Consequences of the constitutive NOX2 activity in living cells: cytosol acidification, apoptosis, and localized lipid peroxidation

ABSTRACT The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O 2 •− ), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91 phox and p22 phox ) forming the catalytic core, three cytosolic proteins (p67 phox , p47 phox and p40 phox ) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47 phox (aa 1-286), p67 phox (aa 1-212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy. Highlights Trimera is a tool to trigger a continuous ROS production in living cells Continuous NOX2 activity causes cytosol acidification and apoptosis ROS overproduction leads to localized oxidation of the membrane lipids Trimera tends to clusterize in the plasma membrane of COSNOX and COS-7 cells