2 antagonism. The current study suggests that PAR-2 activation leads to up-regulation of the DUOX-2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.

Thyroid hormone synthesis requires H2O2, produced by two NADPH oxidases, Duox1 and Duox2. To be fully active at the apical pole of the thyrocytes, these enzymes need additional maturation factors DuoxA1 and DuoxA2. The proteins have been shown to be localized at the cell surface, suggesting that they could form a complex with Duox counterparts. We have generated multiple HEK293 Tet-On3G cell lines that express various combinations of DuoxA upon doxycycline induction, in association with a constitutive expression of the Duox enzyme. We compared Duox specific activity, Duox/DuoxA cell surface interactions and the cellular consequences of sustained H2O2 generation. By normalizing H2O2 extracellular production by Duox or DuoxA membrane expression, we have demonstrated that the most active enzymatic complex is Duox2/DuoxA2, compared to Duox1/DuoxA1. A direct cell surface interaction was shown between Duox1/2 and both DuoxA1 and DuoxA2 using the Duolink® technology, Duox1/DuoxA1 and Duox2/DuoxA2 membrane complexes being more stable than the unpaired ones. A significant increase in DNA damage was observed in the nuclei of Duox2/DuoxA2 expressing cells after doxycycline induction and stimulation of Duox catalytic activity. The maturation and activity of Duox2 were drastically impaired when expressed with the glycosylation-defective maturation factor DuoxA2, while the impact of the unglycosylated DuoxA1 mutant on Duox1 membrane expression and activity was rather limited. The present data demonstrate for the first time that H2O2 produced by the Duox2/DuoxA2 cell surface enzymatic complex could provoke potential mutagenic DNA damage in an inducible cellular model, and highlight the importance of the co-expressed partner in the activity and stability of Duox/DuoxA complexes.

NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.

NADPH oxidases (NOXes) and dual oxidases (DUOXes) generate O2 (.-) and H2O2. Diphenyleneiodonium (DPI) inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog (99m)TcO4 (-) nor of the K(+) cation mimic (86)Rb(+) in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer's disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands.

Inflammation and the excess production of reactive oxygen species (ROS) contribute significantly to the pathogenesis of ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity. We hypothesized that GKT137831, a dual inhibitor of NADPH oxidases (NOX) 1 and NOX4, reduces inflammation in the ischemic retina by dampening the pro-inflammatory phenotype of retinal immune cells as well as macroglial Müller cells and neurons.

Multiple kinds of monoamine-based antidepressants have been shown prophylactic effects in experimentally induced gastric ulcer. The loss of redox homeostasis plays a principle role in the development of peptic mucosal damage. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are one of the most important sources of reactive oxygen species within the gastrointestinal tract. It is unclear whether there are some common NADPH oxidases modulated by monoamine-based antidepressants in different gastric mucosal damage models. We explored the effects of selective serotonin-norepinephrine reuptake inhibitor (SNRI) duloxetine on the reactive oxygen species production and antioxidant capacity in the gastric mucosa of water immersion restraint (WIRS) or indomethacin treated rats, and examined the role of NADPH oxidases in the protective effects. Pretreated duloxetine prevented the increase of gastric mucosal NADPH oxidase activity and NADPH oxidase inhibitor apocynin dose-dependently protected gastric mucosa from damage by the two factors. Furthermore, dual oxidase 2 (DUOX2) and NADPH oxidase4 (NOX4) are involved in the protective effects of duloxetine in both models. We then examined NADPH oxidases expression modulated by the other monoamine-based antidepressants including selective serotonin reuptake inhibitor (SSRIs) fluoxetine, tricyclic agent (TCAs) amitriptyline and monoamine oxidase inhibitor (MAOs) moclobemide in the two models, and all the three antidepressants reduced the DUOX2 expression in the gastric mucosa. So DUOX2 was a common modulator in the preventive effects of all the monoamine-based antidepressants on WIRS- and indomethacin-induced gastric lesion. Our work provided a peripheral joint molecular target for monoamine modulatory antidepressants, which may be helpful to reveal the mechanisms of this kind of drugs more than monoamine regulation.

The biological roles of the dual oxidases, DUOX1 and DUOX2, are dependent upon the tissue in which they are expressed. However, the mechanisms that control DUOX expression in these tissues are largely unexplored. Given the known role of DUOX for host defense in the gut and respiratory tract, we characterized potential mechanisms that control DUOX2 expression in response to interferon gamma (IFNgamma) in respiratory tract epithelium. We discovered that IFNgamma-mediated DUOX2 expression was regulated by a STAT-independent, JAK-independent pathway. These data provide insights into a novel IFNgamma signaling pathway with potential importance for regulation of host defense responses.

Cigarette smoke (CS) exposure and intrinsic factors such as the NADPH oxidases produce high levels of reactive oxygen species (ROS), ensuing inflammatory tissue injury. We previously demonstrated that CS-generated ROS, particularly hydrogen peroxide (H2O2), impaired adenosine stimulated wound repair. We hypothesized that CS exposure modulates expression of Dual oxidase 1 (Duox-1), a NADPH oxidases known to generate H2O2. To test this hypothesis, we used human bronchial epithelial cell line Nuli-1 and C57BL/6 mice. Cells were treated with 5% CS extract (CSE) for various periods of time, and mice were exposed to whole body CS for six weeks. Both CSE and CS treatment induced increased expression of Duox-1, and silencing of Doux-1 improved the rate of cell wound repair induced by CSE treatment. Nuli-1 cells pretreated with thapsigargin but not calcium ionophore exhibited increased Duox-1 mRNA expression. CSE treatment stimulated PKCα activation, which was effectively blocked by pretreatment with diphenylene iodonium, a NADPH oxidase inhibitor. Compared to control, lungs from CS-exposed mice showed a significant increase in PKCα activity and Duox-1 expression. Collectively, the data demonstrated that CS exposure upregulates expression of Duox-1 protein. This further leads to H2O2 production and PKCα activation, inhibiting A2AAR-stimulated wound repair.

As the key producers of reactive oxygen species (ROS), NADPH oxidases (NOXs), also known as respiratory burst oxidase homologs (RBOHs), play crucial roles in various biological processes in plants with considerable evolutionary selection and functional diversity in the entire terrestrial plant kingdom. However, only limited resources are available on the phylogenesis and functions of this gene family in wheat. Here, a total of 46 NOX family genes were identified in the wheat genome, and these NOXs could be classified into three subgroups: typical TaNOXs, TaNOX-likes, and ferric reduction oxidases (TaFROs). Phylogenetic analysis indicated that the typical TaNOXs might originate from TaFROs during evolution, and the TaFROs located on Chr 2 might be the most ancient forms of TaNOXs. TaNOXs are highly expressed in wheat with distinct tissue or organ-specificity and stress-inducible diversity. A large-scale expression and/or coexpression analysis demonstrated that TaNOXs can be divided into four functional groups with different expression patterns under a broad range of environmental stresses. Different TaNOXs are coexpressed with different sets of other genes, which widely participate in several important intracellular processes such as cell wall biosynthesis, defence response, and signal transduction, suggesting their vital but diversity of roles in plant growth regulation and stress responses of wheat.

FAD-dependent lysine-specific demethylase 1 (LSD1) is overexpressed or deregulated in many cancers such as AML and prostate cancer and hence is a promising anticancer target with first inhibitors in clinical trials. Clinical candidates are N-substituted derivatives of the dual LSD1-/monoamine oxidase-inhibitor tranylcypromine (2-PCPA) with a basic amine function in the N-substituent. These derivatives are selective over monoamine oxidases. So far, only very limited information on structure-activity studies about this important class of LSD1 inhibitors is published in peer reviewed journals. Here, we show that N-substituted 2-PCPA derivatives without a basic function or even a polar group are still potent inhibitors of LSD1 in vitro and effectively inhibit colony formation of leukemic cells in culture. Yet, these lipophilic inhibitors also block the structurally related monoamine oxidases (MAO-A and MAO-B), which may be of interest for the treatment of neurodegenerative disorders, but this property is undesired for applications in cancer treatment. The introduction of a polar, non-basic function led to optimized structures that retain potent LSD1 inhibitors but exhibit selectivity over MAOs and are highly potent in the suppression of colony formation of cultured leukemic cells. Cellular target engagement is shown via a Cellular Thermal Shift Assay (CETSA) for LSD1.

Plants respond to oxygen deprivation by activating the expression of a set of hypoxia-responsive genes (HRGs). The master regulator of this process is a small group of transcription factors belonging to group VII of the ethylene response factors (ERF-VIIs). ERF-VIIs are highly unstable under aerobic conditions due to the continuous oxidation of their characteristic Cys residue at the N terminus by plant cysteine oxidases (PCOs). Under hypoxia, PCOs are inactive and the ERF-VIIs activate transcription of the HRGs required for surviving hypoxia. However, if the plant exposed to hypoxia has limited sugar reserves, the activity of ERF-VIIs is severely dampened. This suggests that oxygen sensing by PCO/ERF-VII is fine-tuned by another sensing pathway, related to sugar or energy availability. Here, we show that oxygen sensing by PCO/ERF-VII is controlled by the energy sensor target of rapamycin (TOR). Inhibition of TOR by genetic or pharmacological approaches leads to a much lower induction of HRGs. We show that two serine residues at the C terminus of RAP2.12, a major ERF-VII, are phosphorylated by TOR and are needed for TOR-dependent activation of transcriptional activity of RAP2.12. Our results demonstrate that oxygen and energy sensing converge in plants to ensure an appropriate transcription of genes, which is essential for surviving hypoxia. When carbohydrate metabolism is inefficient in producing ATP because of hypoxia, the lower ATP content reduces TOR activity, thus attenuating the efficiency of induction of HRGs by the ERF-VIIs. This homeostatic control of the hypoxia-response is required for the plant to survive submergence.

In the course of an infection, Salmonella enterica occupies diverse anatomical sites with various concentrations of oxygen (O2) and nitric oxide (NO). These diatomic gases compete for binding to catalytic metal groups of quinol oxidases. Enterobacteriaceae express two evolutionarily distinct classes of quinol oxidases that differ in affinity for O2 and NO as well as stoichiometry of H+ translocated across the cytoplasmic membrane. The investigations presented here show that the dual function of bacterial cytochrome bd in bioenergetics and antinitrosative defense enhances Salmonella virulence. The high affinity of cytochrome bd for O2 optimizes respiratory rates in hypoxic cultures, and thus, this quinol oxidase maximizes bacterial growth under O2-limiting conditions. Our investigations also indicate that cytochrome bd, rather than cytochrome bo, is an intrinsic component of the adaptive antinitrosative toolbox of Salmonella Accordingly, induction of cytochrome bd helps Salmonella grow and respire in the presence of inhibitory NO. The combined antinitrosative defenses of cytochrome bd and the flavohemoglobin Hmp account for a great part of the adaptations that help Salmonella recover from the antimicrobial activity of NO. Moreover, the antinitrosative defenses of cytochrome bd and flavohemoglobin Hmp synergize to promote Salmonella growth in systemic tissues. Collectively, our investigations indicate that cytochrome bd is a critical means by which Salmonella resists the nitrosative stress that is engendered in the innate response of mammalian hosts while it concomitantly allows for proper O2 utilization in tissue hypoxia.

Neurons utilize plasticity of dendritic arbors as part of a larger suite of adaptive plasticity mechanisms. This explicitly manifests with motoneurons in the Drosophila embryo and larva, where dendritic arbors are exclusively postsynaptic and are used as homeostatic devices, compensating for changes in synaptic input through adapting their growth and connectivity. We recently identified reactive oxygen species (ROS) as novel plasticity signals instrumental in this form of dendritic adjustment. ROS correlate with levels of neuronal activity and negatively regulate dendritic arbor size. Here, we investigated NADPH oxidases as potential sources of such activity-regulated ROS and implicate Dual Oxidase (but not Nox), which generates hydrogen peroxide extracellularly. We further show that the aquaporins Bib and Drip, but not Prip, are required for activity-regulated ROS-mediated adjustments of dendritic arbor size in motoneurons. These results suggest a model whereby neuronal activity leads to activation of the NADPH oxidase Dual Oxidase, which generates hydrogen peroxide at the extracellular face; aquaporins might then act as conduits that are necessary for these extracellular ROS to be channeled back into the cell where they negatively regulate dendritic arbor size.

Alternative oxidase (AOX) and plastid terminal oxidase (PTOX) are terminal oxidases of electron transfer in mitochondria and chloroplasts, respectively. Here, taking advantage of the variegation phenotype of the Arabidopsis PTOX deficient mutant (im), we examined the functional relationship between PTOX and its five distantly related homologs (AOX1a, 1b, 1c, 1d, and AOX2). When engineered into chloroplasts, AOX1b, 1c, 1d, and AOX2 rescued the im defect, while AOX1a partially suppressed the mutant phenotype, indicating that AOXs could function as PQH2 oxidases. When the full length AOXs were overexpressed in im, only AOX1b and AOX2 rescued its variegation phenotype. In vivo fluorescence analysis of GFP-tagged AOXs and subcellular fractionation assays showed that AOX1b and AOX2 could partially enter chloroplasts while AOX1c and AOX1d were exclusively present in mitochondria. Surprisingly, the subcellular fractionation, but not the fluorescence analysis of GFP-tagged AOX1a, revealed that a small portion of AOX1a could sort into chloroplasts. We further fused and expressed the targeting peptides of AOXs with the mature form of PTOX in im individually; and found that targeting peptides of AOX1a, AOX1b, and AOX2, but not that of AOX1c or AOX1d, could direct PTOX into chloroplasts. It demonstrated that chloroplast-localized AOXs, but not mitochondria-localized AOXs, can functionally compensate for the PTOX deficiency in chloroplasts, providing a direct evidence for the functional relevance of AOX and PTOX, shedding light on the interaction between mitochondria and chloroplasts and the complex mechanisms of protein dual targeting in plant cells.

Many disorders of the central nervous system (CNS), including alcohol use disorder (AUD), are associated with induction of proinflammatory neuroimmune signalling and neurodegeneration. In previous studies, we found increased expression of Toll-like receptors (TLRs), activated NF-κB p65 (RELA), and other proinflammatory signalling molecules. Proinflammatory NADPH oxidases generate reactive oxygen species, which are linked to neurodegeneration. We tested the hypothesis that AUD increased RELA activation increases NADPH oxidase-oxidative stress and endoplasmic reticulum (ER) stress cell death cascades in association with neuronal cell death in the human orbitofrontal cortex (OFC). In the AUD OFC, we report mRNA induction of several NADPH oxidases, the dual oxidase DUOX2, and the oxidative stress lipid peroxidation marker 4-HNE and the DNA oxidation marker 8-OHdG that correlate with RELA, a marker of proinflammatory NF-κB activation. This was accompanied by increased expression of the ER stress-associated regulator protein glucose-regulated protein 78 (GRP78), transmembrane sensors activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and inositol-requiring kinase/endonuclease 1 (pIRE1), and the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Expression of NADPH oxidase-oxidative stress markers correlate with ER stress-associated molecules. Induction of oxidative stress and ER stress signalling pathways correlate with expression of cell death-associated caspases and neuronal cell loss. These data support the hypothesis that proinflammatory RELA-mediated induction of NADPH oxidase-oxidative stress and ER stress-associated signalling cascades is associated with neuronal cell death in the post-mortem human OFC of individuals with AUD.

Lysyl Oxidase-like 2 (LOXL2), a member of the lysyl oxidase family of amine oxidases is known to be important in normal tissue development and homeostasis, as well as the onset and progression of solid tumors. Here we tested the anti-tumor properties of two generations of novel small molecule LOXL2 inhibitor in the MDA-MB-231 human model of breast cancer. We confirmed a functional role for LOXL2 activity in the progression of primary breast cancer. Inhibition of LOXL2 activity inhibited the growth of primary tumors and reduced primary tumor angiogenesis. Dual inhibition of LOXL2 and LOX showed a greater effect and also led to a lower overall metastatic burden in the lung and liver. Our data provides the first evidence to support a role for LOXL2 specific small molecule inhibitors as a potential therapy in breast cancer.