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)

The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 x 10(-2) nmol/min/10(6) cells (S.E.M. = +/-0.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox and p47-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.
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PMID:Detection of protein and mRNA of various components of the NADPH oxidase complex in an immortalized human chondrocyte line. 918 52

Porcine articular chondrocytes have the capacity to release superoxide in response to the addition of the calcium ionophore ionomycin in a concentration-dependent manner. This activity was not stimulated by the addition of fMetLeuPhe or the kinase activator phorbol myristate acetate (PMA). However, this release of superoxide was inhibited by iodonium diphenyl (IDP), suggesting the involvement of NADPH oxidase. Reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotides designed against the known sequences for the human phagocyte NADPH oxidase showed the expression of p22-phox, p40-phox, and p47-phox mRNA, while Western blot analysis of chondrocyte extracts using polyclonal antisera raised against the human phagocyte NADPH oxidase suggested the presence of the p67-phox polypeptide. These results suggest that porcine articular chondrocytes can release reactive oxygen species using a NADPH oxidase-like complex.
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PMID:Detection of superoxide and NADPH oxidase in porcine articular chondrocytes. 929 50

It has been known that eosinophils produce more superoxide anion (O2-) than neutrophils. To elucidate the mechanism involved in the difference in the superoxide-producing activities, we compared the NADPH oxidase components and the translocation of the cytosolic components between eosinophils and neutrophils. Membrane-bound cytochrome b558, cytosolic p47-phox, p67-phox, and p40-phox were present in both neutrophils and eosinophils, but the amounts of these components were 1.5- to 3.3-fold greater in eosinophils than neutrophils. Upon activation, p47-phox, p67-phox, and p40-phox were translocated to the membranes in both leukocytes, but larger amounts were translocated in eosinophils than in neutrophils. Furthermore, the cross-mixing experiments using membrane and cytosol of eosinophils and neutrophils revealed that more cytosolic components were translocated, and more superoxide-producing activities were obtained using eosinophil fractions. Interestingly, Km values of activated oxidase for NADPH were almost the same in any combination of membrane and cytosol from both leukocytes, indicating that oxidase components are likely similar in both eosinophils and neutrophils. These observations suggest that NADPH oxidase components are more abundant in eosinophils than neutrophils, and, upon activation, larger amounts of NADPH oxidase complex are formed in eosinophils than in neutrophils.
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PMID:Study on the superoxide-producing enzyme of eosinophils and neutrophils--comparison of the NADPH oxidase components. 930 91

The NADPH oxidase generates microbicidal superoxide in phagocytes, and when defective it leads to chronic granulomatous disease (CGD). Oxidase specific proteins in the cytosol, p47phox and p67phox, as well as the small GTP binding protein p21rac are important for activation of superoxide production. Because the activity of this oxidase is normally tightly restricted to the phagocytic vacuole, and its temporal and spatial organisation might be regulated by cytoskeletal proteins, we examined the cytosolic phox proteins for interactions with cytoskeletal elements. p67phox copurified with a 57 kDa protein, identified as coronin, an actin binding protein that is important for movement and phagocytosis in Dictyostelium. Binding studies revealed that coronin attaches to the C-terminal half of p40phox, a binding partner of p67phox. The phox proteins and coronin had a similar distribution in the cell, and both accumulated around the phagocytic vacuole. PMA activation of adherent neutrophils resulted in a major rearrangement of these proteins, and of actin, which were lost from the periphery of the cell and condensed around the nucleus. The rearrangement of F-actin and coronin in adherent cells, were absent, or markedly diminished, in cells from patients lacking p47phox or p67phox in which an abnormally large proportion of the coronin was present as part of a large complex. The cytosolic phox proteins might play a regulatory role in the reorganisation of the cytoskeleton accompanying superoxide generation.
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PMID:Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils. 936 77

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67phox, p47phox and p40phox which translocate to the membrane upon activation. Known mechanisms underlying the translocation of these proteins include polyphosphorylation of p47phox and specific Src homology 3/polyproline motif interactions. In this study, through two-dimensionnal electrophoresis and immunoprecipitation experiments, we show using dimethylsulfoxide-differentiated HL60 promyelocytes that p40phox is in a basal phosphorylated state in resting cells and undergoes further phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol myristate acetate or by the formyl peptide fMet-Leu-Phe-Lys. Moreover, the extent of phosphorylation is strongly correlated with the level of superoxide production. Typically, in cells transiently activated by fMet-Leu-Phe-Lys, onset of superoxide production coincides with the appearance of new phosphorylated species of p40phox and, at the end of the respiratory burst, dephosphorylation of p40phox is observed. In vitro assays show that the kinase(s) involved in the phosphorylation of p40phox differ from those which participate in the phosphorylation of p47phox. This suggests that, in the cell, the phosphorylation of p40phox and of p47phox are under the control of two different kinase pathways.
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PMID:The 40-kDa component of the phagocyte NADPH oxidase (p40phox) is phosphorylated during activation in differentiated HL60 cells. 937 Mar 64

Interleukin-15 (IL-15) is a newly described cytokine that shares biological activities with IL-2. We report here results demonstrating the ability of IL-15 to enhance superoxide production and antifungal activity of human monocytes. After 18 and 48 h of treatment with IL-15, human elutriated monocytes manifested enhanced superoxide production in response to either phorbol myristate acetate or opsonized Candida albicans blastoconidia. Similar results were obtained when monocytes were treated with IL-2, but to a lesser extent. Combination studies with IL-15 and IL-2 showed no additive or synergistic effects. Following incubation of monocytes with IL-15 for 18 h, there was no significant increase in mRNA transcripts for components of the NADPH oxidase complex, p40-phox, p47-phox, and gp91-phox, suggesting a posttranscriptional modulation of enhanced superoxide production. Antibodies against the gamma chain of the IL-2 receptor and, to a lesser extent, against the beta chain partially abrogated the IL-15-mediated enhanced superoxide production. Additionally, human monocytes showed enhanced killing activity against C. albicans after 18 h of incubation with IL-15 or IL-2, but this treatment did not enhance the ability of these cells to phagocytose the organism. In addition, the enhanced fungicidal activity seen after 18 h of treatment was no longer detectable after 48 h of cytokine treatment. Culture supernatants from the IL-15-treated monocytes were assayed for the presence of other proinflammatory cytokines. IL-15 treatment did not induce the release of detectable levels of tumor necrosis factor alpha, IL-1beta, or IL-12. Our results indicate that IL-15 upregulates the microbicidal activity of human monocytes against C. albicans.
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PMID:Interleukin-15 augments superoxide production and microbicidal activity of human monocytes against Candida albicans. 942 51

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67phox and p47phox. Another SH3-domain-containing protein p40phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40phox and its partner in phagocytes, p67phox. Both p40phox and p67phox comprise several protein-binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox.
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PMID:Functional modules and expression of mouse p40(phox) and p67(phox), SH3-domain-containing proteins involved in the phagocyte NADPH oxidase complex. 949 28

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40phox. While the first two factors are indispensable for the activity, p40phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40phox revealed that it plays a critical role in the binding to p67phox. Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways.
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PMID:The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40phox and p67phox, SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase. 949 29

CGD is a rare inherited immunodeficiency syndrome, caused by the phagocytes' inability to produce (sufficient) reactive oxygen metabolites. This dysfunction is due to a defect in the NADPH oxidase, the enzyme responsible for the production of superoxide. It is composed of several subunits, two of which, gp91phox and p22phox, form the membrane-bound cytochrome b558, while its three cytosolic components, p47phox, p67phox and p40phox, have to translocate to the membrane upon activation. This is a tightly and intricately controlled process that involves, among others, several low-molecular weight GTP-binding proteins. Gp91phox is encoded on the X-chromosome and p22phox, p47phox and p67phox on different autosomal chromosomes, and a defect in one of these components leads to CGD. This explains the variable mode of inheritance seen in this syndrome. Clinically CGD manifests itself typically already at a very young age with recurrent and serious infections, most often caused by catalase-positive pathogens. Modern treatment options, including prophylaxis with trimethoprim-sulfamethoxazole and rIFN-gamma as well as early and aggressive anti-infection therapy, have improved the prognosis of this disease dramatically. CGD, as a very well-characterized inherited affection of the hematopoietic stem cells, is predestined to be among the first diseases to profit from the advances in cutting-edge therapeutics, such as gene therapy and in utero stem cell transplantation.
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PMID:The molecular basis of chronic granulomatous disease. 961 66

This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2- was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2- in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.
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PMID:Nicotinamide-adenine dinucleotide phosphate oxidase assembly and activation in EBV-transformed B lymphoblastoid cell lines of normal and chronic granulomatous disease patients. 979 33


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