scholarly journals 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

1995 ◽  
Vol 308 (3) ◽  
pp. 991-994 ◽  
Author(s):  
S Dusi ◽  
M Donini ◽  
F Rossi
Keyword(s):  
Nadph Oxidase ◽  
G Protein ◽  
Oxygen Radicals ◽  
Human Neutrophils ◽  
Enzyme Complex ◽  
Granulomatous Disease ◽  
Activated Neutrophils ◽  
Functional Complex ◽  
Flavocytochrome B ◽  
Nadph Oxidase Activation

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.

scholarly journals Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox

1996 ◽  
Vol 314 (2) ◽  
pp. 409-412 ◽  
Author(s):  
Stefano DUSI ◽  
Marta DONINI ◽  
Filippo ROSSI
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
G Proteins ◽  
Oxygen Radicals ◽  
Cell Membranes ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Functional Complex ◽  
Flavocytochrome B ◽  
Nadph Oxidase Activation

On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox, p47phox, p40phox, as well as the Ras-related G-proteins Rac1 and Rac2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b, forming a functional complex responsible for the production of oxygen radicals in phagocytes. In this paper we show that (a) in neutrophils from a patient with a form of chronic granulomatous disease (CGD) in which p67phox is absent, p47phox and Rac2, but not p40phox and Rac1 were translocated from the cytosol to the membrane on stimulation with formylmethionyl-leucylphenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA); (b) in neutrophils from a patient with a form of CGD in which p47phox is absent, p67phox, p40phox and Rac1 failed to associate with the membrane on stimulation with fMLP or PMA, whereas Rac2 was translocated as in normal neutrophils. We also show that in neutrophils from a patient lacking p67phox, the amount of cytosolic p40phox was decreased by about 40%. These findings indicate that, on neutrophil stimulation, p67phox mediates the translocation of p40phox and Rac1 from the cytosol to cell membranes and that Rac2 associates with the membranes independently of p47phox and p67phox.

scholarly journals Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox

1993 ◽  
Vol 296 (2) ◽  
pp. 367-371 ◽  
Author(s):  
S Dusi ◽  
F Rossi
Keyword(s):  
Nadph Oxidase ◽  
Membrane Fraction ◽  
Direct Evidence ◽  
Disease Patient ◽  
Human Neutrophils ◽  
Maximal Rate ◽  
Granulomatous Disease ◽  
Chronic Granulomatous Disease Patient ◽  
Phosphorylated Form ◽  
Activated Neutrophils

Activation of human neutrophil NADPH oxidase requires the interaction of cytosolic and membrane-associated components. Evidence has been accumulated that in phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils, the translocation to the plasma membrane of the cytosolic components p47phox and p67phox and the phosphorylation of p47phox are essential steps in activation of NADPH oxidase. No direct evidence has been presented to date as to whether p67phox is also phosphorylated. To address this problem we have immunoprecipitated p67phox from neutrophil cytosol and membrane fractions. The results indicate that, very soon after activation with PMA (20 s), p67phox was present in a phosphorylated form in the cytosol and in the membranes. At later times (1-3 min) the extent of p67phox phosphorylation continuously increased both in the cytosol and in the membrane fraction, while oxygen consumption reached the maximal rate within 40 s, and then remained linear. p67phox was also phosphorylated in formyl-methionyl-leucyl-phenylalanine-activated neutrophils. That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the observation that no phosphorylated band of 67 kDa was immunoprecipitated from the cytosol and membranes of PMA-stimulated neutrophils from a p67phox-deficient chronic granulomatous disease patient. In this case, p47phox was normally phosphorylated. These data demonstrate that: (1) the phosphorylation of p67phox is correlated with activation of NADPH oxidase, and (2) continuous phosphorylation of p67phox is required in order to maintain the linearity of the respiratory burst.

Transient Association of the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Subunits p47phox and p67phox With Phagosomes in Neutrophils From Patients With X-Linked Chronic Granulomatous Disease

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3521-3530 ◽  
Author(s):  
Lee-Ann H. Allen ◽  
Frank R. DeLeo ◽  
Annabelle Gallois ◽  
Satoshi Toyoshima ◽  
Kensuke Suzuki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Nicotinamide Adenine Dinucleotide ◽  
Polymorphonuclear Leukocytes ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Actin Binding ◽  
Nicotinamide Adenine ◽  
Cell Periphery ◽  
Granulomatous Disease ◽  
Flavocytochrome B

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (≈5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.

scholarly journals Protein kinase C-β contributes to NADPH oxidase activation in neutrophils

2000 ◽  
Vol 347 (1) ◽  
pp. 285-289 ◽  
Author(s):  
Lodewijk V. DEKKER ◽  
Michael LEITGES ◽  
Gabriel ALTSCHULER ◽  
Nishil MISTRY ◽  
Aileen MCDERMOTT ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Specific Inhibitor ◽  
Human Neutrophils ◽  
Cell Fractionation ◽  
Coated Particles ◽  
Kinase C ◽  
Pkc Β ◽  
Nadph Oxidase Activation

We have analysed the involvement of the β isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-β is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcγ-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-β specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-β, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-β also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-β isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcγ receptors in neutrophils.

Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation

2016 ◽  
Vol 93 ◽  
pp. 190-203 ◽  
Author(s):  
Deepika Awasthi ◽  
Sheela Nagarkoti ◽  
Amit Kumar ◽  
Megha Dubey ◽  
Abhishek Kumar Singh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidized Ldl ◽  
Human Neutrophils ◽  
Trap Formation ◽  
Extracellular Trap ◽  
Nadph Oxidase Activation

scholarly journals A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3309-3315 ◽  
Author(s):  
Juan D. Matute ◽  
Andres A. Arias ◽  
Nicola A. M. Wright ◽  
Iwona Wrobel ◽  
Christopher C. M. Waterhouse ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Oxidase Activity ◽  
Autosomal Recessive ◽  
Premature Stop Codon ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Recessive Mutations ◽  
Px Domain

Abstract Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

scholarly journals Altered expression of neutrophil peripheral benzodiazepine receptor in X-linked chronic granulomatous disease

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 184-188
Author(s):  
F Zavala ◽  
F Veber ◽  
B Descamps-Latscha
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Covalent Binding ◽  
Benzodiazepine Receptor ◽  
Mononuclear Phagocytes ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Peripheral Benzodiazepine Receptor ◽  
Cytochrome B558

This study was aimed at determining whether the peripheral benzodiazepine receptor (PBZDR), which is abundantly expressed on mononuclear phagocytes, is involved in host defense mechanisms depending on phagocyte membrane-associated NADPH-oxidase complex. Analysis by reversible and covalent binding of PBZDR expression on human neutrophils shows that it is modulated during NADPH-oxidase activation with phorbol 12-myristate 13-acetate. Based on a series of 17 patients with chronic granulomatous disease (CGD), results show that PBZDR expression is dramatically impaired in X-linked CGD, an inherited disorder due to a mutation on the gene coding for cytochrome b558 NADPH- oxidase component, whereas it is unaffected in autosomal recessive CGD where cytochrome b558 is normally expressed, suggesting a link between PBZDR and cytochrome b558 expressions. PBZDR can be assigned by covalent binding to an 18-Kd membrane protein. These results suggest that the neutrophil PBZDR, which can accommodate the widely prescribed anxiolytic drug Valium (diazepam), is involved in host defense against pathogens, a function that could be affected by neuroimmune interactions.

scholarly journals The TLR7/8 Agonist CL097 PrimesN-Formyl-Methionyl-Leucyl-Phenylalanine–Stimulated NADPH Oxidase Activation in Human Neutrophils: Critical Role of p47phox Phosphorylation and the Proline Isomerase Pin1

2012 ◽  
Vol 189 (9) ◽  
pp. 4657-4665 ◽  
Author(s):  
Karama Makni-Maalej ◽  
Tarek Boussetta ◽  
Margarita Hurtado-Nedelec ◽  
Sahra Amel Belambri ◽  
Marie-Anne Gougerot-Pocidalo ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Critical Role ◽  
Human Neutrophils ◽  
Nadph Oxidase Activation
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