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)

Expression of the phagocyte cytosolic protein p47(phox), a component of NADPH oxidase, is restricted mainly to myeloid cells. To study the cis-elements and trans-acting factors responsible for its gene expression, we have cloned and characterized the p47(phox) promoter. A predominant transcriptional start site was identified 21 nucleotides upstream of the translation initiation codon. To identify the gene promoter sequences, transient transfections of HL-60 human myeloid cells were performed with a series of 5'-deletion p47(phox)-luciferase reporter constructs that extended as far upstream as -3050 bp relative to the transcriptional start site. The -224 and -86 constructs had the strongest p47(phox) promoter activity, whereas the -46 construct showed a major reduction in activity and the -36 construct a complete loss of activity. DNase I footprint analysis identified a protected region from -37 to -53. This region containing a consensus PU.1 site bound specifically both PU.1 present in nuclear extracts from myeloid cells and PU.1 synthesized in vitro. Mutations of this site eliminated PU.1 binding and abolished the ability of the p47(phox) promoter to direct expression of the reporter gene. The p47(phox) promoter was active in all myeloid cell lines tested (HL-60, THP-1, U937, PLB-985), but not in non-myeloid cells (HeLa, HEK293). Finally, PU.1 trans-activated the p47(phox)-luciferase constructs in HeLa cells. We conclude that, similar to certain other myeloid-specific genes, p47(phox) promoter activity in myeloid cells requires PU.1.
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PMID:PU.1 is essential for p47(phox) promoter activity in myeloid cells. 921 34

The NADPH oxidase cytochrome b558 is a membrane heterodimer comprised of a glycosylated 91-kDa subunit, gp91(phox), and a nonglycosylated 22-kDa subunit, p22(phox). The role of heme in cytochrome b558 biosynthesis was studied using succinyl acetone, an inhibitor of heme synthesis, in PLB-985 myeloid cells undergoing granulocytic differentiation. Succinyl acetone markedly reduced expression of p22(phox) and the mature 91-kDa form of gp91(phox) but not its 65-kDa high mannose precursor, in association with a profound reduction in NADPH oxidase activity. Expression of non-heme-containing cytosolic oxidase components was unaffected. The reduction in cytochrome b558 expression and NADPH oxidase activity was prevented by adding exogenous heme and was reversible upon removal of succinyl acetone. Transgenic expression of gp91(phox) in monkey COS-7 and murine 3T3 cells, both of which lacked endogenous p22(phox) mRNA, demonstrated that p22(phox) was not required for maturation of gp91(phox) carbohydrate to complex oligosaccharides. However, coexpression of transgenic p22(phox) increased the abundance of the mature gp91(phox) glycoprotein. These results suggest that heme incorporation plays an important role in cytochrome b558 assembly and provide further support for the concept that stability of p22(phox) and the mature gp91(phox) subunit is increased by heterodimer formation.
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PMID:Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits. 934 Nov 76

Arachidonic acid (AA) can trigger activation of the phagocyte NADPH oxidase in a cell-free assay. However, a role for AA in activation of the oxidase in intact cells has not been established, nor has the AA generating enzyme critical to this process been identified. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected that lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D3, indicating that cPLA2 is not involved in the differentiation process. Neither cPLA2 nor stimulated [3H]AA release were detectable in differentiated cPLA2-deficient PLB-985 cells, demonstrating that cPLA2 is the major type of PLA2 activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but the addition of exogenous AA fully restores oxidase activity. This establishes an essential requirement of cPLA2-generated AA for activation of phagocyte NADPH oxidase.
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PMID:Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase. 941 1

We have used a human leukemia cell line that, after homologous recombination knockout of the gp91-phox subunit of the phagocyte respiratory-burst oxidase cytochrome b-558, mimics chronic granulomatous disease (X-CGD) to study the role of oxygen radicals in apoptosis. Camptothecin (CPT), a topoisomerase I inhibitor, induced significantly more apoptosis in PLB-985 cells than in X-CGD cells. Sensitivity to CPT was enhanced after neutrophilic differentiation, but was lost after monocytic differentiation. No difference between the two cell lines was observed after treatment with other apoptosis inducers, including etoposide, ultraviolet radiation, ionizing radiation, hydrogen peroxide, or 7-hydroxystaurosporine. After granulocytic differentiation of both cell lines, CPT still induced apoptosis, suggesting independence from replication in fully differentiated and growth-arrested cells. Pyrrolidine dithiocarbamate (an antioxidant inhibitor of NF-kappaB) and catalase partially inhibited CPT-induced DNA fragmentation in granulocytic-differentiated PLB-985 cells, but had no effect in X-CGD cells. Flow cytometry analysis revealed that reactive oxygen intermediates were generated in CPT-treated PLB-985 cells. These data indicate that oxygen radicals generated by NADPH oxidase may contribute directly or indirectly to CPT-induced apoptosis in human leukemia and in neutrophilic-differentiated cells.
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PMID:Role of oxygen radicals generated by NADPH oxidase in apoptosis induced in human leukemia cells. 983 21

The redox center of the phagocyte NADPH oxidase is flavocytochrome b558, a transmembrane protein with two subunits, gp91(phox) and p22(phox). In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91(phox) precursor (p65). Expressing the gp91(phox) cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes. Sucrose density gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91(phox) transgene demonstrated that p65 co-sedimented with the ER marker protein calreticulin and myeloperoxidase precursors. Unexpectedly, the majority of p22(phox) was found in subcellular compartments containing the mature 91-kDa form of gp91(phox) and not with p65, suggesting that heterodimer formation may occur in a post-ER compartment. The heme synthesis inhibitor, succinyl acetone, reduced the abundance of mature gp91(phox) and p22(phox) but had little or no impact on p65. These studies demonstrate (a) gp91(phox) is synthesized as a glycosylated 65-kDa precursor in the ER, (b) heterodimer formation is not a co-translational process, and (c) heme insertion is a determinant in the formation of a stable heterodimer but does not appear to affect the stability of p65.
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PMID:Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum. 993 39

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA(2) in the regulation of both the H(+) channel and the Na(+)/H(+) antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-sensitive alkalization, indicating H(+) channel activity. In contrast, differentiated PLB-D cells failed to activate the H(+) channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) caused significant inhibition of NADPH oxidase activity, suggesting a role of the H(+) channel in regulating oxidase activity. Na(+)/H(+) antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.
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PMID:Essential requirement of cytosolic phospholipase A(2) for activation of the H(+) channel in phagocyte-like cells. 1041 67

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytes are unable to generate superoxide (O(2)(-)) due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X-linked gene for gp91(phox), the large subunit of the flavocytochrome b(558) heterodimer, account for the majority of CGD. An X-CGD patient in which a splice junction mutation results in an in-frame deletion of 30 nucleotides encoding amino acids 488 to 497 of gp91(phox) (delta488-497 gp91(phox)) has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant triangle delta488-497 gp91(phox) to further characterize its functional properties. These cells mimicked the phenotype of the patient's neutrophils with normal expression of a nonfunctional delta488-497 gp91(phox) flavocytochrome. Translocation of p47(phox) and p67(phox) to delta488-497 gp91(phox) PLB-985 plasma membranes was not affected, as determined both in activated intact cells and in the cell-free system. Furthermore, a synthetic peptide corresponding to residues 488-497 of gp91(phox) was relatively ineffective in inhibiting O(2)(-) production in the cell-free oxidase assay (IC50, approximately 500 micromol/L), suggesting that residues 488-497 of gp91(phox) are not directly involved in oxidase assembly. Mutant delta488-497 gp91(phox) flavocytochrome failed to support iodonitrotetrazolium (INT) reduction, showing a disruption of electron transfer from NADPH to the FAD center of gp91(phox). However, the FAD binding capacity of the mutant flavocytochrome was normal, as measured by equilibrium dialysis. Taken together, these results suggest that the delta488-497 deletion in gp91(phox) disrupts electron transfer to FAD, either due to a defect in NADPH binding or to impaired delivery of electrons from NADPH.
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PMID:Functional analysis of NADPH oxidase in granulocytic cells expressing a delta488-497 gp91(phox) deletion mutant. 1049 23

The phagocyte NADPH oxidase is a multicomponent transport chain that generates superoxide, a precursor of microbicidal oxidants, important for host defense. This transport chain is contained mainly in the large membrane subunit of the oxidase (gp91phox), and transfers electrons from cytosolic NADPH, through FAD binding and heme centers, to molecular oxygen (Babior, 1999; Fujii and Kakinuma, 1991; Rotrosen et al., 1992; Segal and Abo, 1993). Cross et al. have recently described a novel NADPH oxidase diaphorase activity present in the membrane fraction of activated neutrophils, using a cell free model (Cross et al., 1994). This diaphorase activity is measured by the artificial electron acceptor 4-iodonitrotetrazolium violet (INT) and is attributed to the reduction of the flavin center of the flavocytochrome (Cross et al., 1994; Li and Guillory, 1997). In the present study we establish a system for detecting diaphorase activity in intact cells. Neutrophils and PLB-985 cells, that were differentiated using 1.25% dimethyl sulfoxide (DMSO) to granulocyte phenotype, were permeabilized by electroporation, and diaphorase activity was determined using INT. Neutrophils and differentiated PLB-985 cells stimulated by PMA or GTP gamma S showed a diaphorase activity that was not present in unstimulated differentiated cells. The diaphorase activity could not be detected in undifferentiated cells and was developed during differentiation. The pattern of diaphorase activity in stimulated parent differentiated PLB cells was similar to that observed in stimulated human neutrophils. The permeabilized-INT cell system offers a unique tool for the evaluation of NADPH oxidase diaphorase activity, in whole cells.
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PMID:The NADPH oxidase diaphorase activity in permeabilized human neutrophils and granulocytic like PLB-985 cells. 1089 13

The NADPH oxidase producing-superoxide is the major mechanism by which phagocytes kill invading pathogens. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected which lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid, DMSO or 1,25 dihydroxyvitamin D3 indicating that cPLA2 is not involved in the differentiation process. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but addition of exogenous arachidonic acid (AA) fully restores oxidase activity. This establishes an essential requirement of cPLA2 generated AA for activation of phagocyte NADPH oxidase. In order to elucidate the mechanism by which cPLA2 regulates the oxidase, the role of cPLA2 in NADPH oxidase associated H+ channel was studied. Activation of differentiated PLB cells resulted in a Zn+2 sensitive alkalization, indicating H+ channel activity. In contrast, differentiated PLB-D cells failed to activate the H+ channel, but addition of exogenous AA fully restored this activity, indicating an essential and specific physiological requirement of cPLA2-generated AA for activation of the H+ channel. The presence of the H+ channel inhibitor, Zn+2, caused significant inhibition of NADPH oxidase activity, suggesting a role of the NADPH oxidase associated H+ channel in regulating oxidase activity.
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PMID:Cytosolic phospholipase A2 is required for the activation of the NADPH oxidase associated H+ channel in phagocyte-like cells. 1089 15

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.
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PMID:A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase. 1093 44


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