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

Durussel, Isabelle
Knisz, Judit
Moskwa, Patryk
Cox, J. A.
Published in The Journal of biological chemistry. 2004, vol. 279, no. 18, p. 18583-18591
Abstract 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.
Keywords Amino Acid SequenceBinding SitesCalcium/ pharmacologyCell LineCell-Free SystemEnzyme Activation/drug effectsHumansKineticsMelitten/metabolism/pharmacologyMembrane Proteins/ metabolismNADPH Oxidase/ metabolismPeptide Fragments/metabolismProtein ConformationSuperoxides/metabolism
PMID: 14982937
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BANFI, Botond et al. Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). In: The Journal of biological chemistry, 2004, vol. 279, n° 18, p. 18583-18591. doi: 10.1074/jbc.M310268200

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Deposited on : 2010-08-27

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