5 November 2014
NADPH oxidase, Cholesterol, Cell-free system, Arachidonic acid activation, Superoxide production, AA, arachidonic acid, PBS, phosphate buffer saline, Cyt b558, cytochrome b558, Cytc, cytochrome c, DTT, dithiotreitol, EDTA, ethylenediaminetetraacetic acid, FAD, flavin adenine dinucleotide, FMLP, formyl-methionyl-leucyl-phenylalanine, GTP, guanosine-5′-triphosphate, HEPES, [4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid, IPTG, isopropylthiogalatoside, LB, Luria Bertoni, LDL, low density lipoprotein, LR, lipid raft, MF, membrane fractions, MβCD, methyl-β-cyclodextrin, NADPH, reduced β-nicotinamide adenine dinucleotide phosphate, PMSF, phenylmethanesulfonyl fluoride, PtdIns(3)P, phosphatidyl-inositol3-phosphate, PtdIns(3,4)P2, phosphatidylinositol(3,4)-bisphosphate, PX, phox homology domain, ROS, reactive oxygen species, SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis
The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O 2 •−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O 2 •−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase.