Identification of the Binding Site for the Regulatory Calcium-Binding Domain in the Catalytic Domain of NOX5

Abstract

NADPH oxidases (NOX) are important superoxide producing enzymes that regulate a variety of physiological and pathological processes such as bacteria killing, angiogenesis, sperm-oocyte fusion, and oxygen sensing. NOX5 is a member of the NOX family but distinct from the others by the fact that it contains a long N-terminus with four EF-hand Ca(2+)-binding sites (NOX5-EF). NOX5 generates superoxide in response to intracellular Ca(2+) elevation in vivo and in a cell-free system. Previously, we have shown that the regulatory N-terminal EF-hand domain interacts directly and in a Ca(2+)-dependent manner with the catalytic C-terminal catalytic dehydrogenase domain (CDHD) of the enzyme, leading to its activation. Here we have characterized the interaction site for the regulatory NOX5-EF in the catalytic CDHD of NOX5 using cloned fragments and synthetic peptides of the CDHD. The interaction was monitored with pull-down techniques, cross-linking experiments, tryptophan fluorescence, hydrophobic exposure, isothermal titration calorimetry, and cell-free system enzymatic assays. This site is composed of two short segments: the 637-660 segment, referred to as the regulatory EF-hand-binding domain (REFBD), and the 489-505 segment, previously identified as the phosphorylation region (PhosR). NOX5-EF binds to these two segments in a Ca(2+)-dependent way, and the superoxide generation by NOX5 depends on this interaction. Controlled proteolysis suggests that the REFBD is autoinhibitory and inhibition is relieved by NOX5-EF.

Nephropathy and Elevated BP in Mice with Podocyte-Specific NADPH Oxidase 5 Expression

“…24,25 Nox5 has a large amino terminal EF handcontaining domain that plays a critical role in its calciumdependent activation along with several phosphorylation sites that alter the sensitivity of Nox5 to intracellular calcium. [26][27][28][29] Because AngII increases intracellular calcium concentrations, it seems to induce renal Nox5-dependent ROS generation, which was shown in human endothelial cells. 23 Here, we show that (1) Nox5 is upregulated in human diabetic glomeruli; (2) AngII stimulates ROS generation in human podocytes in a Nox5-dependent manner, a process associated with actin cytoskeletal reorganization and activation of Rac GTPase, which promotes podocyte motility in vitro; (3) mice that express human Nox5 in a podocyte-specific manner (Nox5b pod+ mice) exhibit renal dysfunction, including albuminuria, podocyte effacement, glomerular basement membrane (GBM) thickening, interstitial fibrosis, and hypertension; and (4) Nox5 pod+ mice subjected to streptozotocin (STZ)-induced diabetes develop a more severe kidney phenotype than nontransgenic littermates.…”

Les NADPH oxydases, Nox

“…Le (Figure 1). Le domaine régulateur serait donc auto-inhibiteur pour l'activité de Nox5, et l'activation serait associée à une levée d'inhibition [38]. Une concentration de 7 μM de calcium permet d'atteindre un niveau d'activité comparable à celui de l'activité induite de Nox2 [8].…”

Novel Nox homologues in the vasculature: focusing on Nox4 and Nox5

“…Binding of Ca 2+ to the EF hand induces a marked conformational change allowing the interaction with a hydrophobic domain on the C-terminal domain [69]. Recently, this hydrophobic domain was characterized in a study by Tirone et al [71], where the EF hands, once activated by Ca 2+ , bind to two specific domains: the REFBD (regulatory EFhand-binding domain) and the PhosR (phosphorylation region). The interaction with the EF hands relieves autoinhibitory mechanisms on REFBD [71].…”

“…Recently, this hydrophobic domain was characterized in a study by Tirone et al [71], where the EF hands, once activated by Ca 2+ , bind to two specific domains: the REFBD (regulatory EFhand-binding domain) and the PhosR (phosphorylation region). The interaction with the EF hands relieves autoinhibitory mechanisms on REFBD [71]. As Nox5 activation is dependent on Ca 2+ , it was initially believed that any stimulus that induces an increase in intracellular levels of Ca 2+ could induce ROS generation through Nox5.…”