Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes-Differential sensitivity to calcium and phosphorylation events.
NADPH oxidase is a crucial element of phagocytes involved in microbicidal mechanisms. It becomes active when membrane-bound cytochrome b(558), the redox core, is assembled with cytosolic p47(phox), p67(phox), p40(phox), and rac proteins to produce superoxide, the precursor for generation of toxic reactive oxygen species. In a previous study, we demonstrated that the potential second intracellular loop of Nox2 was essential to maintaining NADPH oxidase activity by controlling electron transfer from FAD to O(2). Moreover, replacement of this loop by the Nox4-D-loop (D-loop(Nox4)-Nox2) in PLB-985 cells induced superoxide overproduction. In the present investigation, we demonstrated that both soluble and particulate stimuli were able to induce this superoxide overproduction. Superoxide overproduction was also observed after phosphatidic acid activation in a purified cell-free-system assay. The highest oxidase activity was obtained after ionomycin and fMLF stimulation. In addition, enhanced sensitivity to Ca(2+) influx was shown by thapsigargin, EDTA, or BTP2 treatment before fMLF activation. Mutated cytochrome b(558) was less dependent on phosphorylation triggered by ERK1/2 during fMLF or PMA stimulation and by PI3K during OpZ stimulation. The superoxide overproduction of the D-loop(Nox4)-Nox2 mutant may come from a change of responsiveness to intracellular Ca(2+) level and to phosphorylation events during oxidase activation. Finally the D-loop(Nox4)-Nox2-PLB-985 cells were more effective against an attenuated strain of Pseudomonas aeruginosa compared to WT-Nox2 cells. The killing mechanism was biphasic, an early step of ROS production that was directly bactericidal, and a second oxidase-independent step related to the amount of ROS produced in the first step.
Carrichon
Laure
L
Centre Diagnostic et Recherche sur la Granulomatose septique chronique CGD, TheREx-TIMC/Imag UMR CNRS 5525, CHU and Université Joseph Fourier, 38043 Grenoble Cedex 9, France.
Picciocchi
Antoine
A
Debeurme
Franck
F
Defendi
Federica
F
Beaumel
Sylvain
S
Jesaitis
Algirdas J
AJ
Dagher
Marie-Claire
MC
Stasia
Marie-José
MJ
D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D010586:Phagocytes / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D013481:Superoxides / Q000737:chemistry*
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D013481:Superoxides; C036888:cytochrome b558; D006861:Hydrogen Peroxide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D002118:Calcium