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)

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).
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PMID:Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox. 1125 27

Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil. The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O(2)()). With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity. A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67(phox). N-terminal sequence analysis of p29 revealed homology to an open reading frame gene described in a myeloid leukemia cell line. A cDNA for p29 identical to the open reading frame protein was amplified from RNA of neutrophils. Significant interaction between p29 and p67(phox) was demonstrated using a yeast two-hybrid system. A recombinant (rh) p29 was expressed in Sf9 cells resulting in a protein with an apparent molecular weight of 34,000. The rh-p29 showed immunoreactivity with the original rabbit antiserum that detected p47(phox) and p67(phox). In addition, rh-p29 exhibited PLA(2) activity, which was Ca(2+) independent, optimal at low pH, and preferential for phosphatidylcholine substrates. The recombinant protein protected glutathione synthetase and directly inactivated H(2)O(2). By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin. Finally, O(2)() production by plasma membrane and recombinant cytosolic oxidase components in the SDS-activated, cell-free NADPH oxidase system were enhanced by rh-p29. This effect was not inhibited by PLA(2) inhibitors. Thus, p29 is a novel protein that associates with p67 and has peroxiredoxin activity. This protein has a potential role in protecting the NADPH oxidase by inactivating H(2)O(2) or altering signaling pathways affected by H(2)O(2).
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PMID:A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity. 1212 78

Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue-wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to approximately 55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA-cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction.
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PMID:Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b(558). 1248 56

The PX domain of p47phox is thought to be involved in autoinhibition. However, when the domain was deleted, the ability to activate the phagocyte NADPH oxidase was markedly diminished. We have mutated the proline-rich region of the PX domain and examined the mutants for the ability to activate. Substitution of Gln for Pro-73 of p47phox(1-286) (P73Q) resulted in a considerably lower activity than the wild type and P73Q had a much lower affinity for the oxidase complex. Whereas, Gln substitution for Pro-76 (P76Q) showed a slightly enhanced activation and the mutant had a slightly higher affinity for the complex than the wild type. Affinity for p67phox(1-210) was slightly decreased either by P73Q or P76Q. Optimal SDS concentration for the activation was lowered by these mutations. Binding of PX domain with phosphatidylinositol-3,4-bisphosphate was diminished by P73Q mutation. The results in this study suggest that Pro-73 has a role in interaction with the catalytic component cytochrome b558.
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PMID:A new role of Pro-73 of p47phox in the activation of neutrophil NADPH oxidase. 1285 85

Sanguinarine (SA), a member of the benzo[c]phenanthridine isoquinoline alkaloids, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. We examined the effects of SA on oxidative burst in DMSO-differentiated HL-60 cells, an excellent model for studying oxidative burst. SA inhibited both N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst with half-maximal concentration for inhibition (IC(50)) of 1.5 and 1.8 microM, respectively. Despite suggestions of SA antioxidant activity this inhibition cannot be ascribed to radical scavenging property of SA because the IC(50) for superoxide dismutase-like activity in a non-cellular system was 60 microM. TROLOX, a water-soluble vitamin E analog, had IC(50) of 3 microM in the same system. Moreover, cyclic voltammetry measurements show that SA is not an easily oxidizable species, with a peak anodic potential at 700 mV, as compared to TROLOX with peak anodic potential at 200 mV. On the other hand, TROLOX, when used in cell suspension, was much poorer inhibitor of oxidative burst than SA. When testing direct effect of SA on NADPH oxidase in the post-granular fraction of disrupted cells, the IC(50) was found to be 8.3 microM. It is higher than that observed in whole cells, however, the shift may be ascribed to SDS effect on SA activity. We conclude the SA inhibition of oxidative burst is not caused by SA redox activity but most likely is a result of SA affecting the activity of NADPH oxidase directly and in part by preventing the formation of NADPH oxidase protein complex.
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PMID:Sanguinarine is a potent inhibitor of oxidative burst in DMSO-differentiated HL-60 cells by a non-redox mechanism. 1472 51

Neutrophils and other phagocytic cells support host defense by ingesting microbes and destroying them with reactive oxygen species or oxygen independent mechanisms. Production of ROS is initiated by the phagocyte NADPH oxidase (phox), an enzyme system composed of several constituents. During activation of the cell cytosolic phox proteins (p47phox, p67phox, p40phox, and Rac2) translocate to the plasma membrane and specific granules fuse with the plasma membrane increasing the amount of flavocytochrome b(558). The resultant assembly of phox components results in formation of a complete complex and expression of activity. In this study, we evaluated the oxidase activity of specific granules. In the SDS cell-free system, specific granules expressed oxidase activity in the presence of cytosol in a manner similar to plasma membrane. In contrast to plasma membrane, activity of specific granules was latent, diminishing rapidly over time. In addition, this subcellular fraction contained an inhibitor, possibly related to contamination with azurophilic granules explaining previously published discrepant results. Experiments with recombinant p47phox, p67phox, and dilute cytosol or fractionated cytosol as a source of Rac demonstrated that specific granules have requirements identical to specific granules for oxidase activity. Finally, analysis of neutrophils stimulated with PMA demonstrated translocation of p47phox and to p67phox to specific granules as well as plasma membrane. Both plasma membrane and specific granules from PMA stimulated cells expressed oxidase activity with addition of NADPH demonstrating an assembled oxidase complex. These studies establish a critical role for specific granules as a site for assembly and activation of the oxidase enzyme system and an important constituent for the microbicidal activity of the neutrophil.
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PMID:NADPH oxidase activity of neutrophil specific granules: requirements for cytosolic components and evidence of assembly during cell activation. 1505 19

Eosinophils are selectively primed and activated by the cytokine IL-5. The aim of this investigation was to study the effects of IL-5 treatment on stimulation-dependent protein phosphorylations, in human peripheral blood eosinophils. After IL-5 treatment, basal phosphorylation patterns showed increases in the phosphorylation of 67, 80 and 93 kDa proteins. Cell stimulations resulted in the following protein phosphorylation increases: 50, 60, 67, 80 and 93 kDa (PMA); 50, 67, 80 and 93 kDa (STZ); and 67, 80 and 93 kDa (IL-5). The phosphorylation of the 50 and 60 kDa proteins was shown to be MEK-independent and dependent on some PKC isoform/s, whereas that of the 67, 80 and 93 kDa proteins was both MEK- and PKC-alpha, beta, delta, gamma, tau and zeta-independent. A phosphoprotein of 50 kDa was identified as p47(phox) and another of 67 kDa protein as the tyrosine phosphatase SHPTP-1. Incubation with IL-5 followed by cell stimulation increased the total phosphorylation of p47(phox). Bidimensional (IEF-SDS/PAGE) analysis showed that the combination of IL-5 treatment followed by stimulation with either PMA or STZ induced the formation of an additional, hyperphosphorylated form of p47(phox). The presence of this form would explain the higher NADPH oxidase activity normally observed after IL-5 priming.
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PMID:The effect of IL-5 treatment on the stimulation-induced phosphorylation of proteins in blood eosinophils. 1547 55

Cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodeling to heart failure. All-trans retinoic acid (RA), a bioactive vitamin A derivative, prevents stretch- and angiotensin II (Ang II)-induced cardiac hypertrophy. However, the anti-apoptotic potential of RA in the heart remains unexplored. Here, we demonstrate that stretch- and Ang II-induced apoptosis is prevented by RA in neonatal cardiomyocytes. RA improved mitochondrial function by inhibiting the stretch- and Ang II-induced reduction in mitochondrial membrane potential, cytochrome c release and by increasing the Bcl2/Bax ratio. RA inhibited stretch- and Ang II-induced intracellular reactive oxygen species (ROS) generation and upregulated the SOD2 level. Hydrogen peroxide-induced increases in the number of TUNEL-positive cells and percentage of Annexin V positive cells, were dose-dependently inhibited by RA. The thiol antioxidant, N-acetyl cysteine (NAC), completely inhibited stretch- and Ang II-induced apoptosis. Using diazoxide (mitochondrial ATP-sensitive K(+) channel opener) and SDS (NADPH oxidase activator), we confirmed that RA suppressed both mitochondrial- and NADPH oxidase-derived ROS. We also observed that both RAR and RXR were involved in preventing Ang II- and stretch-induced ROS production and apoptosis, by using selective retinoid receptor agonists and antagonists. Our data provide the first evidence that RA prevents Ang II and stretch induced apoptosis, by inhibiting ROS generation and increasing the anti-oxidant defense system, suggesting that RA-mediated signaling may provide a new therapeutic target for the prevention of the cardiac remodeling process.
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PMID:All-trans retinoic acid prevents angiotensin II- and mechanical stretch-induced reactive oxygen species generation and cardiomyocyte apoptosis. 1794 Oct 88

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.
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PMID:Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity. 2217 85

Peroxiredoxins (Prdxs) are a family of proteins which catalyze the reduction of H2O2 through the interaction of active site cysteine residues. Conserved within all plant and animal kingdoms, the function of these proteins is related to protection from oxidation or participation of signaling through degradation of H2O2. Peroxiredoxin 6 (Prdx6), a protein belonging to the class of 1-cys Prdxs, was identified in polymorphonuclear leukocytes or neutrophils, defined by amino acid sequence and activity, and found associated with a component of the NADPH oxidase (Nox2), p67(phox). Prdx6 plays an important role in neutrophil function and supports the optimal activity of Nox2. In this chapter, methods are described for determining the Prdx activity of Prdx6. In addition, the approach for assessing the effect of Prdx6 on Nox2 in the SDS-activated, cell-free system of NADPH oxidase activity is presented. Finally, the techniques for suppressing Prdx6 expression in phox-competent K562 cells and cultured myeloid cells with siRNA and shRNA methods are described. With these approaches, the role of Prdx6 in Nox2 activity can be explored with intact cells. The biochemical mechanisms of the Prdx6 effect on the NADPH oxidase can be investigated with the experimental strategies described.
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PMID:Peroxiredoxin-6 and NADPH oxidase activity. 2383 Jun 30


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