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)

Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.
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PMID:The Ku70 autoantigen interacts with p40phox in B lymphocytes. 991 62

The role of magnesium ions in the activation of NADPH oxidase has been investigated using flavocytochrome b-245 and either neutrophil cytosol or mixtures of recombinant p40phox, p47phox, p67phox and Rac2. Purified flavocytochrome b-245 is highly active (turnover number 120-150 mol of O2(-)/s per mol of cytochrome haem) in the absence of Mg2+, in marked contrast to neutrophil membranes or detergent-solubilized membranes, which have an absolute requirement for Mg2+ for NADPH oxidase activity. It was also found that Mg2+ affected the anionic amphiphile requirement for oxidase activation, and this was dependent on whether neutrophil cytosol or mixtures of recombinant cytosolic proteins were used in the assay. Unexpectedly we found that, using purified flavocytochrome b-245 and recombinant cytosolic proteins, NADPH oxidase undergoes spontaneous activation in the absence of anionic amphiphiles under Mg2+-free conditions. The results suggest that Mg2+ ions play an important role in NADPH oxidase function, perhaps stabilizing the 260 kDa complex of cytosolic phox proteins or the regulation of a guanine nucleotide-binding protein. We provide evidence that if the latter explanation is correct, the identity of the guanine nucleotide-binding protein is unlikely to be Rap1a.
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PMID:Spontaneous activation of NADPH oxidase in a cell-free system: unexpected multiple effects of magnesium ion concentrations. 993 20

Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrome b558 composed of gp91-phox and p22-phox, and four cytosolic components, p47-phox, p67-phox, rac p21 and p40-phox, which translocate to the membrane upon activation. In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as 1 out of 250,000 births. The expected life span of the CGD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47-phox deficiency is lower (less than 10% vs. 23%) and that of gp91-phox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to develop the gene therapy technology for CGD patients as more promising treatment. In the current study we constructed two retrovirus vectors; MFGS-gp91/293 SPA which contains only the therapeutic gp91-phox gene, a bicistronic retrovims pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp91-phox gene connected with an internal ribosome entry site (IRES). We demonstrate high efficiency transduction of gp91-phox to CGD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp91, respectively. We also demonstrate sufficient transduction of gp91-phox to CD34+ haematopoietic stem cell from the patients with gp91-phox deficiency by MFGS-gp91/293 SPA. Our current studies suggest that the combination of the 293-SPA packaging system and the bicistronic retrovirus system inserted MDR1 gene make our CGD gene therapy more feasible for clinical application.
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PMID:[Statistical evaluation of chronic granulomatous disease in Japan and basic studies for gene therapy for CGD patients]. 1044 45

Phagocytic cells possess a tightly regulated multicomponent enzyme complex, the NADPH oxidase, which produces superoxide, a reactive oxygen molecule that is an essential component of host defense against infection. Upon stimulation, a functional NADPH oxidase is assembled when the cytosolic proteins, Rac, p67phox, p47phox, and possibly p40phox, associate with the gp91phox and p22phox transmembrane proteins. Rac is a GTPase that in the GTP-bound state binds p67phox to activate NADPH oxidase. The function of p40phox is not known; it is believed to have a regulatory function in sequestering p67phox and p47phox in a cytosolic complex. We investigated binding interactions between p40phox, p67phox, and Rac and found that Rac1-GTP displaced p67phox bound to p40phox. In contrast, Cdc42, a GTPase homologous to Rac, did not displace p67phox from p40phox. A synthetic peptide corresponding to p67phox amino acids 170-199, a region identified previously as a Rac binding domain, significantly reduced the ability of Rac1-GTP to disrupt p67phox/p40phox binding. We hypothesize that Rac-GTP binds the p67phox N-terminal domain encompassing amino acids 170-199 that transmits a conformational change which causes p40phox to dissociate from its binding site in the p67phox C-terminus.
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PMID:Rac1 disrupts p67phox/p40phox binding: a novel role for Rac in NADPH oxidase activation. 1048 63

NADPH oxidase, a superoxide-producing enzyme in phagocytic cells, consists of membrane-associated cytochrome b558 and cytosolic components (p47-phox, p67-phox, p40-phox, rac 1/2). Activation of NADPH oxidase is accompanied by the phosphorylation of cytosolic components (p47-phox and p67-phox). In this study, we have examined whether p40-phox, a newly identified cytosolic component, is phosphorylated during neutrophil activation, and the relationship between p40-phox phosphorylation and NADPH oxidase activation. When 32P-labeled guinea pig neutrophils were stimulated by phorbol 12-myristate 13-acetate, p40-phox was phosphorylated as p47-phox. It is interesting that phosphorylation of p40-phox was markedly inhibited by protein kinase C inhibitor, H-7, but not by casein kinase II inhibitor, A-3, and H-7 inhibited translocation of p40-phox and activation of NADPH oxidase. Furthermore, purified protein kinase C but not casein kinase II directly phosphorylated p40-phox of p40-phox/p47-phox/p67-phox complex. Together these observations suggest that p40-phox is phosphorylated by protein kinase C during neutrophil activation, and phosphorylation of p40-phox may be important for the activation of NADPH oxidase.
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PMID:Phosphorylation of p40-phox during activation of neutrophil NADPH oxidase. 1057 19

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

Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.
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PMID:Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction. 1067 14

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.
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PMID:P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy. 1092 23

We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion (O(2)(-)). We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [(3)H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O(2)(-) and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH(2)F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-kappaB, and inhibition of this activity by nuclear factor-kappaB decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O(2)(-) produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.
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PMID:Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1. 1109 39

Mastoparan, an amphiphilic cationic tetradecapeptide was previously shown to block activation of the NADPH oxidase in the cell-free system presumably by association with a cytosolic component/s of the enzyme. Blockade of oxidase activation was now demonstrated in the semirecombinant NADPH oxidase system. The structural basis of the inhibitory effect of MP on oxidase assembly was explored employing a variety of truncated and specifically substituted synthetic peptide analogs. The data indicated that an alpha helical fold, positive net charge, hydrophobicity and amphiphilicity were essential for the inhibitory potency and that peptide analogs below eleven residues were inactive. To identify the MP-binding oxidase subunit three different binding assays were carried out utilizing free or immobilized recombinant p47-phox, p67-phox, p40-phox and Rac1 in conjunction with immobilized MP or soluble (125)I-tyr-MP, respectively. The data implicated p67-phox as the main MP-binding component. The binding site on the p67-phox was localized to the 1-238 aminoterminal fragment of the molecule. NADPH oxidase activation supported by this fragment was inhibitable by MP. In addition, SH3 domains of p47-phox and p40-phox and the carboxyterminal SH3 domain of p67-phox exhibited a low affinity towards MP.
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PMID:Structure-function relationship in the interaction of mastoparan analogs with neutrophil NADPH oxidase. 1130 Oct 39


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