title: Coupling of 6-phosphogluconate dehydrogenase with NADPH oxidase in neutrophils: Nox2 activity regulation by NADPH availability.
abstract: It is well known that activation of the phagocyte NADPH oxidase requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac) with the membrane cytochrome b(558), leading to its conformation change. Recently, the phagocyte NADPH oxidase complex was isolated in a constitutively active form. In this complex, 6-phosphogluconate dehydrogenase (6PGDH), an enzyme involved in the production of intracellular NADPH, was identified. This protein was absent from the oxidase complex isolated from B lymphocytes, suggesting a specific interaction with the neutrophil NADPH oxidase. To clarify the implication of 6PGDH in the NADPH oxidase activity, a siRNA approach was conducted in neutrophil-like PLB985 cells. NADPH oxidase activity of siRNA-transfected cells was shown to be decreased. Similar results were obtained in vitro, after reconstitution of oxidase activity with subcellular fractions isolated from siRNA-transfected cells. Interestingly, the Michaelis constant (K(m)) of Nox2 for NADPH increases in 6PGDH-depleted cells. Moreover, 6PGDH coimmunoprecipitated with oxidase cytosolic factors from cytosol of stimulated cells. Data suggested that the affinity of Nox2 for NADPH is increased in the presence of 6PGDH on cell stimulation. The present work proposes a new way of NADPH oxidase activity regulation by modulating Nox2 affinity for NADPH.
authors: Groupe de Recherche et d'Etude du Processus Inflammatoire (GREPI), Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier (UJF), Grenoble, France.
authors: Athan
authors: A
authors: Baillet
authors: Rang
authors: R
authors: Xu
authors: Alexei
authors: A
authors: Grichine
authors: Sylvie
authors: S
authors: Berthier
authors: Françoise
authors: F
authors: Morel
authors: Marie-Hélène
authors: MH
authors: Paclet
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D009249:NADP / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D034622:RNA Interference; D013379:Substrate Specificity
chemical_list: D008562:Membrane Glycoproteins; D009249:NADP; D010734:Phosphogluconate Dehydrogenase; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases