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)

We wished to determine when each of the four NADPH oxidase components p22 phagocytic oxidase (phox), gp91 phox, p47 phox, p67 phox is first expressed embryologically and whether the expression pattern occurs in a consistent temporal sequence or whether the four genes are expressed simultaneously. A deficiency of any one of them results in chronic granulomatous disease (CGD). mRNA transcripts and protein expression for p22 phox, gp91 phox, p47 phox, p67 phox was monitored in murine embryos at time of implantation (E5.5) until E 11.5, and in fetal liver, spleen, and limb bone marrow from E 14 until term (E 19). We observed that mRNA was first expressed for p22 phox at E 5.5, for p67 phox at E 7.0 and for p47 phox at E 7.5 before the onset of yolk sac hematopoiesis (E 8.0). gp91 Phox mRNA was first expressed at E 9.0. However, only p22 phox protein was expressed in circulating hemocytoblast by E 9.0. No other embryonic tissue contained phox proteins either before or after the establishment of hemocytoblastic circulation. The four specific mRNA transcripts and phox proteins were expressed in nests of developing granulocytes in liver by E 14 and the expression continued in the liver at E 16 and E 19. Spleen and limb bone marrow showed inconsistent results. Cord blood neutrophils contained all phox proteins. These studies confirm that the four CGD-related phox mRNA components of NADPH oxidase are expressed early in embryonic development and the expression occurs in a consistent sequential fashion but only p22 phox protein appears in embryonic hemocytoblast.
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PMID:Developmental expression of NADPH phagocytic oxidase components in mouse embryos. 1044 7

Chronic granulomatous disease is a rare inherited disorder caused by non-existent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for superoxide production, NADPH oxidase, involves at least five components. An absence of, or a defect in, any one of four of these proteins (p47(phox), p67(phox), p22(phox) and gp91(phox)) gives rise to the known types of chronic granulomatous disease. The most common form of inheritance is X-linked and is due to mutations in the CYBB gene that encodes gp91(phox), the large subunit of flavocytochrome b, the terminal electron donor of the oxidase. We have recently reported a large number of mutations in this gene revealing a broad range of defects, including large and small deletions, and frameshift, nonsense, missense, splice region and regulatory region mutations. Here we report a patient who has an unusual type of mutation that results in the generation of a 'pseudo-exon' in the gp91(phox) mRNA and an unexpected pattern of splicing.
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PMID:A novel mutation in the CYBB gene resulting in an unexpected pattern of exon skipping and chronic granulomatous disease. 1045 61

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 myeloid-specific transcription factor PU.1 is essential for expression of p47(phox), a component of the superoxide-forming phagocyte NADPH oxidase. The consensus PU.1 binding sequence (GAGGAA) is located on the non-coding strand from position -40 to -45 relative to the transcriptional start site of the p47phox promoter. A promoter construct extending to -46 was sufficient to drive tissue-specific expression of the luciferase reporter gene, but extension of the promoter from -46 to -48 resulted in a significant increase in reporter expression. Mutations of the nucleotides G at -46 and/or T at -47 reduced both reporter expression and PU.1 binding, whereas mutations at -48 had no effect. The PU.1 binding avidity of these sequences correlated closely with their capacity to dictate reporter gene transcription. In parallel studies on the functional PU.1 site in the promoter of CD18, mutations of nucleotides G and T at positions -76 and -77 (corresponding to -46 and -47, respectively, of the p47phox promoter) reduced PU.1 binding and nearly abolished the contribution of this element to promoter activity. We conclude that the immediate flanking nucleotides of the PU.1 consensus motif have significant effects on PU.1 binding avidity and activity and that this region is the dominant cis element regulating p47phox expression.
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PMID:Critical flanking sequences of PU.1 binding sites in myeloid-specific promoters. 1054 90

Severe, malignant osteopetrosis is a disease characterized by osteoclasts that fail to resorb bone. Serious defects in the ability of white blood cells to eradicate infectious agents confound the clinical course. Defective superoxide generation by neutrophils, monocytes, and lymphocytes contributes to this inability to fight infection. To elucidate the mechanisms resulting in the defective superoxide generation observed in osteopetrotic leukocytes, gene expression, translocation, and phosphorylation of the major components that form the functional NADPH oxidase complex were studied in transformed B-lymphocytes. The expression of the p47 subunit of NADPH oxidase was reduced in B-lymphocytes collected from osteopetrotic patients compared to those from controls. Phosphorylation and translocation of p47 to the cell membrane after PMA stimulation was similar in B-lymphocytes from both patients and normal controls. However, total amount of p47 phosphorylation and translocation was reduced in patient samples. This was further supported by the experiment using p47 antisense oligonucleotide. The other major components of the oxidase (p91, p22, p67) were found to be present at normal levels. Thus, the reduction in p47 expression results in reduced ability to assemble a functional NADPH oxidase complex at the membrane of lymphocytes from osteopetrotic patients. This defect translates into reduced superoxide generation and an increased propensity for infection.
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PMID:Superoxide generation in transformed B-lymphocytes from patients with severe, malignant osteopetrosis. 1054 47

GTPgammaS activates the NADPH oxidase and this activity declines rapidly with time after preexposure to streptolysin O. This was not due to loss of p47(phox), p67(phox), or Rac. To identify the component(s) leaking out of the permeabilized cell responsible for loss of activity, a GTPgammaS-dependent reconstitution assay was established. Neutrophil cytosol was subjected to chromatographic fractionation steps for purification of the minimum fraction required to restore activity. The reconstitution of the GTPgammaS-stimulated activity was dependent on ATP. The inhibitors staurosporine and calphostin C greatly reduced the activity in the reconstitution assay, implicating the involvement of a protein kinase C (PKC) pathway. PKC isoforms beta and delta were eliminated as the active factors in the most pure reconstitution fraction. With this novel cell-based reconstitution assay, we have identified the requirement for a protein kinase, or its substrate, for the restoration of GTPgammaS activation of the NADPH oxidase.
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PMID:Reconstitution of GTPgammaS-induced NADPH oxidase activity in streptolysin-O-permeabilized neutrophils by specific cytosol fractions. 1054 86

Activation of the superoxide-producing phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox), which is mediated via the SH3 domains of the former protein. This interaction is considered to be induced by exposure of the domains that are normally masked by an intramolecular interaction with the C-terminal region of p47(phox). Here we locate the intramolecular SH3-binding site at the region of amino acid residues 286-340, where Ser-303, Ser-304, and Ser-328 that are among several serines known to become phosphorylated upon cell stimulation exist. Simultaneous replacement of the three serines in p47(phox) with aspartates or glutamates, each mimicking phosphorylated residues, is sufficient for disruption of the intramolecular interaction and resultant access to p22(phox). The triply mutated proteins are also capable of activating the NADPH oxidase without in vitro activators such as arachidonate under cell-free conditions. In a whole-cell system where expression of the wild-type p47(phox) reconstitutes the stimulus-dependent oxidase activity, substitution of the kinase-insensitive residue alanine for Ser-328 as well as for Ser-303/Ser-304 leads to a defective production of superoxide. These findings suggest that phosphorylation of the three serines in p47(phox) induces a conformational change to a state accessible to p22(phox), thereby activating the NADPH oxidase.
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PMID:Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase. 1055 53

p47(phox) is an essential component of the NADPH oxidase, and phosphorylation of p47(phox) is associated with activation of the enzyme. Here we have used p47(phox) affinity chromatography to extract a p47(phox) kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-beta(I), -beta(II) and -delta. The C-terminus of p47(phox) represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47(phox) takes place in intact cells. However PKC-beta and -delta showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47(phox), we investigated the functional relevance of the interaction between PKC and p47(phox). Subcellular fractionation revealed an abnormal recruitment of PKC-beta(I) and -beta(II), but not PKC-delta, to particulate fractions in p47(phox)-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47(phox)-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47(phox)-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47(phox) affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47(phox) can act as a regulator of PKC in neutrophils.
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PMID:Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils. 1058 74

Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.
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PMID:NADPH oxidase activation and assembly during phagocytosis. 1058 71

Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935), we previously showed that p47(phox), a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22(phox), a subunit of the NADPH oxidase component flavocytochrome b(558), also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22(phox). We identified two protein kinase families capable of phosphorylating p22(phox): 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47(phox) and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47(phox). In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22(phox). These findings suggest that phosphorylation of p22(phox) by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.
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PMID:A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox), an NADPH oxidase component. 1059 61


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