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)

Eosinophils induce tissue injury by releasing granule-associated cytotoxic proteins, lipid mediators and superoxide anions in response to appropriate stimuli. Superoxide generation associated with respiratory burst is largely dependent on the assembly of the NADPH oxidase complex in the membrane, consisting of membrane-bound cytochrome b558 and translocated p47phox and p67phox. The activation of this complex is critically dependent on the translocation of GTP-bound Rac1, or its homologue Rac2, from the cytosol to the membrane in neutrophils. Rac expression has not yet been fully characterized in eosinophils. We proposed that eosinophils translate and express Rac2 and its GDP-dissociation inhibitor, RhoGDI. Furthermore, we hypothesized that Rac2 translocates along with p47phox and p67phox proteins from the cytosol to the plasma membrane during respiratory burst. By reverse transcription-polymerase chain reaction analysis and sequencing of the amplified product, guinea-pig eosinophils were found to express Rac2 mRNA, exhibiting 93% homology with the human Rac2 sequence. Rac1 mRNA was also detected in eosinophils but not its translated product. In contrast, Rac2 protein expression was detected using a specific antibody. In subcellular fractions, Rac2 was found to translocate, along with p47phox and p67phox, from cytosol to plasma membrane-associated fractions following phorbol myristate acetate stimulation, while RhoGDI remained within cytosolic fractions. These findings suggest that Rac2 is preferentially expressed and activated in eosinophils, and is likely to be a crucial regulator of the release of reactive oxygen species from these cells during inflammatory reactions.
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PMID:Expression and translocation of Rac2 in eosinophils during superoxide generation. 1054 Feb 23

Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.
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PMID:NADPH oxidase activation and assembly during phagocytosis. 1058 71

We have evaluated the accumulation of neutrophils in the gut and their infiltration into the intestinal extravascular spaces in rats subjected to a 25% total body surface area scald burn. The accumulation of neutrophils was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates, and the immunohistochemical localization of neutrophil NADPH oxidase component proteins (p47phox and p67phox) within the intestinal extravascular spaces determined neutrophil tissue infiltration. MPO measurements demonstrated a 12- and 21-fold increase above the control value in the intestinal tissue at day 1 and day 3 post-burn, respectively, suggesting that a substantial total tissue accumulation of neutrophils occurs in the gut after burn injury. The immunohistochemical staining procedures showed both a definitive presence of the neutrophil in the intestinal extravascular spaces and an enhanced immunoreactivity in neutrophils accumulating in intestine after burn injury. There was no evidence of either the presence of neutrophils in the extravascular regions or any significant neutrophil immunoreactivity to NADPH oxidase component proteins in the intestines of sham control rats. These findings indicate that burn injury causes an enhanced migration of circulating neutrophils into the intestinal interstitial spaces and an upregulation of NADPH oxidase activity in the infiltrating neutrophils.
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PMID:Enhanced expression of neutrophil NADPH oxidase components in intestine of rats after burn injury. 1058 11

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disease that leads to severe recurrent infections. CGD is caused by defects in the phagocyte NADPH oxidase, a multiprotein enzyme that reduces oxygen to superoxide, a precursor of microbicidal oxidants. Less than 6% of CGD patients have an autosomal recessive form of the disease caused by mutations in NCF-2. This gene encodes p67-phox, a cytosolic oxidase subunit that associates with membrane-bound flavocytochrome b558 and regulates electron transfer. We studied six patients from five families with p67-phox deficiency and identified seven different mutant alleles. Patients from three of the kindreds were homozygous for their respective mutation, although the parents of only one family were known to be related. Five of the mutations have not previously been identified: (1) a missense mutation (383C-->T) in exon 5, (2) a nonsense mutation (196C-->T) in exon 3, (3) a missense mutation (230G-->A) in exon 3, (4) a nonsense mutation (298C-->T) in exon 4, and (5) a dinucleotide deletion (835-836 AC) from exon 9. Phagocytes from each of the patients analyzed failed to generate a measurable respiratory burst and had no detectable p67-phox protein. Our results further demonstrate that there is great heterogeneity among the mutations in p67-phox-deficient CGD patients, with no evidence for mutational hot-spots or a founder effect. Our data also support the hypothesis that the stability of p67-phox is particularly sensitive to missense mutations that cause amino acid substitutions within its N-terminal domain. In contrast, mutations predicting single amino acid changes elsewhere in the protein generally represent benign polymorphisms.
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PMID:Autosomal recessive chronic granulomatous disease caused by novel mutations in NCF-2, the gene encoding the p67-phox component of phagocyte NADPH oxidase. 1059 13

NADPH oxidase is an O2*- -generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes.
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PMID:P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox. 1061 85

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- 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 p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. In whole cells and under certain circumstances in the cell-free system, the phosphorylation of p47phox mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47phox may be an important part of the activation mechanism. The total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47phox was substantially decreased, reflecting on the conformational change that occurs when p47phox was phosphorylated with protein kinase C. We show here that the phosphorylation of p47phox by protein kinase A or mitogen-activated protein kinase, however, had little effect on the intrinsic fluorescence of p47phox. In addition, the present experiments indicate that in the mutant p47phoxS379A, only the single S-->A mutation appears to be a major importance for the function of p47phox, which is able to undergo the change in conformation that takes place when p47phox is phosphorylated by protein kinase C.
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PMID:Kinase-dependent change in the conformation of the leukocyte NADPH oxidase subunit p47phox. 1067 33

The reduced nicotinamide dinucleotide phosphate (NADPH) oxidase complex allows phagocytes to rapidly convert O2 to superoxide anion which then generates other antimicrobial reactive oxygen intermediates, such as H2O2, hydroxyl anion, and peroxynitrite anion. Chronic granulomatous disease (CGD) results from a defect in any of the 4 subunits of the NADPH oxidase and is characterized by recurrent life-threatening bacterial and fungal infections and abnormal tissue granuloma formation. Activation of the NADPH oxidase requires translocation of the cytosolic subunits p47phox (phagocyte oxidase), p67phox, and the low molecular weight GT-Pase Rac, to the membrane-bound flavocytochrome, a heterodimer composed of the heavy chain gp91phox and the light chain p22phox. This complex transfers electrons from NADPH on the cytoplasmic side to O2 on the vacuolar or extracellular side, thereby generating superoxide anion. Activation of the NADPH oxidase requires complex rearrangements between the protein subunits, which are in part mediated by noncovalent binding between src-homology 3 domains (SH3 domains) and proline-rich motifs. Outpatient management of CGD patients relies on the use of prophylactic antibiotics and interferon-gamma. When infection is suspected, aggressive effort to obtain culture material is required. Treatment of infections involves prolonged use of systemic antibiotics, surgical debridement when feasible, and, in severe infections, use of granulocyte transfusions. Mouse knockout models of CGD have been created in which to examine aspects of pathophysiology and therapy. Gene therapy and bone marrow transplantation trials in CGD patients are ongoing and show great promise.
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PMID:Genetic, biochemical, and clinical features of chronic granulomatous disease. 1084 36

An NADPH oxidase complex composed of a membrane-bound flavocytochrome b558 consisting of two subunits (p22phox and gp91phox) and cytosolic activating factors (p47phox and p67phox) generates superoxide anions from oxygen in the respiratory burst of phagocytic cells. Inconsistent results have been previously obtained concerning its additional occurrence in pulmonary artery endothelial cells (PAEC), and this issue was addressed in the present study. PAEC isolated from porcine pulmonary trunk contained mRNA for p22phox and gp91phox as demonstrated by reverse transcription-polymerase chain reaction. Immunohistochemistry demonstrated cytochrome subunits, p22phox, gp91phox, p47phox, and p67phox, both in vitro in isolated PAEC and in situ in endothelial cells in tissue sections of the pulmonary trunk. Isolated PAEC generated reactive oxygen species (ROS; measured by lucigenin-induced chemiluminescence and conversion of dihydrorhodamine 123 into rhodamine 123) in response to stimulation with phorbol 12-myristate 13-acetate. This stimulated ROS production was sensitive to the flavoprotein inhibitor diphenylene-iodonium, and reduced when the superoxide scavenger superoxide dismutase was added. Chemiluminescence measurements of superoxide generation by stimulated PAEC accounted for approximately 1% of that generated by stimulated peritoneal macrophages. The data demonstrate a low-output NADPH oxidase system in porcine PAEC sharing several components with that identified in phagocytic cells.
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PMID:NADPH oxidase subunits and superoxide production in porcine pulmonary artery endothelial cells. 1095 20

We examined the effect of cytochalasin B (CB) or granulocyte colony stimulating factor (GCSF) on superoxide radical (O2-) production of neutrophils by phorbor myristate acetate (PMA)-stimulation. It was observed that O2- generation of intact and GCSF-treated neutrophils by PMA-stimulation showed a lag during the early stage, and was largely inhibited by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (200 microm) or GF109203X (GFX) (0.2 microM), but not by ethanol (1%) and wortmannin (100 nM). In contrast, O2- generation of CB-pretreated neutrophils by PMA-stimulation did not show a lag, but was less than that of intact cells, and was only minimally depressed by the above inhibitors, but was markedly depressed by the simultaneous addition of GFX and ethanol or GFX and wortmannin. Although translocation of p47phox and p67phox to the membrane fraction by PMA-stimulation of intact and GCSF-treated neutrophils occurred in parallel with O2- production, that of CB-treated neutrophils by PMA-stimulation was not always proportional to O2- production. These findings suggest that pretreatment of neutrophils with CB dramatically alters the PMA response of the cells; that is, the protein kinase C-dependent pathway is largely depressed, and a phospholipase D-dependent one for NADPH oxidase activation appears in CB-treated cells.
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PMID:Phorbor myristate acetate induces NADPH oxidase activity of cytochalasin B-primed neutrophils through the protein kinase C-independent pathway. 1099 14

p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac.GTP bound to the N-terminal TPR (tetratricopeptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.
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PMID:Structure of the TPR domain of p67phox in complex with Rac.GTP. 1109 Jun 27


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