Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The yeast two-hybrid system is finding increased use in the study of interactions between proteins. In this method, two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain (bd) and activating domain (ad), respectively. Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters. The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 (comprised of subunits p22-phox and gp91-phox) and three cytosol proteins (p47-phox, p67-phox, and p21rac) that translocate to membrane and bind to cytochrome b558. This is the first report to demonstrate that two of cytosolic components of cytochrome b558, p47-phox binding to p67-phox each other. We encountered several methodological problems in the two-hybrid system which are the focus of this report.
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PMID:Elimination of false negative results in the two-hybrid system in the phagocyte NADPH oxidase. 867 37

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.
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PMID:Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase. 870 27

Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.
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PMID:p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. 879 32

Low-level generation of reactive oxygen species (ROS) by endothelial cells in response to a variety of stimuli has been observed; however, the enzyme system responsible is unknown. Using a variety of techniques, we examined for components of the phagocyte superoxide-generating NADPH oxidase to elucidate whether this enzyme could be a source of endothelial-derived ROS. Superoxide generation on addition of 100 microM NAD(P)H to human umbilical vein endothelial cell (HUVEC) sonicates (using lucigenin-enhanced chemiluminescence) was partially inhibited on addition of the flavoenzyme inhibitor diphenyliodonium (IDP). Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated expression of gp91phox, p22phox, p67phox, and p47phox in four independent HUVEC isolates. Expression of p22phox was also confirmed by Northern blotting. RT-PCR for tumor necrosis factor-alpha was negative, indicating an absence of mononuclear cell contamination (a potential source of NADPH oxidase). Immunoperoxidase staining, using anti-p47phox (JW-1)- and anti-p67phox (JW-2)-specific antibodies, showed protein expression of these cytosolic components. However, heme spectroscopy failed to indicate the presence of the low-potential cytochrome b558. These data indicate that cultured human endothelial cells express both mRNA and protein for cytosolic components of the phagocyte superoxide-generating NADPH oxidase. However, because the cytochrome b558 heme could not be conclusively demonstrated, a contribution of the phagocyte NADPH oxidase to endothelial oxidant generation may be unlikely.
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PMID:Expression of phagocyte NADPH oxidase components in human endothelial cells. 889 60

The human NADPH oxidase is a very intriguing enzyme; although its catalytic unit is retained within cytochrome b558, various additional proteins are required for activity of the NADPH oxidase. In the past few years substantial progress has been made to elucidate the protein-protein interactions and the activation events involved. The following facts have become evident: (1) activation of rac and subsequent interaction with p67-phox is crucial for the interaction of p67-phox with cytochrome b558, and probably with gp91-phox; (2) p47-phox interacts with p22-phox, and phosphorylation of 379Ser of p47-phox is obligatory for this event; (3) p47-phox and p67-phox regulate each other's translocation in a positive sense (see also reference 71). To put it differently: it is vital to gain insight in the intrigues within the phox family and associated characters to fully understand NADPH oxidase activation.
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PMID:Interactions between the components of the human NADPH oxidase: intrigues in the phox family. 890 Feb 89

This study concerns the controversial problem of whether the TNF-alpha (TNF) induces a respiratory burst in human neutrophils in suspension. The results have shown that in these cells TNF induces a classical respiratory burst. In fact, the production of oxygen free radicals 1) is linked to the translocation of NADPH oxidase components from cytosol to the plasma membrane, 2) does not take place in neutrophils from a patient lacking the cytochrome b558, and 3) does not involve other sources such as mitochondrial respiratory chain or xanthine oxidase. Signal transduction studies have demonstrated that this respiratory burst 1) is not accompanied by calcium transients, stimulation of phosphoinositide turnover, and phospholipase D activity (moreover, this burst is associated with the stimulation of the activity of phospholipase A2, but not of sphingomyelinase); 2) is strictly dependent on activation of tyrosine kinases, which is functional to the translocation to the plasma membrane of the cytosolic NADPH oxidase component rac; and 3) is dependent on the integrity of the cytoskeleton because it is completely suppressed by cytochalasin B. The integrity of the cytoskeleton is required for a full translocation of all the NADPH oxidase components and for an optimal activation of tyrosine kinases, but not for phospholipase A2 activation. Taken together, these findings demonstrate that TNF activates the NADPH oxidase through stimulation of tyrosine kinases, whose function is cytoskeleton-dependent, and raise the problem of whether the activation of this respiratory burst involves signals arising from TNF-activated beta2 integrins.
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PMID:Mechanisms of stimulation of the respiratory burst by TNF in nonadherent neutrophils: its independence of lipidic transmembrane signaling and dependence on protein tyrosine phosphorylation and cytoskeleton. 890 41

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 microM) and inhibited by diphenyliodonium (5 microM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or corresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.
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PMID:NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines. 891 65

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.
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PMID:Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes. 894 64

Recent studies suggest that superoxide production by the NADPH/NADH oxidase may be involved in smooth muscle cell growth and the pathogenesis of hypertension. We previously showed that angiotensin II (Ang II) activates a p22phoxbased NADPH/NADH oxidase in cultured rat vascular smooth muscle cells and in animals made hypertensive by infusion of Ang II. To investigate the mechanism responsible for this increased oxidase activity, we examined p22phox mRNA expression in rats made hypertensive by implanting an osmotic minipump that delivered Ang II (0.7 mg/kg per day). Blood pressure began to increase 3 days after the start of Ang II infusion and remained elevated for up to 14 days. Expression of p22phox mRNA in aorta was also increased after 3 days and reached a maximum increase of 338 +/- 41% by 5 days after pump implantation compared with the value after sham operation. This increase in mRNA expression was accompanied by an increase in the content of the corresponding cytochrome (twofold) and NADPH oxidase activity (179 +/- 11% of that in sham-operated rats 5 days after pump implantation). Treatment with the antihypertensive agents losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) inhibited this upregulation of mRNA levels and activity. Furthermore, infusion of recombinant heparin-binding superoxide dismutase decreased both blood pressure and p22phox mRNA expression. In situ hybridization of aortic tissue showed that p22phox mRNA was expressed in medial smooth muscle as well as in the adventitia. These findings suggest that Ang II-induced hypertension activates the NADPH/NADH oxidase system by upregulating mRNA levels of one or several components of this oxidase system, including the p22phox, and that the NADPH/NADH oxidase system is associated with the pathology of hypertension in vivo.
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PMID:p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. 897 21

The superoxide-producing NADPH oxidase consists of membrane-associated cytochrome b558 and cytosolic components, p47-phox and p67-phox. Recently, we have found a novel cytosolic component, p40-phox, which is tightly associated with p67-phox. In this study, we examined the translocation of p40-phox during activation of NADPH oxidase in a cell-free system using the membrane and the purified p47-phox/p67-phox/p40-phox complex. p40-phox was translocated to the membrane by arachidonic acid in a dose-dependent manner. The translocation pattern of p40-phox was similar to those of p47-phox and p67-phox. However, immunoprecipitation assay revealed that p40-phox was dissociated from p47-phox and p67-phox during activation. The translocation of three cytosolic components was not affected by the deletion of GTP-gamma-s from the reaction mixture. Interestingly, a synthetic peptide corresponding to carboxyl-terminus of p40-phox inhibited the activation of NADPH oxidase and translocation of p40-phox, p47-phox, and p67-phox, suggesting that p40-phox might play a role in the activation of NADPH oxidase. These observations suggest that p40-phox is dissociated from p67-phox during activation, and translocates to the membrane by GTP-gamma-s-independent mechanism.
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PMID:Translocation of guinea pig p40-phox during activation of NADPH oxidase. 898 88


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