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)

Two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p40phox, are shown by analyses of their sequences to contain single copies of a novel class of domain, the PX (phox) domain. Homologous domains are demonstrated to be present in the Cpk class of phosphatidylinositol 3-kinase, S. cerevisiae Bem1p, and S. pombe Scd2, and a large family of human sorting nexin 1 (SNX1) homologues. The majority of these domains contains a polyproline motif, typical of SH3 domain-binding proteins. Two further findings are reported. A third NADPH oxidase subunit, p67phox, is shown to contain four tetratricopeptide repeats (TPRs) within its N-terminal RaclGTP-binding region, and a 28 residue motif in p40phox is demonstrated to be present in protein kinase C isoforms iota/lambda and zeta, and in three ZZ domain-containing proteins.
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PMID:Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains? 893 Nov 54

NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.
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PMID:Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes. 894 60

Chronic granulomatous disease (CGD) is a syndrome characterized by failure of the NADPH oxidase of phagocytes that generates superoxide, which is central to the microbicidal process. Cytosolic components of this oxidase system include the proteins p67phox and p47phox, deficiencies of which cause the autosomal recessive form of CGD, whereas the X-linked form of the disease is characterized by a deficiency in the plasma membrane component gp91phox. Components of the oxidase system have been reported to be associated with the cytoskeleton and neutrophils from CGD patients have been reported to have a defective chemotactic response in Boyden chambers. Using a chamber that permits the direct observation of cell behaviour in a linear gradient of a chemoattractant, we have analysed the chemotactic response of neutrophils from a patient lacking p67phox; from another lacking p47phox and from a third lacking gp91phox. The results of measuring the speeds and directions of locomotion of the cells show that their speeds are undiminished relative to cells from healthy control subjects and that their directions of migration are at least as strongly biased in the direction of the gradient as those of the control cells. We conclude that these definitive aspects of the chemotactic response are not abnormal in either the autosomal recessive or the X-linked forms of CGD and that they are therefore not factors in the predisposition to infection that characterizes the syndrome.
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PMID:Deficiency of p67phox, p47phox or gp91phox in chronic granulomatous disease does not impair leucocyte chemotaxis or motility. 905 62

The NADPH oxidase that produces superoxide in professional phagocytic cells is a flavocytochrome b electron transport chain in the membrane, a heterodimer of gp91phox and p22phox, that is activated by a number of cytosolic proteins, including p47phox, p67phox, and the small GTP-binding protein p21rac, which translocate to the membrane and attach to the flavocytochrome on activation. The components of this oxidase were localized on the cytoplasmic surface of the plasma membrane of adherent unroofed neutrophils by immunolabeling. Components of the NADPH oxidase and p21rac were found together in punctate clusters occupying 0.03-0.1 microm2 of the cytoplasmic surface of the plasma membrane where the density of labeling of the cytosolic components was increased after stimulation with phorbol myristate acetate.
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PMID:Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane. 906 Apr 53

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.
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PMID:Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox. 906 49

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47phox, p67phox, and p22phox). p47phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22phox and cytosolic p67phox. To understand the specific roles of each SH3 domain of p47phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr167 or Tyr237-->Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47phox-p22phox interaction and plays a predominant role in oxidase activity and p47phox membrane assembly, while the second p47phox SH3 domain interacts with the NH2-terminal domain of p67phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.
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PMID:Specificity of p47phox SH3 domain interactions in NADPH oxidase assembly and activation. 912 67

The differential expression of protein kinase C (PKC) isozymes and small GTP-binding proteins, and their relation to O2 generation and phospholipase D (PLD) activation were analyzed during the differentiation of human promyelocytic HL60 cells to neutrophil-like cells induced by either retinoic acid (RA) or dibutyryl cyclic AMP (dbcAMP). In response to either one of the inducers, nitroblue tetrazolium (NBT) reduction activity time-dependently increased. Although PLD activity was upregulated by dbcAMP-treatment, only a slight increase was observed in RA-treated cells. Small GTP-binding proteins Rac1, Rap1, and RhoA, which are reported to be implicated in O2- generation or PLD activation, were already expressed in undifferentiated HL60 cells and their significant changes were not detected during differentiation. The mRNAs of the cytosolic components of NADPH oxidase system, p47phox and p67phox, were present in trace amounts in undifferentiated cells. However, they rapidly increased in response to RA or dbcAMP. In response to either RA or dbcAMP, the increases were observed in cPKC isozymes (alpha, beta I, beta II) but not in other subtypes (delta, epsilon, theta, zeta) by both RT-PCR and Western blot analyses. In dbcAMP-treated cells PKC alpha increased remarkably, whereas PKC beta I and beta II mainly elevated in RA-treated cells. These results suggest the possibility that cPKCs are closely related to cell differentiation and that PKC alpha is involved in PLD activation.
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PMID:Differential expression of protein kinase C isozymes and small GTP-binding proteins during HL60 cell differentiation by retinoic acid and cyclic AMP: relation with phospholipase D (PLD) activation. 914 35

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-bound flavohemoprotein, to assemble the active oxidase. An essential element of the activation process is the phosphorylation of p47phox, an event that accompanies oxidase activation in whole cells and can activate the oxidase in a cell-free system. We show here that the phosphorylation of p47phox leads to a substantial decrease in the reactivity of cysteine C378 toward N-ethylmaleimide, indicating the occurrence of a conformational change involving the C-terminal region of p47phox. A similar conformational change occurs when p47phox is exposed to arachidonate, one of a number of anionic detergents that activate the oxidase in the cell-free system. We propose that this change in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.
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PMID:Activation of the leukocyte NADPH oxidase subunit p47phox by protein kinase C. A phosphorylation-dependent change in the conformation of the C-terminal end of p47phox. 920 Jun 96

Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro, however, Rac2 is largely predominant in human phagocytes. NADPH oxidase is a plasma membrane enzyme of phagocytes, generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme, minimally requires assembly at the membrane with flavocytochrome b258 of cytosolic components p47phox, p67phox and Rac proteins. Using the yeast two hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants which are known to be inactive in NADPH oxidase lose their interaction with p67phox. Finally, we observe that p67phox interacts six fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac, and particularly Rac2, can regulate superoxide production by NADPH oxidase of phagocytic cells through direct interaction with p67phox subunit. Recently published data suggest that Rac proteins could transduce mitogenic signals in non-phagocytic cells through superoxide production by a phagocytic-related NADPH oxidase enzymatic system which remains to be determined. NADPH oxidase regulation by Rac proteins in phagocytes could then be used as a model to understand the molecular mechanisms underlying Rac functions in various cell types.
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PMID:[Signal transduction by Rac small G proteins in phagocytes]. 925 50

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


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