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:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.
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PMID:Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation. 1568 31

Reactive oxygen species (ROS) trigger programmed cell death in neonatal sympathetic neurons that have been deprived of nerve growth factor (NGF), however, the source of these oxygen intermediates has not been established. Using laser scanning confocal microscopy (LSCM), the intracellular distribution of the subunits of the ROS-generating enzyme NADPH oxidase was examined in sympathetic neurons of the superior cervical ganglion (SCG). Optical sectioning using LSCM showed that gp91-phox and p22-phox co-localize in neurons at the cell membrane, while the p47-phox and p67-phox subunits are found uniformly distributed in the cytoplasm of neurons maintained in the presence of NGF. Within 4h after NGF deprivation, both the p47-phox and p67-phox subunits exhibit punctate staining in the cytoplasm and at the membrane. Furthermore, a sub-population of the cytosolic p47-phox appeared to co-localize with the membrane-bound gp91-phox in NGF-deprived neurons. These data provide support for the presence of NADPH oxidase in sympathetic neurons and suggest that this enzyme may become activated following the withdrawal of NGF.
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PMID:Localization of NADPH oxidase subunits in neonatal sympathetic neurons. 1572 79

Chronic granulomatous disease (CGD) is a rare inherited disorder in which antimicrobial activity of phagocytes is impaired due to the lack of reactive oxygen species, or oxidative burst, produced by NADPH oxidase. The X-linked form of CGD, representing approximately 70% of all cases, is caused by mutations in the cytochrome b beta subunit (CYBB) gene, which maps to chromosome Xp21.1. CYBB encodes the gp91-phox protein, a necessary component in the NADPH oxidase pathway. A wide variety of mutations have been identified in X-linked CGD patients, all of which lead to deletion of the functional protein and no oxidative burst activity. The mutations vary from single nucleotide substitutions to deletions of the entire gene. In this article, we report a mutation detection method for probands of female relatives at risk for carrier status of large deletions of the CYBB gene. Through fluorescent in situ hybridization of metaphase chromosomes, we were able to consistently distinguish carriers from noncarriers using polymerase chain reaction-derived, labeled DNA specific for exons 2 to 13 of the CYBB region at Xp21.1.
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PMID:Long polymerase chain reaction-based fluorescence in situ hybridization analysis of female carriers of X-linked chronic granulomatous disease deletions. 1585 41

Chronic granulomatous disease (CGD) is a rare congenital disorder in which the patients' phagocytes fail to kill ingested microbes due to an inability to generate superoxide and other microbicidal oxygen derivatives. This inability is caused by mutations in one of the four components of the phagocyte-specific NADPH oxidase. A small subgroup of CGD patients has mutations in the CYBA gene that encodes the p22-phox subunit of the NADPH oxidase. This subunit forms, together with gp91-phox, a flavocytochrome b(558) heterodimer in the phagocyte plasma membrane. Expression of both subunits is required for normal expression of this heterodimer. Here, we report an autosomal recessive CGD patient with neutrophils that did not express flavocytochrome b(558) and did not generate superoxide upon activation. Analysis of genomic DNA revealed a 4-bp deletion at the exon-1/intron-1 boundary in CYBA (IVS1+4_7delAGTG). In the patient's cDNA, we found a low expression of an abnormal product, containing exon 1 extended by 79 nucleotides from intron 1, joined to exon 2. This extension is apparently caused by the activation of a cryptic donor splice site with a GT sequence at position 84-85 in intron 1. Both parents of the patient had the same mutation in their genomic DNA, in heterozygous form, but their cDNA contained exclusively the wild-type p22-phox cDNA sequence, indicating that the mutant mRNA was labile. This is, as far as we know, the first description of the molecular and clinical consequences of a donor splice site mutation in intron 1 of any gene reported so far.
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PMID:A donor splice site mutation in intron 1 of CYBA, leading to chronic granulomatous disease. 1615 92

The aim of this work was to analyze the effect of Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in human colostrum macrophages (CM), peripheral blood monocytes (PBM), and myelomonocytic THP-1 cells. We also investigated the effect of IFN-gamma on the release of TNF-alpha by these cells. Our results show that under basal culture conditions, CM release more superoxide than PBM and THP-1 cells (p < 0.05). The addition of IFN-gamma, alone or in combination with TNF-alpha, increased spontaneous superoxide release by PBM and THP-1 cells (p < 0.05) and increased phorbol myristate acetate (PMA)-stimulated superoxide release by CM, PBM, and THP-1 cells (p < 0.05). The NADPH oxidase activity of THP-1 cells consistently remained lower than that of CM or PBM, despite a dramatic response to IFN-gamma and TNF-alpha. Under basal conditions, gp91-phox gene expression was significantly higher in CM and PBM compared with THP-1 cells (p < 0.05). The addition of IFN-gamma alone or in combination with TNF-alpha caused a dramatic increase in gp91-phox gene expression in THP-1 cells (p < 0.05) but not in CM or PBM. Under basal conditions or in the presence of IFN-gamma, CM released more TNF-alpha than PBM or THP-1 cells (p < 0.05). In addition, PBM released more TNF-gamma than THP-1 cells (p < 0.05). IFN-gamma did not significantly augment the release of TNF-alpha by these cells (p > 0.05). Thus, IFN-gamma and TNF-alpha induced equivalent gp91-phox gene expression in THP-1 cells compared with CM or PBM but did not bring about equivalent NADPH oxidase activity. TNF-alpha release was higher in more mature cells. This partial divergence of gp91- phox gene expression, NADPH oxidase activity, and TNF-alpha release is probably a consequence of different events of myeloid cell biology and relates at least in part to cell differentiation state.
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PMID:The effect of IFN-gamma and TNF-alpha on the NADPH oxidase system of human colostrum macrophages, blood monocytes, and THP-1 cells. 1618 Oct 54

Osteopontin (OPN) is a cytokine upregulated in diabetic vascular disease. To better understand its role in vascular remodeling, we assessed how OPN controls metalloproteinase (MMP) activation in aortic adventitial myofibroblasts (AMFs) and A7r5 vascular smooth muscle cells (VSMCs). By zymography, OPN and tumor necrosis factor (TNF)-alpha preferentially upregulate pro-matrix metalloproteinase 9 (pro-MMP9) activity. TNF-alpha upregulated pro-MMP9 in AMFs isolated from wild-type (OPN(+/+)) mice, but pro-MMP9 induction was abrogated in AMFs from OPN(-/-) mice. OPN treatment of VSMCs enhanced pro-MMP9 activity, and TNF-alpha induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F(2) alpha-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-alpha, 8-IsoP preferentially activated pro-MMP9. Superoxide, 8-IsoP, and NADPH oxidase 2 (Nox2) subunits were reduced in OPN(-/-) AMFs. Treatment of A7r5 VSMCs with OPN upregulated NADPH oxidase subunit accumulation. OPN structure/function studies mapped these activities to the SVVYGLR heptapeptide motif in the thrombin-liberated human OPN N-terminal domain (SLAYGLR in mouse OPN). Treatment of aortic VSMCs with SVVYGLR upregulated pro-MMP9 activity and restored TNF-alpha activation of pro-MMP9 in OPN(-/-) AMFs. Injection of OPN-deficient OPN(+/-) mice with SVVYGLR peptide upregulated pro-MMP9 activity, 8-IsoP levels, and Nox2 protein levels in aorta and increased panmural superoxide production (dihydroethidium staining). At equivalent hyperglycemia and dyslipidemia, 8-IsoP levels and aortic pro-MMP9 were reduced with complete OPN deficiency in a model of diet-induced diabetes, achieved by comparing OPN(-/-)/LDLR(-/-) versus OPN(+/-)/LDLR(-/-) siblings. Thus, OPN provides a paracrine signal that augments vascular pro-MMP9 activity, mediated in part via superoxide generation and oxylipid formation.
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PMID:An osteopontin-NADPH oxidase signaling cascade promotes pro-matrix metalloproteinase 9 activation in aortic mesenchymal cells. 1679 91

The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with cytochrome b558, thus affecting NADPH access to its binding site.
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PMID:Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly. 1706 Mar 62

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.
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PMID:New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody. 1739 83

Chronic granulomatous disease (CGD) is a hereditary illness generally occurring in childhood, in the form of recurrent severe infections. The main pathogens are staphylococci and aspergilli. It results from a failure of professional phagocytes, and particularly neutrophils, to produce superoxide ions O2- and their derivatives, which protect cells from bacterial, invasion through an oxidative and toxic defence mechanism. At an infection site. contact between the neutrophils and microorganisms or an inflammatory mediator triggers a respiratory burst, which results in the activation of the NADPH oxidase enzyme complex. NADPH depletes surrounding oxygen to yield O2-. In its active form. NADPH oxidase is an assembly of two components, namely the membrane cytochrome b558 (consisting o two subunits, gp91-phox and p22-phox) and soluble protein factors present in the resting neutrophil cytoplasm. Transfer of these cytosolic factors and their anchorage to cytochrome b558 determines the activity of NADPH oxidase. The respiratory burst lasts no more than a few minutes, but the precise mechanisms underlying its termination are not well known. In chronic granulomatous disease, neutrophils have lost their bactericidal capacity The most frequent form is hereditary and X-linked; in this case, the affected gene is CYBB, which encodes gp91-phox, the catalytic subunit of cytochrome b558. In autosomal and recessive forms of CGD the mutations affect the genes encoding p22-phox, p67-phox or p47-phox. We have unraveled the assembly mechanisms of the NADPH oxidase complex and have demonstrated that the cytosolic factor p67-phox is the determining element: it triggers both the assembly and the activation of NI4DPH oxidase. Binding of p67-phox to cytochrome b558 induces a gradual conformational change of cytochrome b558, which then becomes capable of transferring electrons produced in the cytoplasm from NADPH to oxygen, reducing the latter to O2-. The isolation of NADPH oxidase in its active and assembled form has allowed us to identify the activation partners of the oxidase complex. We also demonstrated that calcium-binding myeloid-related proteins (MRP). that are abundant in neutrophil cytoplasm, play a fundamental role in this activation. CGD patient management is essentially based on long-term high-dose prophylactic antibiotic administration. Gene therapy is promising but some distance away from practical application. We are currently investigating a new therapeutic concept that consists of transferring cytochrome b558 protein directly into deficient cells (initially the PLB 985 X cell line), encapsulated in proteoliposomes, which are hydrophobic.
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PMID:[Molecular aspects of chronic granulomatous disease. "the NADPH oxidase complex"]. 1796 55

The interleukin 4 (IL-4)/IL-4 receptor (IL-4R) system in promyelocytes is not well documented. Here, we used promyelocytic leukaemia PLB-985 cells differentiated with dimethylsulfoxide (PLB-985D) toward neutrophil-like phenotype to investigate the IL-4/IL-4R system. PLB-985 cells did not express CD132 (gammac) but expressed the complete IL-4 type II receptor (IL-4Ralpha and IL-13Ralpha1). Moreover, PLB-985 cells lost surface expression of IL-13Ralpha1 during differentiation, resulting in PLB-985D cells expressing only IL-4Ralpha fully responsive to IL-4, as judged by activation of mitogen-activated protein (MAP) kinases and Janus kinase 1. IL-4 also increased suppressor of cytokine signalling 3 (SOCS3) protein level in the presence of the proteasome inhibitor MG132 exclusively in PLB-985D cells. As the IL-4Ralpha chain has been associated with a component of the phagocyte NADPH oxidase, we used PLB-985-gp91(phox) deficient cells (mimicking chronic granulomatous disease, X-CGD), to investigate the IL-4/IL-4R system in X-CGD-D cells. IL-4 was found to activate MAP kinases in X-CGD-D cells but did not up-regulate SOCS3, in contrast to granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and IL-6. Utilization of catalase, cycloheximide and genistein inhibitors showed that IL-4 induced SOCS3 by a mechanism dependent on a complete NADPH oxidase complex, protein synthesis and tyrosine phosphorylation, but independent of production of reactive oxygen species. We conclude that IL-4 induces cell signalling in promyelocytes expressing only IL-4Ralpha.
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PMID:Investigation of the interleukin (IL)-4/IL-4 receptor system in promyelocytic leukaemia PLB-985 cells during differentiation toward neutrophil-like phenotype: mechanism involved in IL-4-induced SOCS3 protein expression. 1800 66


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