Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites

2001 ◽  
Vol 358 (2) ◽  
pp. 315-324 ◽  
Author(s):  
Tosti J. MANKELOW ◽  
Lydia M. HENDERSON
Keyword(s):  
Nadph Oxidase ◽  
Human Neutrophils ◽  
Closed Channel ◽  
Superoxide Generation ◽  
Free System ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Target Sites ◽  
A Cell ◽  
Membrane Components

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91phox to function as an H+ pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H+ flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H+ channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67phox and p47phox, to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b558 spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H+ pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.

scholarly journals Phosphatidic acid and diacylglycerol synergize in a cell-free system for activation of NADPH oxidase from human neutrophils.

1993 ◽  
Vol 268 (32) ◽  
pp. 23843-23849
Author(s):  
D Qualliotine-Mann ◽  
D.E. Agwu ◽  
M.D. Ellenburg ◽  
C.E. McCall ◽  
L.C. McPhail
Keyword(s):  
Nadph Oxidase ◽  
Phosphatidic Acid ◽  
Human Neutrophils ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Cross Talk between IRAK4 and the NADPH Oxidase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3081-3081
Author(s):  
Sergio D. Catz ◽  
Sandrine Pacquelet ◽  
Beverly A. Ellis ◽  
Jennifer L. Johnson
Keyword(s):  
Nadph Oxidase ◽  
Fungal Infections ◽  
Interleukin 1 ◽  
Human Neutrophils ◽  
Toll Like Receptor 4 ◽  
Free System ◽  
Rich Domain ◽  
A Cell ◽  
The Relationship ◽  
Cytosolic Factor

Abstract The ability of human neutrophils to combat bacterial and fungal infections depends on their production of reactive oxygen species. The enzymatic complex NADPH oxidase which is responsible for the generation of superoxide anion, is essential for the microbicidal activity of neutrophils, since patients with chronic granulomatous disease, whose NADPH oxidase is inactive, suffer recurrent infections. The exposure of neutrophils to lipopolysaccharide (LPS) amplifies their oxidative response to formylated-peptides (priming). However, the relationship between the signaling downstream of the toll-like receptor 4 after LPS stimulation and the activation of the oxidase has not been elucidated. The phosphorylation of the NADPH oxidase cytosolic factor p47phox is an essential step during the oxidase activation. Here, we test the hypothesis that Interleukin-1 receptor-associated kinase-4 (IRAK4) regulates the NADPH oxidase through phosphorylation of p47phox. We first show that p47phox is a substrate for IRAK4. We showed that IRAK4-phosphorylated p47phox could be subsequently phosphorylated by PKC suggesting that they phosphorylate p47phox at different residues. IRAK4 phosphorylated p47phox to a similar extent as PKC, however, while p47phox was phosphorylated by PKC only in serines, IRAK4 phosphorylated p47phox also in threonines as determined by two-dimensional electrophoresis. Importantly, IRAK4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system (significantly different from the unphosphorylated control, p<0.05). Furthermore, cophosphorylation of p47phox by IRAK4 and PKC significantly potentiated the activity of the NADPH oxidase when compared to PKC phosphorylation alone (p<0.01). Finally, we identified, by mass spectrometry, the residues in p47phox that are targets for IRAK4 phosphorylation. We found that IRAK4 phosphorylates p47phox at a threonine rich domain which possibly constitutes a novel regulatory domain.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

scholarly journals Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation [see comments]

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2646-2653 ◽  
Author(s):  
Y Liel ◽  
A Rudich ◽  
O Nagauker-Shriker ◽  
T Yermiyahu ◽  
R Levy
Keyword(s):  
Gaucher Disease ◽  
Reticuloendothelial System ◽  
Type I ◽  
Dependent Manner ◽  
Superoxide Generation ◽  
Free System ◽  
Nitroblue Tetrazolium Reduction ◽  
Nadph Oxidase Complex ◽  
A Cell ◽  
Nbt Reduction

Abstract Gaucher disease patients are occasionally affected by chronic or fulminant infections. Since Gaucher cells originate from tissue phagocytes, we studied the functional implications of glucocerbroside accumulation on phagocytes in Gaucher disease patients. Circulating monocytes and granulocytes from nine type I Gaucher disease patients, and matched controls, were studied. Evaluation of phagocytic activity included (1) maximal superoxide generation rates following stimulation by phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucylphenylalanine (FMLP); (2) nitroblue tetrazolium reduction test (NBT); (3) chemotaxis toward FMLP; (4) phagocytosis of OZ particles; and (5) killing activity against Staphylococcus aureus. Superoxide generation in monocytes following PMA, OZ, and FMLP stimulation was significantly suppressed at 52% +/- 15%, 39% +/- 8%, and 51% +/- 11% of control, respectively. Superoxide generation in granulocytes was normal. NBT reduction, staphylococcal killing, and phagocytosis were also markedly decreased in monocytes, and normal in granulocytes. Mean chemotaxis rates were normal in both monocytes and granulocytes; however, decreased chemotactic rates were observed in some patients. The abnormality of superoxide generation could be reproduced in a dose- and time-dependent manner in normal circulating monocytes incubated with glucocerebroside. Superoxide generation in glucocerebroside-conditioned normal monocytes in a cell-free system showed normal superoxide generation, reflecting the integrity of the NADPH oxidase complex itself. These results demonstrate markedly compromised phagocytic functions in circulating monocytes in Gaucher disease patients. These abnormalities can be attributed to accumulation of glucocerebroside, since it could be reproduced in normal monocytes incubated with glucocerebroside. Similar abnormalities in Gaucher cells throughout the reticuloendothelial system could impair host defense, and may be of particular importance in the pathogenesis of osteomyelitis in Gaucher disease patients.

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