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Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5).
J Biol Chem 2004; 279(18):18583-91JB

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.

Authors+Show Affiliations

Department of Biochemistry, University of Geneva, CH-1211 Genève, Switzerland.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14982937

Citation

Bánfi, Botond, et al. "Mechanism of Ca2+ Activation of the NADPH Oxidase 5 (NOX5)." The Journal of Biological Chemistry, vol. 279, no. 18, 2004, pp. 18583-91.
Bánfi B, Tirone F, Durussel I, et al. Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). J Biol Chem. 2004;279(18):18583-91.
Bánfi, B., Tirone, F., Durussel, I., Knisz, J., Moskwa, P., Molnár, G. Z., ... Cox, J. A. (2004). Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). The Journal of Biological Chemistry, 279(18), pp. 18583-91.
Bánfi B, et al. Mechanism of Ca2+ Activation of the NADPH Oxidase 5 (NOX5). J Biol Chem. 2004 Apr 30;279(18):18583-91. PubMed PMID: 14982937.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). AU - Bánfi,Botond, AU - Tirone,Fabiana, AU - Durussel,Isabelle, AU - Knisz,Judit, AU - Moskwa,Patryk, AU - Molnár,Gergely Zoltán, AU - Krause,Karl-Heinz, AU - Cox,Jos A, Y1 - 2004/02/24/ PY - 2004/2/26/pubmed PY - 2004/6/21/medline PY - 2004/2/26/entrez SP - 18583 EP - 91 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 279 IS - 18 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/14982937/Mechanism_of_Ca2+_activation_of_the_NADPH_oxidase_5__NOX5__ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=14982937 DB - PRIME DP - Unbound Medicine ER -