Identification of the Binding Site for the Regulatory Calcium-Binding Domain in the Catalytic Domain of NOX5 (original) (raw)
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Mechanism of Ca2+ Activation of the NADPH Oxidase 5 (NOX5)
Journal of Biological Chemistry, 2004
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.
Biochimie, 2007
NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca 2þ elevations. However the C-terminal region of NOX5 also contains putative phosphorylation sites. In this study we used HEK cells stably expressing NOX5 to analyze the size and subcellular localization of the NOX5 protein, its mechanisms of activation, and the characteristics of the ROS released. We demonstrate that NOX5 can be activated both by the protein kinase C activating phorbol esther PMA and by the Ca 2þ ionophore ionomycin. The PMA-but not the ionomycin-dependent activation can be inhibited by protein kinase C inhibitors. NOX5 activity is inhibited by submicromolar concentrations of diphenyl iodonium (DPI), but not by apocynin. Western blot analysis showed a lower (w70 kDa) than expected (82 kDa) molecular mass. Two arguments suggest that NOX5 is at least partially expressed on the plasma membrane: (i) the membrane-impermeant superoxide was readily detected by extracellular probes, and (ii) immunofluorescent labeling of NOX5 detected a fraction of the NOX5 protein at the plasma membrane. In summary, we demonstrate that NOX5 can be found intracellularly and at the cell surface. We also describe that it can be activated through protein kinase C, in addition to its Ca 2þ activation.
FEBS Journal, 2019
It is now accepted that reactive oxygen species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS-controlled production is played by the NADPH oxidases (NOXs), a group of seven membranebound enzymes (NOX1-5 and DUOX1-2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family member present in cyanobacteria, protists, plants, fungi, and the animal kingdom. We show that the calmodulin-like regulatory EF-domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C-terminal lobe of the EF-domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase (DH) domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the DH domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded-tofolded transition of the EF-domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation.
On the mechanism of calcium‐dependent activation of NADPH oxidase 5 (NOX5)
The FEBS Journal, 2019
It is now accepted that reactive oxygen species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS-controlled production is played by the NADPH oxidases (NOXs), a group of seven membranebound enzymes (NOX1-5 and DUOX1-2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family member present in cyanobacteria, protists, plants, fungi, and the animal kingdom. We show that the calmodulin-like regulatory EF-domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C-terminal lobe of the EF-domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase (DH) domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the DH domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded-tofolded transition of the EF-domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation.
Journal of Biological Chemistry, 2005
Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91 phox /Nox2 and Nox1 is known to absolutely require both an organizer protein (p47 phox or Noxo1) andanactivatorprotein(p67 phox orNoxa1);forthep47 phoxdependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91 phox and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22 phox ; Nox3 physically interacts with and stabilizes p22 phox , and the Nox3-dependent superoxide production is totally dependent on p22 phox . The organizers p47 phox and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22 phox , further indicating a link between Nox3 and p22 phox . The p47 phoxenhanced Nox3 activity is further facilitated by p67 phox or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91 phox activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22 phox as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.
Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl
Free Radical Biology and Medicine, 2008
We investigated the mechanism of H 2 O 2 activation of the Ca 2+-regulated NADPH oxidase NOX5. H 2 O 2 induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H 2 O 2 is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H 2 O 2. H 2 O 2-NOX5 activation was inhibited by extracellular Ca2+ chelators, a pharmacological inhibitor of c-Abl, and over-expression of kinase-dead c-Abl. Transfected kinase-active GFP-c-Abl co-localized with vesicular sites of superoxide production in a Ca 2+-dependent manner. In contrast to H 2 O 2 , the Ca2+ ionophore ionomycin induced NOX5 activity independently of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H 2 O 2 treatment. Furthermore, H 2 O 2-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be co-immunoprecipitated with GFP-Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca 2+-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.
Journal of Biological Chemistry, 2005
Assembly of cytosolic factors p67 phox and p47 phox with cytochrome b 558 is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop 191 TSSTKTIRRS 200 and the C-terminal 484 DESQANHFAVHHDEEKD 500 of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47 phox translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop Nox4-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/ R/G mutants of the ␣-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47 phox membrane translocation. This suggests that the ␣-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.
Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5)
Journal of Physiology and Biochemistry
NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•−) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsen...
Journal of Biological …, 2004
Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprec...
The E-loop Is Involved in Hydrogen Peroxide Formation by the NADPH Oxidase Nox4
Journal of Biological Chemistry, 2011
In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O 2 . ), Nox4 produces hydrogen peroxide (H 2 O 2 ). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H 2 O 2 versus L-012 and cytochrome c for O 2 . . The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H 2 O 2 to O 2 . generation while preserving expression and intracellular localization. In the presence of an NO donor, the O 2 . -producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O 2 . . In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against