scholarly journals p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains

1993 ◽  
Vol 296 (3) ◽  
pp. 557-561 ◽  
Author(s):  
F B Wientjes ◽  
J J Hsuan ◽  
N F Totty ◽  
A W Segal
Keyword(s):  
Nadph Oxidase ◽  
Sequence Similarity ◽  
Granulomatous Disease ◽  
Phagocytic Cells ◽  
Src Homology 3 ◽  
N Terminus ◽  
Phagocyte Oxidase ◽  
Src Homology ◽  
Membrane Bound ◽  
Flavocytochrome B

The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.

scholarly journals Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox

1996 ◽  
Vol 317 (3) ◽  
pp. 919-924 ◽  
Author(s):  
Frans B. WIENTJES ◽  
George PANAYOTOU ◽  
Emer REEVES ◽  
Anthony W. SEGAL
Keyword(s):  
Nadph Oxidase ◽  
Binding Assays ◽  
Phagocytic Cells ◽  
Sh3 Domains ◽  
Cytosolic Proteins ◽  
Src Homology 3 ◽  
Vitro Binding ◽  
Src Homology ◽  
Flavocytochrome B

The NADPH oxidase of neutrophils and other bone-marrow-derived phagocytic cells is a multi-component system consisting of a flavocytochrome b in the plasma membrane and at least four cytosolic proteins. Three of the cytosolic proteins contain src homology 3 (SH3) domains, two each in p47phox and p67phox, and one in p40phox. All three translocate from the cytosol to the flavocytochrome in the membrane upon stimulation of the cells. A small G-protein, p21rac, is also involved in activation of the oxidase. The three cytosolic phox proteins occur as a complex in the cytosol and the strongest interaction appeared to be between p67phox and p40phox. We have investigated the interaction between p40phox and the other two cytosolic phox proteins by in vitro binding assays. An affinity-bead approach was used as well as a biosensor technique (surface plasmon resonance). We observed the strongest attachment between p40phox and p67phox where the binding was between the N-terminal half of p67phox and the C-terminal half of p40phox, and did not appear to involve SH3 domains and proline-rich sequences. p40phox also bound p47phox but more weakly than it did p67phox.

Topics in Chronic Granulomatous Disease

PEDIATRICS ◽  
1991 ◽  
Vol 88 (1) ◽  
pp. 183-185
Author(s):  
SHIGENOBU UMEKI
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Superoxide Anion ◽  
Fungal Infections ◽  
Regulatory Elements ◽  
Granulomatous Disease ◽  
Phagocytic Cells ◽  
Oxidase System ◽  
Membrane Bound

To the Editor.— Such phagocytic cells as neutrophils and macrophages are crucial elements in the host defense against bacterial [See table in the PDF file] and fungal infections. Microbicidal activity depends to a large extent on NADPH oxidase system, which can be activated by stimuli (bacteria, fungi) and which generates the superoxide anion and other highly reactive forms of reduced oxygen.1,2 The neutrophil NADPH oxidase system is composed functionally of membrane-bound catalytic components (which consist of at least two constituents, the low potential cytochrome b5583-5 and flavoprotein5) and soluble cytosolic components6,7 which participate as either catalytic or regulatory elements.

Phosphorylation of the leucocyte NADPH oxidase subunit p47phox by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity

2001 ◽  
Vol 358 (3) ◽  
pp. 783-790 ◽  
Author(s):  
Hee-Sae PARK ◽  
S. Min LEE ◽  
Jin Hyup LEE ◽  
Yun-Sook KIM ◽  
Young-Seuk BAE ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Conformational Changes ◽  
Oxidase Activity ◽  
Serine Threonine Kinase ◽  
Src Homology 3 ◽  
Oxidase Subunit ◽  
Putative Site ◽  
Src Homology ◽  
Membrane Bound ◽  
Eukaryotic Organisms

The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O−2 at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47phox becomes phosphorylated on several serines and migrates to the plasma membrane. Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms. The leucocyte NADPH oxidase subunit p47phox has several putative CK2 phosphorylation sites. In the present study, we report that CK2 is able to catalyse the phosphorylation of p47phoxin vitro. Phosphoamino acid analysis of phosphorylated p47phox by CK2 indicated that the phosphorylation occurs on serine residues. CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47phox. Dependence of phosphorylation on the conformation of p47phox is supported by the finding that p47phox undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47phox. In addition, 5,6-dichloro-1-β-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the p47phox kinase, and that phosphorylation of p47phox by CK2 regulates the deactivation of NADPH oxidase.

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 Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.

1996 ◽  
Vol 184 (3) ◽  
pp. 893-902 ◽  
Author(s):  
S Tsunawaki ◽  
S Kagara ◽  
K Yoshikawa ◽  
L S Yoshida ◽  
T Kuratsuji ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Inhibitory Effect ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Amino Terminal ◽  
Src Homology 3 ◽  
Phagocyte Nadph Oxidase ◽  
A Cell ◽  
Src Homology ◽  
Tight Association

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.

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 Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid

2003 ◽  
Vol 373 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Guihong PENG ◽  
Jin HUANG ◽  
Mellonie BOYD ◽  
Michael E. KLEINBERG
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Free System ◽  
Cell Free System ◽  
Src Homology 3 ◽  
Rich Domain ◽  
Phagocyte Nadph Oxidase ◽  
Px Domain ◽  
Src Homology

In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b558. In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1–guanosine 5′-[γ-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.

scholarly journals Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease

Blood ◽  
2010 ◽  
Vol 116 (9) ◽  
pp. 1570-1573 ◽  
Author(s):  
Felix Meissner ◽  
Reinhard A. Seger ◽  
Despina Moshous ◽  
Alain Fischer ◽  
Janine Reichenbach ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Mononuclear Phagocytes ◽  
Biologically Active ◽  
Inflammasome Activation ◽  
Granulomatous Disease ◽  
Caspase 1 ◽  
Inflammatory Conditions ◽  
Asymptomatic Patients ◽  
Phagocyte Oxidase

Abstract Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent infections and deregulated inflammatory responses. CGD is caused by mutations in subunits of the NADPH oxidase, an enzyme that generates reactive oxygen species in phagocytes. To elucidate the contribution of the proinflammatory protease caspase-1 to aberrant inflammatory reactions in CGD, we analyzed cells isolated from patients with defects in the phagocyte oxidase subunits p22phox, p47phox or gp91phox. We report that mononuclear phagocytes from CGD patients activated caspase-1 and produced biologically active interleukin-1β (IL-1β) in response to danger signals. Notably, caspase-1 activation and IL-1β secretion from CGD monocytes was elevated in asymptomatic patients and strongly increased in patients with noninfectious inflammatory conditions. Treatment with IL-1 receptor antagonist reduced IL-1 production in monocytes ex vivo and during medical therapy. Our results identify phagocyte oxidase defective monocytes as a source of elevated IL-1 and provide a potential therapeutic option to ameliorate inflammatory conditions associated with CGD.

scholarly journals A mutation located at the 5' splice junction sequence of intron 3 in the p67phox gene causes the lack of p67phox mRNA in a patient with chronic granulomatous disease

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 242-249 ◽  
Author(s):  
LC Tanugi-Cholley ◽  
JP Issartel ◽  
J Lunardi ◽  
F Freycon ◽  
F Morel ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Splice Junction ◽  
Granulomatous Disease ◽  
Free System ◽  
Junction Sequence ◽  
Functional Defect ◽  
A Cell ◽  
Membrane Bound ◽  
Main Components

Chronic granulomatous disease (CGD) is due to a functional defect of the O2(-)-generating NADPH oxidase of neutrophils. Mutations resulting in CGD have been shown to occur in only four genes, thus identifying the main components of the oxidase complex, namely the two subunits of a membrane-bound cytochrome b and two cytosolic factors of activation of 67 kD (p67phox) and 47 kD (p47phox). The present study deals with the biochemical and genetic analysis of the defect in a patient suffering from a p67phox-deficient form of CGD. The p67phox deficiency was ascertained by immunochemistry and the ability of recombinant p67phox to restore NADPH oxidase activity using a cell-free system of oxidase activation. The cellular extracts from the proband contained no p67phox protein and no p67phox mRNA when assayed by Western and Northern blot analysis. However, reverse transcription of mRNA and subsequent cDNA amplification by polymerase chain reaction using specific p67phox primers showed that trace amounts of a p67phox mRNA deleted for exon 3 were synthesized in the patient immortalized B lymphocytes. Sequence analysis of the genomic DNA showed a T-to-C transition at position +2 of intron 3. This point mutation in the consensus 5′ splice site of the intron 3 was probably responsible for lack of accumulation of mRNA and also for the skipping of exon 3 detected in the few mRNA molecules that escaped cellular degradation.

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