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)

NADPH oxidase is one of the major components of the innate immune system and is used by phagocytes to generate microbicidal reactive oxygen species. Activation of the enzyme requires the participation of a minimum of five proteins, p22(phox), gp91(phox) (together forming flavocytochrome b(558)), p47(phox), p67(phox) and the GTP-binding protein, Rac2. A sixth protein, p40(phox), has been implicated in the control of the activity of NADPH oxidase principally based on its sequence homology to, and physical association with, other phox components, and also the observation that it is phosphorylated during neutrophil activation. However, to date its role in regulating the activity of the enzyme has remained obscure, with evidence for both positive and negative influences on oxidase activity having being reported. Data are presented here using the cell-free system for NADPH oxidase activation that shows that p40(phox) can function to promote oxidase activation by increasing the affinity of p47(phox) for the enzyme approx. 3-fold.
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PMID:p40(phox) Participates in the activation of NADPH oxidase by increasing the affinity of p47(phox) for flavocytochrome b(558). 1086 Dec 18

To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide-producing activity using HL-60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91(phox), p67(phox), and p47(phox) were expressed after myelocyte stages, whereas p22(phox), p40(phox), and rac-2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO-induced HL-60 cells indicated that gp91(phox), p67(phox), and p47(phox) were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22(phox), p40(phox), and rac-2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO-induced HL-60 cells were obtained using human bone-marrow cells by immunocytochemical staining and NBT assay, except that p22(phox) was detected by immunocytochemical staining after myelocyte stages in bone-marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.
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PMID:Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage. 1094 66

Bacterial type III secretion system drives the translocation of virulence factors into the cystosol of host target cells. In phagocytes and in Epstein-Barr virus immortalized B lymphocytes, NADPH oxidase generates O(-2) through an electron transfer chain the activity of which depends on the assembly of three, p67(phox), p47(phox) and p40(phox) cytosolic activating factors with Rac 1/2 and a membrane redox component, cytochrome b(558). In p67(phox) deficient chronic granulomatous disease (CGD) patients, p67-phox is missing and NADPH oxidase activity is abolished. ExoS is a virulence factor of Pseudomonas aeruginosa which is secreted via the type III secretion system: it was fused with p67(phox). Pseudomonas aeruginosa synthesized and translocated the hybrid ExoS-p67(phox) fusion protein into the cytosol of B lymphocytes via the type III secretion system. Purified ExoS-p67(phox) hybrid protein was as efficient as normal recombinant p67(phox) in cell-free reconstitution of NADPH oxidase activity. Therefore, ExoS-p67(phox) was transferred via the type III secretion system of Pseudomonas aeruginosa into the cytosol of B lymphocytes from a p67(phox)-deficient CGD patient and functionally reconstituted NADPH oxidase activity. In the complementation process, ExoS acted as a molecular courier for protein delivery: the reconstitution of an active NADPH oxidase complex suggests type III secretion system to be a new approach for cellular therapy.
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PMID:Protein delivery by Pseudomonas type III secretion system: Ex vivo complementation of p67(phox)-deficient chronic granulomatous disease. 1097 11

Oxygen radical generation by stimulation with phorbol myristate acetate (PMA) was evaluated in bottlenose dolphin neutrophils. A Cypridina luciferin analog-dependent chemiluminescent assay demonstrated that dolphin neutrophils generate superoxide by the addition of PMA, and that its superoxide-forming activity is completely suppressed by diphenylene iodonium, a specific inhibitor of NADPH oxidase. These results indicate that dolphin neutrophils possess NADPH oxidase activity. Furthermore, the NADPH oxidase activity (hydrogen peroxide production) in dolphin neutrophils, as well as in human neutrophils, was greater at 37 degrees C than at a lower temperature. RT-PCR with specific primers revealed that dolphin neutrophils expressed the mRNAs of the major NADPH oxidase components, which included membrane-associated flavocytochrome b (gp91(phox) and p22(phox)) and cytosolic factors (p40(phox), p47(phox), and p67(phox)), implying the existence of these protein homologues in dolphin neutrophils.
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PMID:Oxygen radical generation and expression of NADPH oxidase genes in bottlenose dolphin (Tursiops truncatus) neutrophils. 1098 Mar 19

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).
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PMID:Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly. 1100 80

p40(phox) of the phagocyte NADPH oxidase forms a complex with p67(phox) in cytosol, and coincidentally decreases in patients who lack p67(phox). Here we investigated the mode of translocation of p40(phox) to the membrane, its cytoskeletal localization on activation of the NADPH oxidase, and the dependency of its expression relative to that of p67(phox). When human polymorphonuclear leukocytes (PMNs) were stimulated with phorbol myristate acetate (PMA), p40(phox) was translocated to the membrane along with p67(phox), and not was released into the cytosol. Studies with resting PMNs using Triton X-100 revealed the exclusive localization of p67(phox) in the cytoskeletal fraction. Unexpectedly, however, about half of p40(phox), which is deemed to be fully associated with p67(phox), was recovered in the non-cytoskeletal fraction. Unlike p47(phox), the association of p40(phox) with cytoskeleton was not induced by the PMA-stimulation. These results indicate not only that p40(phox) associates with cytoskeleton via a molecule of p67(phox), but also that there are distinct states of p40(phox) that can be manipulated with Triton X-100. Lastly, Western-blot analysis of hematopoietic cells revealed no correlation between p40(phox) and p67(phox) in their protein expressions during cell differentiation, and also that p40(phox) can be stably present alone in cells, unless in the case of mature PMNs. In this regard, definitive proof was obtained with Epstein-Barr virus-transformed B cells of a p67(phox)-deficient patient, in which p40(phox) was normally expressed.
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PMID:Relationships of p40(phox) with p67(phox) in the activation and expression of the human respiratory burst NADPH oxidase. 1105 90

The bottle-nosed dolphin NADPH oxidase cytosolic components, p40(phox), p47(phox) and p67(phox) cDNA's were cloned from mitogen stimulated peripheral white blood cell mRNA utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that dolphin p40(phox), p47(phox) and p67(phox) clones contained open reading frames encoding predicted polypeptides of 339, 391 and 526 amino acids, respectively. Analysis of the p47(phox) and p67(phox) amino acid sequences showed two potential Src homology three domains and p40(phox) one. Comparison of the deduced amino acids showed that dolphin p40(phox) sequence shared 88.8% similarity with the human p40(phox), that dolphin p47(phox) sequence shared 87.7% similarity with the bovine p47(phox), and that dolphin p67(phox) shared 88.1% similarity with the bovine p67(phox). Western blot analysis using anti-human p40(phox), p47(phox) and p67(phox) antibodies demonstrated that dolphin neutrophil possesses p40(phox), p47(phox) and p67(phox) with similar molecular masses and structures, to each counterpart in human neutrophils, except for the p67(phox) COOH-terminus. These results suggest that dolphin NADPH oxidase cytosolic components have functional activities equivalent to those of human.
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PMID:Molecular cloning and identification of bottle-nosed dolphin p40(phox), p47(phox) and p67(phox). 1118 45

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47(PHOX), p67(PHOX), p40(PHOX), and Rac1 or Rac2, with the membrane-bound cytochrome b(558). Whereas the interaction of p47(PHOX) with cytochrome b(558) is well established, an interaction between p67(PHOX) and cytochrome b(558) has never been investigated. We report here a direct interaction between p67(PHOX) and cytochrome b(558). First, labeled p67(PHOX) recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b(558)-deficient patient. Second, p67(PHOX) binds to cytochrome b(558) that has been bound to nitrocellulose. Third, GTP-p67(PHOX) bound to glutathione agarose is able to pull down cytochrome b(558.) Rac1-GTP or Rac1-GDP increased the binding of p67(PHOX) to cytochrome b(558), suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67(PHOX) and cytochrome b(558).
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PMID:Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558. 1124 21

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

Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylori lipopolysaccharide (LPS) and Escherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O(2)(-))/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type I H. pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor beta-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O(2)(-) production 10-fold. In contrast, none of these events occurred with H. pylori LPS from complete or partial deletion mutants of the cag pathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity. H. pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H. pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells.
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PMID:Type I Helicobacter pylori lipopolysaccharide stimulates toll-like receptor 4 and activates mitogen oxidase 1 in gastric pit cells. 1140 77


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