Regulation of NADPH Oxidase Activity in Phagocytes

Debeurme, Franck; Picciocchi, Antoine; Dagher, Marie-Claire; Grünwald, Didier; Beaumel, Sylvain; Fieschi, Franck; Stasia, Marie José

doi:10.1074/jbc.m110.151555

Cited by 41 publications

(36 citation statements)

References 43 publications

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“…Clones were selected by limiting dilution in the presence of 1.5 mg/ml geneticin. Positive clones expressing WT or mutated Nox2 were selected by flow cytometry using monoclonal antibody 7D5 directed against Nox2 as described previously (28). In this way, we selected three positive clones of each mutation from 15 to 20 tested clones that were stored in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

“…Relative luminescence units were recorded at 37°C over a time course of 60 min in a luminoscan luminometer (Labsystems, Helsinki, Finland) connected to a computer. Results were expressed as the sum of relative luminescence detected during the time course (28).…”

Section: Methodsmentioning

confidence: 99%

“…Polyclonal anti-p47 phox and anti-p67 phox antibodies were used as primary stain after Triton X-100 solubilization. After fluorescent second antibodies treatment as described previously (28), cells were examined with a confocal laser scanning microscope and analyzed using Leica confocal software. Cellular nuclei were visualized by Hoechst 33258 staining.…”

Section: Detection Of P47 Phox and P67 Phox Translocation In Transfecmentioning

confidence: 99%

“…The binding of p67 phox within these regions could stabilize the FAD-binding site. In addition, the C-terminal aromatic residue analogous to Phe-570 in Nox2 is a crucial residue in the whole ferredoxin-NADP ϩ reductase family that controls electron transfer from NADPH to FAD (28,38) and is involved in the structural stability of Nox2 (14). The D-loop was demonstrated to be involved in the control of electron transfer from FAD to molecular oxygen (12) and could also be located close to the dehydrogenase domain.…”

mentioning

confidence: 99%

“…The intracytosolic B-loop was recently proposed to interact with the dehydrogenase domain providing the interface between the transmembrane and the dehydrogenase domains of Nox2 during NADPH oxidase complex activation (21). Arg-91 and Arg-92 in the B-loop (19) and Cys-369 centered in a sequence between the NADPH-and the FAD-binding sites (28) are docking sites for p67…”

mentioning

confidence: 99%

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Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

Picciocchi

Debeurme

Beaumel

et al. 2011

Journal of Biological Chemistry

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Flavocytochrome b 558 (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22 phox and gp91phox . The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/ NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane ␣-helices coordinating two hemes. We studied the role of the second transmembrane ␣-helix, which contains a "hot spot" for mutations found in rare X ؉ and X ؊ chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b 558 for p67 phox . By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Detection Of P47 Phox and P67 Phox Translocation In Transfecmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

Picciocchi

Debeurme

Beaumel

et al. 2011

Journal of Biological Chemistry

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Binding of p67phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369Cys-Gly-Cys371 triad in Nox2 and in p67phox

Fradin

Bechor

Berdichevsky

et al. 2018

Journal of Leukocyte Biology

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A central event in the activation of the phagocyte NADPH oxidase involves binding of p67 to the dehydrogenase region of Nox2. The identity of the binding site in Nox2 is unknown. By measuring binding of p67 to synthetic Nox2 peptides, we previously identified a sequence corresponding to Nox2 residues 357-383, as a potential binding site. A key role was attributed to a Cys-Gly-Cys triad, shared by peptides 357-371 (peptide 24) and 369-383 (peptide 28). In this study, we show that (1) oxidation of cysteines in peptides 24 and 28 by a variety of oxidants markedly enhances the binding of p67 ; (2) replacing cysteines by arginine abolishes the response to oxidants and the enhanced binding of p67 ; (3) oxidants act by generating an intramolecular disulfide bond linking cysteines 369 and 371, generating such bond during peptide synthesis reproduces the effect of oxidants; (4) for the disulfide bond to lead to enhanced binding, cysteines must be separated by an intervening residue; bonds joining adjacent cysteines, or cysteines located on two peptides, do not enhance binding; (5) dissociating disulfide bonds by reducing agents abolishes enhanced binding; (6) treating p67 with the alkylating agent N-ethylmaleimide suppresses binding; and (7) mutating all nine cysteines in p67 to serines abolishes binding and diminishes the ability of p67 to support NADPH oxidase activation in vitro. Results show that the primary interaction of p67 with Nox2 is followed by a stabilizing step, based on the establishment of disulfide bonds between cysteine(s) in the Cys-Gly-Cys triad and cysteine(s) in p67 .

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The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis

Beaumel¹,

Stasia²

2019

Methods in Molecular Biology

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Regulation of NADPH Oxidase Activity in Phagocytes

Cited by 41 publications

References 43 publications

Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

Binding of p67phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369Cys-Gly-Cys371 triad in Nox2 and in p67phox

The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis

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