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:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane-integrated protein gp91phox, existing as a heterodimer with p22phox, functions as the catalytic core of the phagocyte NADPH oxidase, which plays a crucial role in host defence. The oxidase, dormant in resting cells, becomes activated to produce superoxide, a precursor of microbicidal oxidants, by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. In the past few years, several proteins homologous to gp91phox were discovered as superoxide-producing NAD(P)H oxidases (Nox's) in non-phagocytic cells; however, regulatory mechanisms for the novel oxidases have been largely unknown. Current identification of proteins highly related to p47phox and p67phox, designated Noxol (Nox organizer 1) and Noxal (Nox activator 1), respectively, has shed lights on common and distinct mechanisms underlying activations of Nox family oxidases.
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PMID:Molecular mechanism for activation of superoxide-producing NADPH oxidases. 1474 14

NADPH oxidase activation involves the assembly of membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67phox and full-length Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47phox. We subjected chimera p67phox-(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67phox and Rac1, in the p67phox-(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67phox. This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67phox to the membrane; the other is to induce an "activating" conformational change in p67phox.
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PMID:Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras. 1476 78

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b558, in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase.
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PMID:The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly. 1476 28

TPCK (tosylphenylalanylchloromethane), first discovered as a serine protease inhibitor, has been described to affect in diverse systems a number of physiological events probably unrelated to its antiprotease effect, such as proliferation, apoptosis and tumour formation. In the present study, we focus on its inhibition of the neutrophil respiratory burst, an important element of non-specific immunological defence. The superoxide anion-producing enzyme, NADPH oxidase, is quiescent in resting cells. Upon cell stimulation, the redox component, membrane-bound flavocytochrome b558, is activated when the cytosolic factors (p47phox, p67phox and p40phox, as well as the small GTPase Rac) associate with it after translocating to the membrane. This requires the phosphorylation of several p47phox serine residues. The signal transduction events leading to enzyme activation are not completely understood. In the past, the use of diverse protease inhibitors suggested that proteases were involved in NADPH oxidase activation. We suggested previously that TPCK could prevent enzyme activation by the phorbol ester PMA, not due to inhibition of a protease, but possibly to inhibition of the cytosolic factor translocation [Chollet-Przednowed and Lederer (1993) Eur. J. Biochem. 218, 83-93]. In the present work, we show that TPCK, when added to cells before PMA, prevents p47phox phosphorylation and hence its translocation; moreover, when PMA-stimulated cells are incubated with TPCK, p47phox is dephosphorylated and dissociates from the membrane. These results are in line with previous suggestions that the respiratory burst is the result of a series of continuous phosphorylation and dephosphorylation events. They suggest that TPCK leads indirectly to activation of a phosphatase or inactivation of a kinase, and provide the first clue towards understanding the steps leading to its inhibition of NADPH oxidase activation.
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PMID:Another biological effect of tosylphenylalanylchloromethane (TPCK): it prevents p47phox phosphorylation and translocation upon neutrophil stimulation. 1549 25

The phagocyte NADPH oxidase is dormant in resting cells but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defence. The catalytic core of this enzyme comprises the two membranous subunits gp91phox/Nox2 and p22phox. The oxidase activation requires the small GTPase Rac and the SH3 domain-containing proteins p47phox and p67phox; they normally exist in the cytoplasm and translocate upon cell stimulation to the membrane. The translocation depends on a stimulus-induced conformational change of p47phox, which leads to the SH3 domain-mediated interaction with p22phox, a binding required for the gp91phox/Nox2-dependent superoxide production. Activation of Nox1, an oxidase that is likely involved in host defence at the colon, requires novel proteins homologous to p47phox and p67phox, designated Noxo1 and Noxa1, respectively. Noxo1 and Noxa1, both expressed abundantly in the colon, are capable of constitutively activating Nox1. The constitutive activation may be due to the property of Noxo1: in contrast with p47phox, Noxo1 seems to normally associate with p22phox, which is required for the Nox1 activation. We will also describe the mechanism underlying regulation of the third oxidase Nox3, which exits in fetal kidney and inner ears.
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PMID:Molecular mechanism underlying activation of superoxide-producing NADPH oxidases: roles for their regulatory proteins. 1550 62

Interaction of p50 Rho GTPase-activating protein (p50RhoGAP) with Rho family small GTPases was investigated in a yeast two-hybrid system, by radioactive GAP assay, and in a Rac-regulated enzymatic reaction, through superoxide production by the phagocytic NADPH oxidase. The yeast two-hybrid system revealed an interaction between the C-terminal GAP domain and the N-terminal part of p50RhoGAP. The first 48 amino acids play a special role both in the stabilization of the intramolecular interaction and in recognition of the prenyl tail of small GTPases. The GAP assay and the NADPH oxidase activity indicate that the GTPase-activating effect of full-length p50RhoGAP is lower on non-prenylated than on prenylated small GTPase. Removal of amino acids 1-48 and 169-197 of p50RhoGAP increases the GAP effect on non-prenylated Rac, whereas prenylated Rac reacts equally well with the full-length and the truncated proteins. We suggest that p50RhoGAP is in an autoinhibited conformation stabilized by the stretches 1-48 and 169-197 and the prenyl group of the small GTPase plays a role in releasing this intramolecular restraint.
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PMID:Autoinhibition of p50 Rho GTPase-activating protein (GAP) is released by prenylated small GTPases. 1559 40

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.
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PMID:The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators. 1582 3

During phagocytosis, surface receptors on neutrophils interact with pathogens opsonized with complement factor C3b/iC3b and in some cases with antibodies. In human immune sera antibodies directed against surface-bound M proteins mediated killing of Streptococcus pyogenes by neutrophils. Surprisingly, blocking of the Fc receptors had little effect on the killing. In contrast, inhibition of C3b/iC3b generation, or blocking of the major neutrophil iC3b receptor CD11b/CD18, enabled S. pyogenes to grow efficiently in immune sera. Inhibition of CD11b/CD18, but not of CD32, the major neutrophil signaling Fc receptor, prevented Streptococcus-induced NADPH oxidase-dependent respiratory burst, and blocking of C3b/iC3b formation inhibited Streptococcus-induced activation of Cdc42, a small GTPase critically involved in transmitting pro-inflammatory signals to the cytoskeleton. Consequently, ligation of CD11b/CD18 by bacteria-bound iC3b is necessary for inducing a neutrophil response leading to elimination of S. pyogenes in immune human serum.
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PMID:Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum. 1583 98

Both transforming growth factor-beta (TGF-beta)-induced expression of biglycan (BGN) and activation of p38 MAPK have been implicated in cellular adhesion and migration. Here, we analyzed the role of adhesive events and the small GTPase Rac1 in TGF-beta regulation of BGN. TGF-beta1 induction of BGN expression and activation of p38 was abolished or strongly reduced when cells were kept in suspension or exposed to either the actin cytoskeleton-disrupting agent cytochalasin D or a specific chemical Rac1 inhibitor. Ectopic expression of a dominant negative mutant (T17N) of Rac1 abrogated both TGF-beta-induced p38 MAPK activation and BGN up-regulation but did not affect TGF-beta-induced phosphorylation of Smad3 or transcriptional induction of Growth Arrest DNA Damage 45beta, previously shown to be crucial for TGF-beta regulation of BGN. Overexpression of wild type Rac1 greatly enhanced the TGF-beta effect on BGN in adherent cells, whereas ectopic expression of constitutively active Rac1 (Q61L) activated p38 and in the presence of exogenous TGF-beta was able to rescue BGN expression in nonadherent cells. Endogenous Rac1 was activated by TGF-beta treatment in PANC-1 cells in an adhesion-dependent fashion. Like Rac1-T17N, the NADPH oxidase inhibitor diphenylene iodonium and the tyrosine kinase inhibitor herbimycin A blocked TGF-beta-induced p38 activation and BGN expression, suggesting that Rac1 exerts its effect on BGN and p38 through increasing NADPH oxidase activity and subsequent production of reactive oxygen species. These results show that the TGF-beta effect on BGN is dependent on cell adhesion and that activated Rac1, presumably acting through NADPH oxidase(s), is necessary but not sufficient for TGF-beta-induced BGN expression.
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PMID:Adhesion and Rac1-dependent regulation of biglycan gene expression by transforming growth factor-beta. Evidence for oxidative signaling through NADPH oxidase. 1605 7

The cytoplasmic tyrosine kinase Abl is a Src substrate required for platelet-derived growth factor (PDGF) receptor signaling leading to Myc expression and DNA synthesis. Abl targets are, however, ill defined. Here we report that the small GTPase Rac is an important effector of its mitogenic function. PDGF-induced Rac activation was impaired in cells with inactive Abl and active Rac overcame the mitogenic defects found in these cells. Rac function required both a Jun N-terminal kinase (JNK) and a NADPH oxidase (Nox) pathway. Furthermore, co-activation of JNK and Nox were sufficient to mimic the Rac mitogenic rescue. Abl also regulated PDGF-induced JNK and Nox activation. Finally, we found that Myc is an important target of this signaling cascade: Myc induction was sensitive to small inhibitors of JNK and Nox activities and forced expression of Myc overcame the G1 block induced by dominant interfering mutants of mitogen-activated protein kinase kinase 4 (MKK4) and Nox2 activating subunit. We concluded that cytoplasmic Abl operates on a Rac/JNK and a Rac/Nox pathway for PDGF-induced Myc induction and DNA synthesis.
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PMID:Abl tyrosine kinase regulates a Rac/JNK and a Rac/Nox pathway for DNA synthesis and Myc expression induced by growth factors. 1607 3


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