Conversion of NOX2 into a constitutive enzyme in vitro and in living cells, after its binding with a chimera of the regulatory subunits

• Published in: Free radical biology & medicine Vol. 113; pp. 470 - 477
• Main Authors: Masoud, Rawand, Serfaty, Xavier, Erard, Marie, Machillot, Paul, Karimi, Gilda, Hudik, Elodie, Wien, Frank, Baciou, Laura, Houée-Levin, Chantal, Bizouarn, Tania
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2017
• Subjects: Animals; Arachidonic Acid - metabolism; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell-Free System; Cercopithecus aethiops; COS Cells; COS7gp91-p22 cells; Gene Expression; Humans; Kinetics; NADP - metabolism; NADPH oxidase; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; Neutrophils - cytology; Neutrophils - metabolism; Nox2; Phosphoproteins - genetics; Phosphoproteins - metabolism; Protein Multimerization; Protein Subunits - genetics; Protein Subunits - metabolism; rac1 GTP-Binding Protein - genetics; rac1 GTP-Binding Protein - metabolism; Superoxide anions; Superoxides - metabolism; AA; NADPH; PBS; CGD; SDS; PMA; Cell-free system; SRCD; LiDS; SOD; cyt c; cyt b558; COS7gp91-p22 cells; CCCP; phox; VUV; NADPH oxidase; ROS; MF; PI; DISCO; DPI; Superoxide anions; Nox2; COS7(gp91-p22) cells
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2017.10.376

During the phagocytosis of pathogens by phagocyte cells, the NADPH oxidase complex is activated to produce superoxide anion, a precursor of microbial oxidants. The activated NADPH oxidase complex from phagocytes consists in two transmembrane proteins (Nox2 and p22phox) and four cytosolic proteins (p40phox, p47phox, p67phox and Rac1-2). In the resting state of the cells, these proteins are dispersed in the cytosol, the membrane of granules and the plasma membrane. In order to synchronize the assembly of the cytosolic subunits on the membrane components of the oxidase, a fusion of the cytosolic proteins p47phox, p67phox and Rac1 named trimera was constructed. The trimera investigated in this paper is composed of the p47phox segment 1–286, the p67phox segment 1–212 and the mutated Rac1(Q61L). We demonstrate that the complex trimera-cyt b558 is functionally comparable to the one containing the separated subunits. Each of the subunits p47phox, p67phox and Rac1Q61L has kept its own activating property. The trimera is produced in an activated conformation as seen by circular dichroism. However, the presence of amphiphile is still necessary in a cell-free system to trigger superoxide anion production. The COS7gp91-p22 cells expressing the trimera produce continuously superoxide anion at high rate. This constitutive activity in cells can be of particular interest for understanding the NADPH oxidase functioning independently of signaling pathways. [Display omitted] •Circular dichroism shows that recombinant trimera is produced in an activated state.•COS7Nox2-p22 cells expressing the trimera produce superoxide anion at high rate.•Nox2-trimera complex is constitutively active in COS7 cells.