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)

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47(phox), p67(phox), p40(phox), and Rac2) translocate to the membranes, where they associate with flavocytochrome b(558), which is composed of gp91(phox)/NOX2 and p22(phox), to form the active system. During neutrophil stimulation, p47(phox), p67(phox), p40(phox), and p22(phox) are phosphorylated; however, the phosphorylation of gp91(phox)/NOX2 and its potential role have not been defined. In this study, we show that gp91(phox) is phosphorylated in stimulated neutrophils. The gp91(phox) phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91(phox), which confirms that this phosphoprotein is gp91(phox). The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91(phox), and protein kinase C (PKC) phosphorylated the recombinant gp91(phox)- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91(phox)-cytosolic tail on the same peptides that were phosphorylated on gp91(phox) in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91(phox) flavoprotein cytosolic domain and its binding to Rac2, p67(phox), and p47(phox). These results demonstrate that gp91(phox) is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91(phox)/NOX2 is a novel mechanism of NADPH oxidase regulation.
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PMID:Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox. 1902 40

The neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47(phox) promotes its translocation with p67(phox) and p40(phox), followed by membrane interaction and assembly with flavocytochrome b(558) into a functional complex. To characterise p47(phox) conformational changes during activation, we used wild-type and the S303/304/328E triple mutant mimicking the phosphorylated state. Hydrogen/deuterium exchange and limited proteolysis coupled to mass spectrometry were used to discriminate between the various structural models. An increase in deuteration confirmed that p47(phox) adopts an open and more flexible conformation after activation. Limited proteolysis correlated this change with increased auto-inhibitory region (AIR) accessibility. These results establish a structural link between the AIR release and the exposure of the Phox homology (PX) domain.
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PMID:Conformational changes in p47(phox) upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis. 1919 78

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.
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PMID:Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. 1928 53

1. Additive beneficial effects on cardiovascular disease have been reported for amlodipine and atorvastatin. However, it is still unclear whether the combination of amlodipine and atorvastatin has additive beneficial effects on the regression of advanced cardiac hypertrophy in hypertension. In the present study, the effects of the drug combination on advanced cardiac hypertrophy were investigated in elderly spontaneously hypertensive rats (SHR). 2. Elderly SHR (36 weeks old) were randomly allocated into four groups of 12: (i) a vehicle-treated control group; (ii) an amlodipine (10 mg/kg per day)-treated group; (iii) an atorvastatin (10 mg/kg per day)-treated group; and (iv) a group treated with a combination of amlodipine and atorvastatin (both at 10 mg/kg per day). Drugs were administered by oral gavage every morning for a period of 12 weeks before hearts were harvested for analysis. 3. Combined administration of amlodipine and atorvastatin significantly suppressed cardiomyocyte hypertrophy, interstitial fibrosis and upregulation of hypertrophic and profibrotic genes, and also improved left ventricular diastolic dysfunction to a greater extent than did amlodipine monotherapy. Further beneficial effects of combination therapy on advanced cardiac hypertrophy were associated with a greater reduction of NADPH oxidase-mediated increases in cardiac reactive oxygen species (ROS), rather than decreased blood pressure and serum cholesterol levels. 4. To elucidate the underlying molecular mechanisms, we examined cardiovascular NADPH oxidase subunits and found that amlodipine clearly attenuated the expression of p47(phox) and p40(phox) and slightly but significantly reduced p22(phox) and Rac-1 levels in heart tissue. Combination treatment with amlodipine plus atorvastatin led to a further reduction in p22(phox), p47(phox) and Rac-1 protein levels compared with amlodipine alone. 5. In conclusion, combined amlodipine and atorvastatin treatment has a greater beneficial effect on advanced cardiac hypertrophy compared with amlodipine monotherapy. The benefits are likely to be related to the additive effects of the drugs on the suppression of NADPH oxidase-mediated ROS generation.
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PMID:Additive beneficial effects of amlodipine and atorvastatin in reversing advanced cardiac hypertrophy in elderly spontaneously hypertensive rats. 1941 92

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O(2)(-)) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPgammaS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40(phox), p47(phox) and p67(phox), phosphorylation of Ser residues in p47(phox), association between p47(phox) and p22(phox), and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47(phox) were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47(phox), moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110gamma membrane translocation. The membrane recruitment of protein kinase C (PKC)-alpha, -betaI, -betaII, -delta and -zeta, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca(2+) concentration ([Ca(2+)](i)) elevation in the presence but not in the absence of extracellular Ca(2+), and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.
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PMID:Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils. 1944 20

In pulmonary neuroepithelial bodies (NEB), presumed airway chemoreceptors, classical NADPH oxidase (gp91 phox, NOX2) is co-expressed with O(2) sensitive K(+) channels (K(+)O(2)) and functions as an O(2) sensor. Here we examined related NADPH oxidase homologues "novel oxidases "(NOX 1, 3&4) and their possible involvement in O(2) sensing. For immunolocalization we used specific antibodies against various NADPH components and K(+) (O(2)) subunits to label NEB in rat /rabbit lung and NEB related H146 tumor cell line. For gene expression profiling of NEB cells microdissected from human lung, and H146 cells, we used custom MultiGene-12TM RT-PCR array that included NADPH oxidase components and homologues /accessory proteins (NOX1-4, phox-p22, p40, p47, p67, Rac1, NOXO1 and NOXA1) and K(+)O(2) channels (Kv -1.2, 1.5, 2.1, 3.1, 3.3, 3.4, 4.2, 4.3;TASK1-3). In rat lung, NOX2, NOX4, p22phox, Kv3.3 (and Kv3.4 in rabbit) and TASK1 localized to the apical plasma membrane of NEB cells, and membrane or sub-membrane regions in H146 cells. NEB and H146 cells expressed all NOX proteins except NOX3, as well as all K(+)O(2) channels, except Kv1.5 and Kv4.3. Co-immunoprecipitation using Western blot multicolor Quantum dot labeling showed NOX2 molecular complexes with Kv but not with TASK, while NOX4 associated with TASK1 but not with Kv channel proteins. Hypoxia -induced serotonin release was inhibited in H 146 cells by siRNA to NOX2, while siRNA to NOX4 had only a partial effect, implicating NOX 2 as the predominant NEB cell O(2) sensor. Present findings support NEB cell specific plasma membrane model of O(2) sensing, and suggest unique NOX/K(+)O(2) channel combinations for diverse physiological NEB functions.
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PMID:The role of NOX2 and "novel oxidases" in airway chemoreceptor O(2) sensing. 1953 8

Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91(phox) and p22(phox), which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47(phox) and p67(phox). A fifth subunit, p40(phox), plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40(phox), in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40(phox)R105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40(phox)-deficient granulocytes, with premature loss of p40(phox)R105Q from phagosomes. Thus, p40(phox) binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.
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PMID:A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. 1969 3

The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47(phox), p67(phox) and p40(phox). p67(phox), due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67(phox) is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67(phox). Our data reveals that p67(phox) behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67(phox). We suggest that the dual character of p67(phox) conformation in solution is central to ensure the numerous interactions to be accommodated.
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PMID:NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers. 1972 83

Oxidative stress in the rostral ventrolateral medulla (RVLM) increases sympathetic nervous system activity (SNA). Oral treatment with atorvastatin decreases SNA through antioxidant effects in the RVLM of stroke-prone spontaneously hypertensive rats (SHRSP). We aimed to examine whether centrally administered atorvastain reduces SNA in SHRSP and, if so, to determine whether it is associated with the reduction of oxidative stress induced by alteration of activities of nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase and superoxide dismutase (SOD) in the RVLM of SHRSP. SHRSP received atorvastatin (S-ATOR) or vehicle (S-VEH) by continuous intracerebroventricular infusion for 14 days. Mean blood pressure, heart rate, and SNA were significantly lower in S-ATOR than in S-VEH. Oxidative stress, Rac1 activity, NAD(P)H oxidase activity, Rac1, gp91(phox) and p22(phox) expression in the membrane fraction, and p47(phox) and p40(phox) expression in the cytosolic fraction in the RVLM were significantly lower in S-ATOR than in S-VEH. Rac1 expression in the cytosolic fraction and Mn-SOD activity, however, were significantly higher in S-ATOR than in S-VEH. Our findings suggest that centrally administered atorvastatin decreases SNA and is associated with decreasing NAD(P)H oxidase activity and upregulation of Mn-SOD activity in the RVLM of SHRSP, leading to suppressing oxidative stress.
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PMID:Sympathoinhibition induced by centrally administered atorvastatin is associated with alteration of NAD(P)H and Mn superoxide dismutase activity in rostral ventrolateral medulla of stroke-prone spontaneously hypertensive rats. 2004 Aug 88

We examine whether alveolar cells can control release of O(2)(-) through regulated NADPH oxidase (NOX) 2 (NOX2) activity to maintain lung fluid homeostasis. Using FACS to purify alveolar epithelial cells, we show that type 1 cells robustly express each of the critical NOX components that catalyze the production of O(2)(-) (NOX2 or gp91(phox), p22(phox), p67(phox), p47(phox), and p40(phox) subunits) as well as Rac1 at substantially higher levels than type 2 cells. Immunohistochemical labeling of lung tissue shows that Rac1 expression is cytoplasmic and resides near the apical surface of type 1 cells, whereas NOX2 coimmunoprecipitates with epithelial sodium channel (ENaC). Since Rac1 is a known regulator of NOX2, and hence O(2)(-) release, we tested whether inhibition or activation of Rac1 influenced ENaC activity. Indeed, 1 microM NSC23766 inhibition of Rac1 decreased O(2)(-) output in lung cells and significantly decreased ENaC activity from 0.87 +/- 0.16 to 0.52 +/- 0.16 [mean number of channels (N) and single-channel open probability (P(o)) (NP(o)) +/- SE, n = 6; P < 0.05] in type 2 cells. NSC23766 (10 microM) decreased ENaC NP(o) from 1.16 +/- 0.27 to 0.38 +/- 0.10 (n = 6 in type 1 cells). Conversely, 10 ng/ml EGF (a known stimulator of both Rac1 and O(2)(-) release) increased ENaC NP(o) values in both type 1 and 2 cells. NP(o) values increased from 0.48 +/- 0.21 to 0.91 +/- 0.28 in type 2 cells (P < 0.05; n = 10). In type 1 cells, ENaC activity also significantly increased from 0.40 +/- 0.15 to 0.60 +/- 0.23 following EGF treatment (n = 7). Sequestering O(2)(-) using 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) compound prevented EGF activation of ENaC in both type 1 and 2 cells. In conclusion, we report that Rac1-mediated NOX2 activity is an important component in O(2)(-) regulation of ENaC.
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PMID:Rac1-mediated NADPH oxidase release of O2- regulates epithelial sodium channel activity in the alveolar epithelium. 2009 36


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