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)

Neutrophils possess a multicomponent NADPH oxidase system capable of producing large quantities of superoxide in a process known as the respiratory burst (1). Upon stimulation of a phagocytic cell, two cytosolic components of the oxidase, p67phox and p47phox, associate with a membrane-bound flavocytochrome b and a small GTP-binding protein to form a functional enzyme complex. Each of the Phox proteins contains two src homology 3 (SH3) domains, which are of unknown function but are potential mediators of protein-protein interactions between components of the activated oxidase. We have isolated a 47-kDa protein from lysates of differentiated HL60 cells that specifically bound to the carboxyl-terminal SH3 domain of p67phox and not to any other SH3 domain tested. This protein was identified as p47phox, and the putative SH3 domain binding site was located to a carboxyl-terminal proline-rich region. Proline-rich synthetic peptides based on this carboxyl-terminal region specifically inhibited the binding of p47phox to the carboxyl-terminal SH3 domain of p67phox, and sequential truncation defined a unique minimal sequence, which, although similar, does not match the consensus sequence defined for other SH3-binding proteins.
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PMID:An SH3 domain and proline-rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex. 818 50

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.
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PMID:Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. 820 90

Activation of human neutrophil NADPH oxidase requires the interaction of cytosolic and membrane-associated components. Evidence has been accumulated that in phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils, the translocation to the plasma membrane of the cytosolic components p47phox and p67phox and the phosphorylation of p47phox are essential steps in activation of NADPH oxidase. No direct evidence has been presented to date as to whether p67phox is also phosphorylated. To address this problem we have immunoprecipitated p67phox from neutrophil cytosol and membrane fractions. The results indicate that, very soon after activation with PMA (20 s), p67phox was present in a phosphorylated form in the cytosol and in the membranes. At later times (1-3 min) the extent of p67phox phosphorylation continuously increased both in the cytosol and in the membrane fraction, while oxygen consumption reached the maximal rate within 40 s, and then remained linear. p67phox was also phosphorylated in formyl-methionyl-leucyl-phenylalanine-activated neutrophils. That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the observation that no phosphorylated band of 67 kDa was immunoprecipitated from the cytosol and membranes of PMA-stimulated neutrophils from a p67phox-deficient chronic granulomatous disease patient. In this case, p47phox was normally phosphorylated. These data demonstrate that: (1) the phosphorylation of p67phox is correlated with activation of NADPH oxidase, and (2) continuous phosphorylation of p67phox is required in order to maintain the linearity of the respiratory burst.
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PMID:Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox. 825 26

The NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the beta-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32P]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2(-)-generating activity and caused considerable translocation of the cytosolic components p47phox and p67phox. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and approximately 32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the beta-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91phox-deficient X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system.
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PMID:NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558. 827 Aug 71

The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.
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PMID:p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. 828 52

Phagocytic white blood cells contain a multicomponent oxidase that generates microbicidal products by catalyzing electron transfer from NADPH to molecular oxygen. Activation of this oxidase requires interactions of a unique membrane flavocytochrome with the cytosolic proteins p47phox, p67phox, and p21Rac. This flavocytochrome, designated cytochrome b558, is a heteromer comprising a 22-kDa alpha-subunit (p22phox) and a glycosylated approximately 91-kDa beta-subunit (gp91phox). Cytochrome b558 was expressed in Sf9 insect cells coinfected with recombinant baculoviruses carrying cDNAs for p22phox and gp91phox. Membranes of these cells contained a b-type cytochrome with a dithionite-reduced minus oxidized difference spectrum similar to that of neutrophil cytochrome b558. The recombinant cytochrome b558 beta-subunit was heterogeneously N-glycosylated as demonstrated by its susceptibility to cleavage with endoglycosidases F and H. In contrast to the neutrophil cytochrome b558, a portion of the N-linked oligosaccharide was of the high mannose type. Recombinant cytochrome b558 supported superoxide production in a cell-free assay containing recombinant p47phox, p67phox, and p21Rac. The enzymatic turnover of the partially purified recombinant cytochrome b558 and neutrophil cytochrome b558 were similar (approximately 100-160 mol of superoxide generated/s/mol of cytochrome heme, range of two experiments) and the native and recombinant cytochromes showed similar requirements for NADPH and exogenous FAD. These studies represent the first reconstitution of the NADPH oxidase solely from recombinant proteins and define a model system to explore the structure and function of cytochrome b558.
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PMID:Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins. 831 88

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.
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PMID:Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca(2+)-depleted human neutrophils. 843 86

The NADPH oxidase is an electron transport chain found in lymphocytes and in the wall of the endocytic vacuole of 'professional' phagocytic cells. It is so called because NADPH is used as an electron donor to reduce oxygen to superoxide and hydrogen peroxide. The redox components are provided by a very unusual flavocytochrome b from the membrane, which is dependent upon cytosolic factors (including two specialized proteins, p47phox and p67phox) for activation. The small GTP-binding protein, p21rac, is also implicated in this system, possibly as the switch that triggers electron transport. This system provides a key to our understanding of the way in which these GTP-binding proteins function.
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PMID:The biochemical basis of the NADPH oxidase of phagocytes. 848 57

Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).
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PMID:Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins. 850 89

NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b558 of cytosolic components p47phox, p67phox, and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67phox; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67phox. Finally, we observe that p67phox interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67phox and that Rac2 is the main p67phox-interacting GTPase in human cells.
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PMID:The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1. 855 Jun 29


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