Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide (NO) and reactive oxygen species exert multiple modulating effects on inflammation and play a key role in the regulation of immune responses. They affect virtually every step of the development of inflammation. Low concentrations of nitric oxide produced by constitutive and neuronal nitric oxide synthases inhibit adhesion molecule expression, cytokine and chemokine synthesis and leukocyte adhesion and transmigration. Large amounts of NO, generated primarily by iNOS can be toxic and pro-inflammatory. Actions of nitric oxide are however not dependent primarily on the enzymatic source, but rather on the cellular context, NO concentration (dependent on the distance from NO source) and initial priming of immune cells. These observations may explain difficulties in determining the exact role of NO in Th1 and Th2 lymphocyte balance in normal immune responses and in allergic disease. Similarly superoxide anion produced by NAD(P)H oxidases present in all cell types participating in inflammation (leukocytes, endothelial and other vascular cells etc) may lead to toxic effects, when produced at high levels during oxidative burst, but may also modulate inflammation in a far more discrete way, when continuously produced at low levels by NOXs (non-phagocytic oxidases). The effects of both nitric oxide and superoxide in immune regulation are exerted through multiple mechanisms, which include interaction with cell signalling systems like cGMP, cAMP, G-protein, JAK/STAT or MAPK dependent signal transduction pathways. They may also lead to modification of transcription factors activity and in this way modulate the expression of multiple other mediators of inflammation. Moreover genetic polymorphisms exist within genes encoding enzymes producing both NO and superoxide. The potential role of these polymorphisms in inflammation and susceptibility to infection is discussed. Along with studies showing increasing role of NO and free radicals in mediating inflammatory responses drugs which interfere with these systems are being introduced in the treatment of inflammation. These include statins, angiotensin receptor blockers, NAD(P)H oxidase inhibitors, NO-aspirin and others. In conclusion in this mini-review we discuss the mechanisms of nitric oxide and superoxide dependent modulation of inflammatory reactions in experimental animals and humans. We also discuss potential roles of nitric oxide as a mediator of allergic inflammation.
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PMID:Nitric oxide and superoxide in inflammation and immune regulation. 1472 4

The membrane-integrated protein gp91phox, existing as a heterodimer with p22phox, functions as the catalytic core of the phagocyte NADPH oxidase, which plays a crucial role in host defence. The oxidase, dormant in resting cells, becomes activated to produce superoxide, a precursor of microbicidal oxidants, by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. In the past few years, several proteins homologous to gp91phox were discovered as superoxide-producing NAD(P)H oxidases (Nox's) in non-phagocytic cells; however, regulatory mechanisms for the novel oxidases have been largely unknown. Current identification of proteins highly related to p47phox and p67phox, designated Noxol (Nox organizer 1) and Noxal (Nox activator 1), respectively, has shed lights on common and distinct mechanisms underlying activations of Nox family oxidases.
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PMID:Molecular mechanism for activation of superoxide-producing NADPH oxidases. 1474 14

Insulin stimulation of target cells elicits a burst of H(2)O(2) that enhances tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins as well as distal signaling events in the insulin action cascade. The molecular mechanism coupling the insulin receptor with the cellular oxidant-generating apparatus has not been elucidated. Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells. Adenovirus-mediated expression of Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains acted in a dominant-negative fashion in differentiated 3T3-L1 adipocytes and attenuated insulin-stimulated H(2)O(2) generation, insulin receptor (IR) and IRS-1 tyrosine phosphorylation, activation of downstream serine kinases, and glucose uptake. Transfection of specific small interfering RNA oligonucleotides reduced Nox4 protein abundance and also inhibited the insulin signaling cascade. Overexpression of Nox4 also significantly reversed the inhibition of insulin-stimulated IR tyrosine phosphorylation induced by coexpression of PTP1B by inhibiting PTP1B catalytic activity. These data suggest that Nox4 provides a novel link between the IR and the generation of cellular reactive oxygen species that enhance insulin signal transduction, at least in part via the oxidative inhibition of cellular protein-tyrosine phosphatases (PTPases), including PTP1B, a PTPase that has been previously implicated in the regulation of insulin action.
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PMID:The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. 1496 67

NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.
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PMID:Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). 1498 37

Several non-phagocytic cells can actively generate the superoxide anion by NAD(P)H oxidases resembling the enzymatic complex typical of phagocytes. Overexpression of periplasmic Cu,ZnSOD rescues invasive E. coli strains from killing within epithelial cells, suggesting that superoxide generation by such cells can oxidatively damage invading bacteria. Pre-treatment of HeLa cells with diphenyl iodonium or 4'-hydroxy-3'-methoxyacetophenone, two inhibitors of NAD(P)H oxidase, significantly enhances intracellular survival of wild type invasive E. coli cells. On the contrary, these inhibitors have no effect on the intracellular survival of an invasive E. coli strain engineered to overexpress Cu,ZnSOD. These results support the hypothesis that superoxide generation by a NAD(P)H oxidase-like complex can limit bacterial survival within epithelial cells and suggest that the role of periplasmic Cu,ZnSOD in bacterial infections is not simply that of conferring protection against the phagocytic oxidative burst.
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PMID:Involvement of reactive oxygen species in bacterial killing within epithelial cells. 1500 Aug 69

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H(2)O(2) production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H(2)O(2) production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H(2)O(2) production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.
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PMID:Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure. 1506 85

Oxidative stress contributes to the pathogenesis of atherosclerosis. p22phox-based NAD(P)H oxidases exist in the vessel wall, acting as important superoxide-generating systems in the vasculature. Some studies have identified reduced atherosclerosis in the presence of the C242T CYBA polymorphism, whereas others have not. Because vascular p22phox is identical to neutrophil p22phox, we studied the association between the C242T, A640G, and -930A/G CYBA polymorphisms and the quantity of superoxide produced from neutrophils isolated from healthy adults to determine if these polymorphisms had any functional impact on NADPH oxidase function. Neutrophils were isolated from 90 subjects by Percoll density gradient centrifugation. Genotypes were determined by polymerase chain reaction (PCR) and restriction mapping, as well as real-time PCR. The oxidative burst was stimulated with phorbol 12-myristate 13-acetate. Superoxide was quantified using the superoxide dismutase inhibitable oxidation of the spin probe hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine, detected by electron paramagnetic resonance. Superoxide production was significantly affected by the C242T polymorphism, being 8.7+/-0.7, 7.9+/-0.6, and 5.9+/-1.2 micromol/L per minute per 10(6) neutrophils for the C242T CC, CT, and TT genotypes, respectively (P<0.05). In contrast, the A640G and the -930A/G polymorphisms did not alter the neutrophil respiratory burst. Phagocytic respiratory burst activity in homozygous individuals with the T allele of the C242T CYBA polymorphism is significantly lower than of wild-type carriers and heterozygous individuals. Because p22phox exists in both the neutrophil and vessel wall, vascular oxidative stress is likely diminished in individuals with this polymorphism.
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PMID:C242T CYBA polymorphism of the NADPH oxidase is associated with reduced respiratory burst in human neutrophils. 1507 63

Oxidative stress may be involved in the development of vascular complications associated with diabetes; however, the molecular mechanism responsible for increased production of free radicals in diabetes remains uncertain. Therefore, we examined whether acute hyperinsulinemia increases the production of free radicals and whether this condition affects proliferative extracellular signal-regulated kinase (ERK-1 and -2) signaling in human fibroblasts in vitro. Insulin treatment significantly increased intracellular superoxide anion (O(2)(-)) production, an effect completely abolished by Tiron, a cell-permeable superoxide dismutase (SOD) mimetic and by polyethylene glycol (PEG)-SOD, but not by PEG catalase. Furthermore, insulin-induced O(2)(-) production was attenuated by the NAD(P)H inhibitor apocynin, but not by rotenone or oxypurinol. Inhibition of the phosphatidylinositol 3'-kinase (PI 3'-kinase) pathway with LY294002 blocked insulin-stimulated O(2)(-) production, suggesting a direct involvement of PI 3'-kinase in the activation of NAD(P)H oxidase. The insulin-induced free radical production led to membranous translocation of p47phox and markedly enhanced ERK-1 and -2 activation in human fibroblasts. In conclusion, these findings provided direct evidence that elevated insulin levels generate O(2)(-) by an NAD(P)H-dependent mechanism that involves the activation of PI 3'-kinase and stimulates ERK-1- and ERK-2-dependent pathways. This effect of insulin may contribute to the pathogenesis and progression of cardiovascular disease in the insulin resistance syndrome.
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PMID:Insulin generates free radicals by an NAD(P)H, phosphatidylinositol 3'-kinase-dependent mechanism in human skin fibroblasts ex vivo. 1511 5

The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and chrome b5 reductase cytodomains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = approximately 0.05 and approximately 1 s(-1), respectively. The redox potential at the heme center of NCB5OR is -108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse.
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PMID:NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum. 1513 Nov 10

Proliferation of endothelial cells plays a crucial role in the process of atherosclerotic plaque destabilization. The major component of oxidized low-density lipoprotein lysophosphatidylcholine (LPC) has been shown to promote endothelial proliferation by increasing the production of reactive oxygen species (ROS). Since K(+) channels are known to control the cell cycle, we investigated the role of Ca(2+)-activated K(+) channels (BK(Ca)) in the regulation of LPC-induced endothelial proliferation and ROS generation. A significant increase of cell growth induced by LPC (20 micromol/l; cell counts (CCs): +87%, thymidin incorporation: +89%; n = 12, P < 0.01) was observed, which was inhibited by the BK(Ca) inhibitor iberiotoxin (IBX; 100 nmol/l), by the NAD(P)H-oxidase inhibitor diphenyleneiodonium (5 micromol/l) and by transfection with antisense (AS) oligonucleotides against NAD(P)H oxidase, whereas N(G)-monomethyl-l-arginine (l-NMMA) further increased LPC-induced cell growth. Using the patch-clamp technique a significant increase of BK(Ca) open-state probability (control: 0.004 +/- 0.002; LPC: 0.104 +/- 0.035; n = 21, P < 0.05) by LPC was observed. Using dichlorofluorescein fluorescence microscopy a significant increase of ROS induced by LPC was reported, that was blocked by IBX and Ca(2+) antagonists. Intracellular Ca(2+) measurements revealed a capacitative Ca(2+) influx caused by LPC. Bioactivity of nitric oxide (NO) was measured using a [(3)H]-cGMP radioimmunoassay. LPC significantly decreased acetylcholine-induced NO synthesis. LPC significantly increased cGMP levels in endothelial cells transfected with AS, which was blocked by IBX. In conclusion, our results demonstrate that LPC activates BK(Ca) thereby increasing ROS production which induces endothelial proliferation. In addition LPC-induced BK(Ca)-activation contributes to increased cGMP levels, if ROS production is prevented by AS.
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PMID:Lysophosphatidylcholine-induced modulation of Ca(2+)-activated K(+)channels contributes to ROS-dependent proliferation of cultured human endothelial cells. 1513 62


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