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)

Chronic granulomatous disease (CGD) is a syndrome characterized by failure of the NADPH oxidase of phagocytes that generates superoxide, which is central to the microbicidal process. Cytosolic components of this oxidase system include the proteins p67phox and p47phox, deficiencies of which cause the autosomal recessive form of CGD, whereas the X-linked form of the disease is characterized by a deficiency in the plasma membrane component gp91phox. Components of the oxidase system have been reported to be associated with the cytoskeleton and neutrophils from CGD patients have been reported to have a defective chemotactic response in Boyden chambers. Using a chamber that permits the direct observation of cell behaviour in a linear gradient of a chemoattractant, we have analysed the chemotactic response of neutrophils from a patient lacking p67phox; from another lacking p47phox and from a third lacking gp91phox. The results of measuring the speeds and directions of locomotion of the cells show that their speeds are undiminished relative to cells from healthy control subjects and that their directions of migration are at least as strongly biased in the direction of the gradient as those of the control cells. We conclude that these definitive aspects of the chemotactic response are not abnormal in either the autosomal recessive or the X-linked forms of CGD and that they are therefore not factors in the predisposition to infection that characterizes the syndrome.
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PMID:Deficiency of p67phox, p47phox or gp91phox in chronic granulomatous disease does not impair leucocyte chemotaxis or motility. 905 62

The NADPH oxidase that produces superoxide in professional phagocytic cells is a flavocytochrome b electron transport chain in the membrane, a heterodimer of gp91phox and p22phox, that is activated by a number of cytosolic proteins, including p47phox, p67phox, and the small GTP-binding protein p21rac, which translocate to the membrane and attach to the flavocytochrome on activation. The components of this oxidase were localized on the cytoplasmic surface of the plasma membrane of adherent unroofed neutrophils by immunolabeling. Components of the NADPH oxidase and p21rac were found together in punctate clusters occupying 0.03-0.1 microm2 of the cytoplasmic surface of the plasma membrane where the density of labeling of the cytosolic components was increased after stimulation with phorbol myristate acetate.
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PMID:Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane. 906 Apr 53

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.
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PMID:Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox. 906 49

The p47phox-/- mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus-based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox-/- marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox-/- mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% +/- 0.9% of phorbol myristate acetate-stimulated peripheral blood neutrophils at 4 weeks and 2.6% +/- 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox-/- mice were significantly lower than untreated p47phox-/- mice (0.89 +/- 0.30 colonies v 237.7 +/- 83.6 colonies at 24 hours, P < .02; 4.0 +/- 2.0 colonies v 110.2 +/- 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox-/- mice (P < .001). Thus, stem-cell-directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.
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PMID:Enhanced host defense after gene transfer in the murine p47phox-deficient model of chronic granulomatous disease. 911 68

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47phox, p67phox, and p22phox). p47phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22phox and cytosolic p67phox. To understand the specific roles of each SH3 domain of p47phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr167 or Tyr237-->Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47phox-p22phox interaction and plays a predominant role in oxidase activity and p47phox membrane assembly, while the second p47phox SH3 domain interacts with the NH2-terminal domain of p67phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.
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PMID:Specificity of p47phox SH3 domain interactions in NADPH oxidase assembly and activation. 912 67

The differential expression of protein kinase C (PKC) isozymes and small GTP-binding proteins, and their relation to O2 generation and phospholipase D (PLD) activation were analyzed during the differentiation of human promyelocytic HL60 cells to neutrophil-like cells induced by either retinoic acid (RA) or dibutyryl cyclic AMP (dbcAMP). In response to either one of the inducers, nitroblue tetrazolium (NBT) reduction activity time-dependently increased. Although PLD activity was upregulated by dbcAMP-treatment, only a slight increase was observed in RA-treated cells. Small GTP-binding proteins Rac1, Rap1, and RhoA, which are reported to be implicated in O2- generation or PLD activation, were already expressed in undifferentiated HL60 cells and their significant changes were not detected during differentiation. The mRNAs of the cytosolic components of NADPH oxidase system, p47phox and p67phox, were present in trace amounts in undifferentiated cells. However, they rapidly increased in response to RA or dbcAMP. In response to either RA or dbcAMP, the increases were observed in cPKC isozymes (alpha, beta I, beta II) but not in other subtypes (delta, epsilon, theta, zeta) by both RT-PCR and Western blot analyses. In dbcAMP-treated cells PKC alpha increased remarkably, whereas PKC beta I and beta II mainly elevated in RA-treated cells. These results suggest the possibility that cPKCs are closely related to cell differentiation and that PKC alpha is involved in PLD activation.
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PMID:Differential expression of protein kinase C isozymes and small GTP-binding proteins during HL60 cell differentiation by retinoic acid and cyclic AMP: relation with phospholipase D (PLD) activation. 914 35

1. The ability of acetylshikonin to inhibit the respiratory burst in rat neutrophils was characterized and the underlying mechanism of action was also assessed in the present study. 2. Acetylshikonin caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.48 +/- 0.03 and 0.39 +/- 0.03 microM, respectively. Acetylshikonin also inhibited the O2 consumption in neutrophils in response to fMLP/CB as well as to PMA. 3. Acetylshikonin did not scavenge the generated O2.- in the xanthine-xanthine oxidase system or during dihydroxyfumaric acid (DHF) autoxidation but, on the contrary, acetylshikonin enhanced the O2.- generation in these cell-free oxygen radical generating systems. 4. Acetylshikonin inhibited the formation of inositol trisphosphate (IP3) (39.0 +/- 7.8% inhibition at 10 microM, P < 0.05) in neutrophils in response to fMLP. 5. Both the neutrophil cytosolic protein kinase C (PKC) activity and the PMA-induced PKC associated with the membrane were unaffected by acetylshikonin. 6. Acetylshikonin did not affect the porcine heart protein kinase A (PKA) activity. Upon exposure to acetylshikonin, the cellular cyclic AMP level was decreased in neutrophils in response to fMLP. 7. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP/CB were inhibited by acetylshikonin (60.1 +/- 7.3 and 63.2 +/- 10.5% inhibition, respectively, at 10 microM, both P < 0.05). Moreover, acetylshikonin attenuated the fMLP/CB-induced protein tyrosine phosphorylation (about 90% inhibition at 1 microM). 8. In PMA-activated neutrophil particulate NADPH oxidase preparations, acetylshikonin did not inhibit, but enhanced, the O2.- generation in the presence of NADPH. However, acetylshikonin decreased the membrane associated p47phox in PMA-activated neutrophils (about 60% inhibition at 1 microM). 9. Collectively, these results suggest that the attenuation of protein tyrosine phosphorylation and a failure in the assembly of a functional NADPH oxidase complex probably contribute predominantly to the inhibition of respiratory burst in neutrophils by acetylshikonin. In contrast, the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways play only a minor role in this respect.
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PMID:Investigation of the inhibition by acetylshikonin of the respiratory burst in rat neutrophils. 917 81

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as 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-bound flavohemoprotein, to assemble the active oxidase. An essential element of the activation process is the phosphorylation of p47phox, an event that accompanies oxidase activation in whole cells and can activate the oxidase in a cell-free system. We show here that the phosphorylation of p47phox leads to a substantial decrease in the reactivity of cysteine C378 toward N-ethylmaleimide, indicating the occurrence of a conformational change involving the C-terminal region of p47phox. A similar conformational change occurs when p47phox is exposed to arachidonate, one of a number of anionic detergents that activate the oxidase in the cell-free system. We propose that this change in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.
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PMID:Activation of the leukocyte NADPH oxidase subunit p47phox by protein kinase C. A phosphorylation-dependent change in the conformation of the C-terminal end of p47phox. 920 Jun 96

This investigation was undertaken to clarify the mechanisms of superoxide anion (O2-) generation in rat peritoneal mast cells. Compound 48/80, a typical histamine liberator mediated by calcium influx, elicited O2- generation from the mast cells in a dose-dependent fashion. It was demonstrated by immunohistochemical study and Western blot analysis that the mast cells contained the 47-kDa phagocyte oxidase (p47phox) protein, which was one cytosolic component of the NADPH oxidase system. Arachidonic acid stimulated O2- generation in the mast cells, but other unsaturated fatty acids had no effect. On the other hand, 48/80-induced O2- generation was inhibited by phospholipase A2 inhibitors, such as arachidonyl trifluoromethyl ketone and manoalide. Forskolin, isoprenaline, and dibutyryl cyclic AMP inhibited the O2- generation, and KT-5720, a cyclic AMP-dependent protein kinase (A-kinase) inhibitor, markedly enhanced the O2- generation. These findings suggest that O2- is generated by a NADPH oxidase-like enzyme system in mast cells and that this enzyme system is activated by arachidonic acid released by cytosolic phospholipase A2. Thus, it is regulated by the cyclic AMP-A kinase system.
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PMID:The mechanisms of compound 48/80-induced superoxide generation mediated by A-kinase in rat peritoneal mast cells. 923 5

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. 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 b258 of cytosolic components p47phox, p67phox and Rac proteins. 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. Finally, we observe that p67phox interacts six fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac, and particularly Rac2, can regulate superoxide production by NADPH oxidase of phagocytic cells through direct interaction with p67phox subunit. Recently published data suggest that Rac proteins could transduce mitogenic signals in non-phagocytic cells through superoxide production by a phagocytic-related NADPH oxidase enzymatic system which remains to be determined. NADPH oxidase regulation by Rac proteins in phagocytes could then be used as a model to understand the molecular mechanisms underlying Rac functions in various cell types.
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PMID:[Signal transduction by Rac small G proteins in phagocytes]. 925 50


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