期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 279, 期 18, 页码 18583-18591出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M310268200
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资金
- NIAID NIH HHS [AI20866, R01 AI020866] Funding Source: Medline
- NIA NIH HHS [R01 AG019519, AG19519] Funding Source: Medline
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, Ca2+-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca2+ elevations. In this study, we have analyzed the mechanism of Ca2+ activation of NOX5. In a cell-free system, Ca2+ elevations triggered superoxide production by NOX5 (K-m = 1.06 muM) 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 Ca2+. Flow dialysis experiments showed that NOX5 N terminus contained four Ca2+-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 Ca2+, whereas the more C-terminal pair bound Ca2+ with high affinity. Ca2+-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 Ca2+-dependent way. Our results indicate that the Ca2+-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 Ca2+-activated enzymes.
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