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)

Acute lung injury represents a wide spectrum of pathologic processes, the most severe end of the spectrum being the acute respiratory distress syndrome. Reactive oxygen intermediates have been implicated as important in the pathobiochemistry of acute lung injury. The endogenous sources that contribute to the generation of reactive oxygen intermediates in acute lung injury are poorly defined but probably include the molybdenum hydroxylases, NAD(P)H oxidoreductases, the mitochondrial electron transport chain, and arachidonic acid-metabolizing enzymes. Our laboratory has focused, in particular, on the regulation of two of these enzyme systems, xanthine oxidoreductase (XDH/XO) and NAD(P)H oxidase. We observe that gene expression of XDH/XO is regulatory in a cell-specific manner and is markedly affected by inflammatory cytokines, steroids, and physiologic events such as hypoxia. Posttranslational processing is also important in regulating XDH/XO activity. More recently, the laboratory has characterized an NAD(P)H oxidase in vascular cells. The cytochrome components of the oxidase, gp91 and p22, appear similar to the components present in phagocytic cells that contribute to their respiratory burst. In human vascular endothelial and smooth muscle cells, oncostatin M potently induces gp91 expression. We believe that regulation of gp91 is a central controlling factor in expression of the vascular NAD(P)H oxidase. In summary, the studies support the concept that the oxidoreductases of vascular cells are expressed in a highly regulated and self-specific fashion.
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PMID:Lung injury and oxidoreductases. 978 4

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O-2 from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47(phox) and p67(phox) migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate (SDS) or arachidonic acid, as an activating agent. It has been proposed that conformational changes in the protein structure of cytosolic factor p47(phox) may be an important part of the activation mechanism. The purpose of the present study was to develop an approach to directly monitor conformational changes in p47(phox) when treated with amphiphiles. Cysteines in recombinant p47(phox) were covalently labeled with a sulfhydryl-reactive, environmentally sensitive, fluorescent probe N, N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethyleneamine (IANBD). A series of mutant p47(phox) proteins in which the individual cysteine (C98, C111, C196, and C378) was replaced with alanine revealed that all four cysteines of p47(phox) are reactive to IANBD. We found that anionic amphiphiles elicited a dose-dependent increase in fluorescence at an emission maximum of 537 nm from IANBD-labeled p47(phox). Furthermore, a blue shift of emission maximum and a decrease in quenching by the ionic quencher, potassium iodide, were observed in the presence of amphiphiles. These results indicate that the amphiphile-mediated increase in fluorescence from IANBD-labeled p47(phox) is due to the conformational change as seen in the leukocyte NADPH oxidase activation. We propose that this alteration in conformation results in the appearance of a binding site through which p47(phox) interacts with cytochrome b558 during the activation process. In addition, recombinant p67(phox) or a peptide containing proline-rich sequence of p22(phox) (residues 149-162) induces the attenuation of the amphiphile-mediated enhancement of fluorescence from IANBD-labeled p47(phox). This supports the notion that both p67(phox) and p22(phox) influence the conformation of p47(phox).
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PMID:Fluorescent labeling of the leukocyte NADPH oxidase subunit p47(phox): evidence for amphiphile-induced conformational changes. 985 27

-Recent reports suggest that the increased production of reactive oxygen species (ROS) in the vascular wall may contribute to the functional and structural changes associated with hypertension and atherosclerosis. Although glucocorticoid therapy can promote atherosclerosis, protective effects of these compounds on vascular lesion formation have been reported. In the present study, we investigated whether ROS production in cultured human aortic smooth muscle cells (HSMCs) can be modulated by glucocorticoids. Pretreatment of HSMCs with dexamethasone for 24 hours attenuated the basal and platelet-derived growth factor (PDGF)-AB- and angiotensin II-induced superoxide anion (O2. -) production. PDGF-AB-stimulated O2. - production was also inhibited by prednisolone and hydrocortisone but not by other steroids, such as testosterone and norgestrel. Incubation of HSMCs with glucocorticoids for 24 hours decreased 2',7'-dichlorodihydrofluorescein (DCHF) oxidation, an indicator of intracellular ROS levels. Dexamethasone decreased the mRNA expression of p22 phox, one of the components of NADPH oxidase, but had no effect on the activity of superoxide dismutase. The effects of dexamethasone on DCHF oxidation, and p22 phox mRNA expression and PDGF-AB-stimulated O2. - production were inhibited by the glucocorticoid receptor antagonist RU486. These results indicate that glucocorticoids decrease O2. - production by HSMCs via a receptor-dependent pathway. This effect is likely to be mediated by a decrease in the generating system, such as downregulation of p22 phox mRNA, rather than an increased inactivation of O2. -. The inhibition of ROS production might contribute to the local protective effects that glucocorticoids have on vascular lesion formation.
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PMID:Glucocorticoids inhibit superoxide anion production and p22 phox mRNA expression in human aortic smooth muscle cells. 985 78

The redox center of the phagocyte NADPH oxidase is flavocytochrome b558, a transmembrane protein with two subunits, gp91(phox) and p22(phox). In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91(phox) precursor (p65). Expressing the gp91(phox) cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes. Sucrose density gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91(phox) transgene demonstrated that p65 co-sedimented with the ER marker protein calreticulin and myeloperoxidase precursors. Unexpectedly, the majority of p22(phox) was found in subcellular compartments containing the mature 91-kDa form of gp91(phox) and not with p65, suggesting that heterodimer formation may occur in a post-ER compartment. The heme synthesis inhibitor, succinyl acetone, reduced the abundance of mature gp91(phox) and p22(phox) but had little or no impact on p65. These studies demonstrate (a) gp91(phox) is synthesized as a glycosylated 65-kDa precursor in the ER, (b) heterodimer formation is not a co-translational process, and (c) heme insertion is a determinant in the formation of a stable heterodimer but does not appear to affect the stability of p65.
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PMID:Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum. 993 39

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that phosphorylation of p47(phox) with protein kinase C induces conformational changes, as reflected by a fluorescence change of N, N'-di-methyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD)-labeled p47(phox). We propose that this alteration in conformation results in the appearance of a binding site through which p47(phox) interacts with cytochrome b558 during the activation process. In addition, the present study indicates that other oxidase components, such as p67(phox) and p22(phox), influence the conformation of p47(phox).
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PMID:Phosphorylation induces conformational changes in the leukocyte NADPH oxidase subunit p47(phox). 1033 12

Recently, we showed that cultured guinea pig gastric pit cells possess a phagocyte NADPH oxidase-like activity, which was up-regulated by Helicobacter pylori lipopolysaccharide. We demonstrate here that these cells express all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phoxes). Treatment with lipopolysaccharide increased the expression of gp91-, p22-, and p67-phoxes, but not that of p47- and p40-phoxes. Intriguingly, the p67-phox expression consistently correlated with up-regulation of superoxide anion-producing ability. Thus, the gastric pit cell NADPH oxidase may play an important role in regulation of the inflammatory response associated with H. pylori infection.
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PMID:Helicobacter pylori lipopolysaccharide enhances the expression of NADPH oxidase components in cultured guinea pig gastric mucosal cells. 1038 99

Activation of phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox). p47(phox) in resting phagocytes does not bind p22(phox). Phosphorylation of serines in the p47(phox) C terminus enables binding to the p22(phox) C terminus by inducing a conformational change in p47(phox) that unmasks the SH3A domain. We report that an arginine/lysine-rich region in the p47(phox) C terminus binds the p47(phox) SH3 domains expressed in tandem (SH3AB) but does not bind the individual N-terminal SH3A and C-terminal SH3B domains. Peptides matching amino acids 301-320 and 314-335 of the p47(phox) arginine/lysine-rich region block the p47(phox) SH3AB/p22(phox) C-terminal and p47(phox) SH3AB/p47(phox) C-terminal binding and inhibit NADPH oxidase activity in vitro. Peptides with phosphoserines substituted for serines 310 and 328 do not block binding and are poor inhibitors of oxidase activity. Mutated full-length p47(phox) with aspartic acid substitutions to mimic the effects of phosphorylations at serines 310 and 328 bind the p22(phox) proline-rich region in contrast to wild-type p47(phox). We conclude that the p47(phox) SH3A domain-binding site is blocked by an interaction between the p47(phox) SH3AB domains and the C-terminal arginine/lysine-rich region. Phosphorylation of serines in the p47(phox) C terminus disrupts this interaction leading to exposure of the SH3A domain, binding to p22(phox), and activation of the NADPH oxidase.
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PMID:Activation of the phagocyte NADPH oxidase protein p47(phox). Phosphorylation controls SH3 domain-dependent binding to p22(phox). 1039 14

The rapid response to hypoxia in the pulmonary artery (PA), carotid body, and ductus arteriosus is partially mediated by O2-responsive K+ channels. K+ channels in PA smooth muscle cells (SMCs) are inhibited by hypoxia, causing membrane depolarization, increased cytosolic calcium, and hypoxic pulmonary vasoconstriction. We hypothesize that the K+ channels are not themselves "O2 sensors" but rather respond to the reduced redox state created by hypoxic inhibition of candidate O2 sensors (NADPH oxidase or the mitochondrial electron transport chain). Both pathways shuttle electrons from donors, down a redox gradient, to O2. Hypoxia inhibits these pathways, decreasing radical production and causing cytosolic accumulation of unused, reduced, freely diffusible electron donors. PASMC K+ channels are redox responsive, opening when oxidized and closing when reduced. Inhibitors of NADPH oxidase (diphenyleneiodonium) and mitochondrial complex 1 (rotenone) both inhibit PASMC whole-cell K+ current but lack the specificity to identify the O2-sensor pathway. We used mice lacking the gp91 subunit of NADPH oxidase [chronic granulomatous disease (CGD) mice] to assess the hypothesis that NADPH oxidase is a PA O2-sensor. In wild-type lungs, gp91 phox and p22 phox subunits are present (relative expression: macrophages > airways and veins > PASMCs). Deletion of gp91 phox did not alter p22 phox expression but severely inhibited activated O2 species production. Nonetheless, hypoxia caused identical inhibition of whole-cell K+ current (in PASMCs) and hypoxic pulmonary vasoconstriction (in isolated lungs) from CGD vs. wild-type mice. Rotenone vasoconstriction was preserved in CGD mice, consistent with a role for the mitochondrial electron transport chain in O2 sensing. NADPH oxidase, though a major source of lung radical production, is not the pulmonary vascular O2 sensor in mice.
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PMID:O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase. 1039 27

We wished to determine when each of the four NADPH oxidase components p22 phagocytic oxidase (phox), gp91 phox, p47 phox, p67 phox is first expressed embryologically and whether the expression pattern occurs in a consistent temporal sequence or whether the four genes are expressed simultaneously. A deficiency of any one of them results in chronic granulomatous disease (CGD). mRNA transcripts and protein expression for p22 phox, gp91 phox, p47 phox, p67 phox was monitored in murine embryos at time of implantation (E5.5) until E 11.5, and in fetal liver, spleen, and limb bone marrow from E 14 until term (E 19). We observed that mRNA was first expressed for p22 phox at E 5.5, for p67 phox at E 7.0 and for p47 phox at E 7.5 before the onset of yolk sac hematopoiesis (E 8.0). gp91 Phox mRNA was first expressed at E 9.0. However, only p22 phox protein was expressed in circulating hemocytoblast by E 9.0. No other embryonic tissue contained phox proteins either before or after the establishment of hemocytoblastic circulation. The four specific mRNA transcripts and phox proteins were expressed in nests of developing granulocytes in liver by E 14 and the expression continued in the liver at E 16 and E 19. Spleen and limb bone marrow showed inconsistent results. Cord blood neutrophils contained all phox proteins. These studies confirm that the four CGD-related phox mRNA components of NADPH oxidase are expressed early in embryonic development and the expression occurs in a consistent sequential fashion but only p22 phox protein appears in embryonic hemocytoblast.
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PMID:Developmental expression of NADPH phagocytic oxidase components in mouse embryos. 1044 7

Chronic granulomatous disease is a rare inherited disorder caused by non-existent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for superoxide production, NADPH oxidase, involves at least five components. An absence of, or a defect in, any one of four of these proteins (p47(phox), p67(phox), p22(phox) and gp91(phox)) gives rise to the known types of chronic granulomatous disease. The most common form of inheritance is X-linked and is due to mutations in the CYBB gene that encodes gp91(phox), the large subunit of flavocytochrome b, the terminal electron donor of the oxidase. We have recently reported a large number of mutations in this gene revealing a broad range of defects, including large and small deletions, and frameshift, nonsense, missense, splice region and regulatory region mutations. Here we report a patient who has an unusual type of mutation that results in the generation of a 'pseudo-exon' in the gp91(phox) mRNA and an unexpected pattern of splicing.
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PMID:A novel mutation in the CYBB gene resulting in an unexpected pattern of exon skipping and chronic granulomatous disease. 1045 61


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