NOX5, a calcium-regulated NADPH oxydase : biochemical and biophysical characterization

Les formes réactives de l'oxygène (FRO) sont des médiateurs importants impliqués dans un grand nombre de processus physiologiques et pathologiques. Les NADPH oxydases (NOX) sont des enzymes spécialisées dans la production des FRO. La première NOX mise en évidence et la mieux étudiée est la NADPH oxydase de phagocytes (NOX2), composée d'un complexe catalytique transmembranaire, gp91phox et p22phox et de quatre sous unités cytosoliques régulatrices, p47phox, p67phox, p40phox et Rac2. La NOX2 est impliquée dans l'immunité innée. Récemment, six homologues de la NOX2 ont été identifiés dans diffèrents tissus et organes: NOX1, NOX3, NOX4, NOX5, DUOX1 et DUOX2. Toutes ces enzymes ont en commun un long domaine C-terminal qui contient les sites d'interaction pour le NADPH et le FAD et six hélices transmembranaires qui contiennent les sites d'ancrage pour deux hèmes. NOX5 contient en plus un long domaine N-terminal régulateur composé de trois sites de type "EF-hand " liant le Ca2+. DUOX1 et DUOX2 contiennent dans leur domaine N-terminal deux motifs EF-hand, une hélice transmembranaire supplémentaire et un domaine peroxydase extracellulaire. Cette étude porte sur la NADPH oxydase 5 (NOX5)

Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5)

NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes