Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues

Klinger, E; Aviram, Irit

doi:10.1042/bj2850635

Cited by 10 publications

(16 citation statements)

References 32 publications

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“…It has been recently reported that p47 binds covalently GTP analogues (Klinger and Aviram, 1992) and that both p47 and p67 have the ability to bind GTP (Mizunari et al, 1993). Experiments were therefore carried out to examine whether oxidase activation could be stimulated with recombinant p47 and p67 loaded with GTP.…”

Section: Compared Efficiencies Of Purified Prenylated and Unprocessedmentioning

confidence: 99%

Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Fuchs

Dagher

Jouan

et al. 1994

European Journal of Biochemistry

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The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotidebinding regulatory (G) protein Racl , were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Racl , the latter was not detected in the p47-p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Racl in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell-free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KC1-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.Phagocytes, among which neutrophils are the most studied, respond to soluble or particulate stimuli, by a large increase in oxygen consumption with concomitant production of the superoxide anion 0;. The enzyme complex which catalyzes the monoelectronic reduction of 0, to 0; is termed NADPH oxidase and is located in the plasma membrane. Oxidase activation corresponds to the functional assembly of a number of components including an heterodimeric redox protein of hydrophobic nature, the flavocytochrome b,,,, and water soluble proteins referred to as cytosolic factors, p47 and p67. The monomeric guanosine-nucleotide-binding regulatory (G) proteins, Rac and possibly RaplA, are also involved in the modulation of oxidase activation in a manner which is not yet clear (for review see Babior, 1992; Morel et al., 1991 ;Segal and Abo, 1993).The observation that addition of GTP to a cell-free system of oxidase activation significantly enhanced the elicited oxidase activity (Seifert and Schultz, 1987;Gabig et al., 1987;Ligeti et al., 1988) was the beginning of a series of investigations that ended with in vivo experiments in which Correspondence to A. Fuchs,

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Section: Compared Efficiencies Of Purified Prenylated and Unprocessedmentioning

confidence: 99%

Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Fuchs

Dagher

Jouan

et al. 1994

European Journal of Biochemistry

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“…Human PMNs were isolated from fresh buffy coats by standard procedures of dextran sedimentation, hypotonic lysis of erythrocytes, and Ficoll-Hypaque density gradient centrifugation [8,12].…”

Section: Isolation Of Neutrophilsmentioning

confidence: 99%

Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation

Tal

Sharabani

Aviram

1998

Journal of Leukocyte Biology

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We have previously reported inhibition of cell-free activation of the neutrophil superoxidegenerating NADPH oxidase by a soluble cationic protein of neutrophil granules and by low concentrations of human defensin. Subcellular fractionation carried out in the current study indicated that the inhibitory substance was derived from azurophilic granules, was released into the medium on cell stimulation, and was resistant to phenylmethylsulfonyl fluoride (PMSF). Phorbol ester was the most effective stimulus for the release of the blocking activity. The possibility was raised that granule protein(s) act in vivo as negative modulators of superoxide production. Gel filtration of granule extract revealed a markedly retarded protein peak exhibiting oxidase-blocking activity and containing lysozyme as the main protein. Because lysozyme did not exert inhibitory effects on oxidase activation, association of the inhibitory protein with lysozyme was assumed. Indeed a column of immobilized lysozyme retained a fraction of the granule extract's oxidase-blocking activity. Elution with a low-pH buffer recovered a component capable of inhibition of the NADPH oxidase in stimulated neutrophils and in the cell-free system. The main 29-kDa protein band in the eluted fraction was identified as proteinase 3, a serine protease of azurophilic granules. Enzymatically active as well as PMSF-blocked conventionally purified proteinase 3 interfered with phorbol myristate acetateinduced superoxide release. These findings support the hypothesis that exocytosed granule constituents may prevent excessive activation of the NADPH oxidase.

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“…Human neutrophils were isolnted from fresh buffy coats by standard procedures of dextran sedimentation, Ficol-Hypayue (Pharmacia) gradient centrifugation and hypotonic lysis of red blood cells. Cells (108/ml) in 10 mM potassium phosphate pH 7.0, 131 mM NaCl (buffer B) containing, 1 mM EGTA, 3.5 mM phenylmethylsulfonyl fluoride (PhMeS0,F) and 15 pg/ml leupeptin were disrupted by three 20-s cycles of sonication at 4°C by a microprobe sonicator (Klinger and Aviram, 1992). Unbroken cells and nuclei were pelleted at 500 g for 5 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…The reaction mixture (0.8 ml) consisted of 10 mM 131 mM NaCI, 10 mM Hepes, pH6.7, 1 0 p M FAD, 80pM cytochrome c, 1 mM EGTA (Klinger and Aviram, 1992). Neutrophil membranes and cytosol at 2X cells/ml and 4X 10' celldml, respectively (corresponding to ==I0 pg and 120 pg membrane and cytosolic proteinlml) were incubated with SDS for 5 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

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Activation of Signaling Pathways in HL60 Cells and Human Neutrophils by Farnesylthiosalicylate

Tisch

Halpern

Marciano

et al. 1996

European Journal of Biochemistry

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Effects of the farnesylcysteine mimetic, farnesylthiosalicylate on the activation of myeloid cells were studied. In dimethyl-sulfoxide-differentiated HL60 cells and in human neutrophils farnesylthiosalicylate (G20 pM) dose-dependently elevated cytosolic Ca" concentrations, suggesting phospholipase-c-mediated release of the ion from intracellular stores. In human neutrophils, in addition to the production of inositol trisphosphate, farnesylthiosalicylate induced activation of the NADPH oxidase and translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane. The calcium signal, inositol-trisphosphate production and superoxide generation elicited by farnesylthiosalicylate were partially blocked by treatment of the cells with pertussis toxin, consistent with participation of pertussistoxin-sensitive and pertussis-toxin-resistant elements. In HL60 cells, farnesylthiosalicylate ( c 2 0 pM) did not activate NADPH oxidase but dose-dependently augmented PMA-elicited activity of the enzyme. This effect was resistant to pertussis-toxin treatment. In vitro augmentation of PKC-mediated phosphorylation of histone and cytosolic p47-phox by farnesylthiosalicylate and the finding that downregulation of PKC abrogated potentiation of NADPH oxidase activity by farnesylthiosalicylate were compatible with the involvement of PKC in the response of HL60 cells to farnesylthiosalicylate. It is suggested that the effects of farnesylthiosalicylate on myeloid cells reflect interaction of the analog with prenylcysteine-docking sites on cellular signaling elements.Keywords: neutrophil ; HL60 cells ; NADPH oxidase ; farnesylcysteine; farnesylthiosalicylate.Neutrophils constitute the first line of defense against invading microorganisms. They respond to exogenous signals by chemotaxis, activation of the respiratory-burst oxidase, exocytosis of their cytoplasmic granules, and phagocytosis of foreign particles. Studies of responses of neutrophils to extracellular stimuli contributed to a better understanding of diverse signal-transduction pathways and their elements. In many studies, the promyelocytic leukemia cell line HL60 (Collins, 1987) was employed as a model of neutrophils. HL60 cells induced to myelocytic differentiation by dimethyl sulfoxide (Me,SO) or retinoic acid acquire most of the features of circulating blood neutrophils.Activation of the neutrophil NADPH oxidase (Morel et al., 1991 ;Chanock et al., 1994) in response to exogenous stimuli is characterized by a rapid onset and is amenable to easy experimental analysis. The release of superoxide radicals reflects assembly at the cell membrane of the multicomponent enzymatic

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Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues

Cited by 10 publications

References 32 publications

Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation

Activation of Signaling Pathways in HL60 Cells and Human Neutrophils by Farnesylthiosalicylate

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Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues

Cited by 10 publications

References 32 publications

Activation of the O–2‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Activation of the O–2‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation

Activation of Signaling Pathways in HL60 Cells and Human Neutrophils by Farnesylthiosalicylate

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Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System