Role of the phospholipid binding sites, PX of p47 phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2 - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochimica et Biophysica Acta:Biomembranes Année : 2023

Role of the phospholipid binding sites, PX of p47 phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

Résumé

In phagocytes, superoxide anion (O2 •-), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40 phox , p47 phox , p67 phox , and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme. To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47 phox. We also used a fused chimera consisting of p47 phox (aa 1-286), p67 phox (aa 1-212) and Rac1Q61L, as well as mutated versions in the p47 phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.-production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.
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Dates et versions

hal-04129544 , version 1 (15-06-2023)

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Dina Al Abyad, Xavier Serfaty, Pauline Lefrançois, Stéphane Arbault, Laura Baciou, et al.. Role of the phospholipid binding sites, PX of p47 phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2. Biochimica et Biophysica Acta:Biomembranes, 2023, 1865 (7), pp.184180. ⟨10.1016/j.bbamem.2023.184180⟩. ⟨hal-04129544⟩
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