Mechanism of Ca2+ Activation of the NADPH Oxidase 5 (NOX5)

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 EFhands, 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 M) 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 EFhands 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.

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…NOX5 and DUOX1/2 differ from the other NOX homologs and contain additional intracellular Ca 2+ -binding EF-hand domain regions. Consequently, these homologs can be activated by Ca 2+ -mobilizing stimuli and do not rely on association with p22phox or with cytosolic co-factors (9)(10)(11)(12). In this regard, NOX5 and DUOX1/2 relate closely to NADPH oxidases in plants and in protists (Fig.…”

Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

“…Using primers to Nox1 and Nox2, we were able to measure copy numbers as low as 10 per reaction, but detected minimal Nox1 and no Nox2 mRNA in HASMCs. Five variants of Nox5 have been reported: α, β, γ, δ, and a short form that is missing exon 6 [24,31,32]. We used reverse transcriptase PCR using primers specific to the α/γ or β/ δ isoforms to amplify products that were then cloned and sequenced.…”

NADPH Oxidase 5 Is a Pro‐Contractile Nox Isoform and a Point of Cross‐Talk for Calcium and Redox Signaling‐Implications in Vascular Function

“…Nox5 is the only Nox isoform that has been crystalized 16. Unique to Nox5 is its long N‐terminal extension that contains 4 calcium (Ca 2+ )‐binding EF hands, which undergoes conformational change upon Ca 2+ binding 17, 18. Increased intracellular free Ca 2+ concentration ([Ca 2+ ] i ) is essential for the activity of Nox5,18, 19, 20 as evidenced in cell‐culture studies where ROS are not synthesized in Ca 2+ ‐free buffer or from cells with truncation of the N‐terminal region of Nox5 21.…”

“…Unique to Nox5 is its long N‐terminal extension that contains 4 calcium (Ca 2+ )‐binding EF hands, which undergoes conformational change upon Ca 2+ binding 17, 18. Increased intracellular free Ca 2+ concentration ([Ca 2+ ] i ) is essential for the activity of Nox5,18, 19, 20 as evidenced in cell‐culture studies where ROS are not synthesized in Ca 2+ ‐free buffer or from cells with truncation of the N‐terminal region of Nox5 21. This is of particular significance in vascular smooth muscle cells (VSMCs), given that increased [Ca 2+ ] i is the major trigger for vascular contraction 22, 23.…”