NoxO1 Determines the Level of ROS Formation by the Nox1-Centered NADPH Oxidase.

The Nox1-centered NADPH oxidase complex facilitates the transfer of electrons from intracellular NADPH across the cell membrane to extracellular molecular oxygen, resulting in the formation of superoxide. The complex is comprised of two membrane-bound subunits, namely Nox1 and p22phox, and the cytosolic subunits, namely NoxA1 and NoxO1. The presence of NoxO1 facilitates the proximity of all components, thereby enabling the complex to exhibit constitutive activity. Despite the theoretical sufficiency of all subunits in a 1:1 ratio, the precise composition of the Nox1-centered NADPH oxidase remains unknown. Analyses of mRNA expression in different cell lines revealed an unequal expression of the components, with an excess of NoxO1. Furthermore, plasmid-based overexpression of individual components of the Nox1-centered NADPH oxidase resulted in an excess of NoxO1 mRNA. The objective of this study was to analyze the ability of NoxO1 to control the level of ROS formation by the Nox1 complex. To this end, we generated Hek293 cells for constitutive expression of Nox1 and NoxA1, which were then transfected with increasing concentrations of NoxO1. The data presented herein suggests that ROS formation by the Nox1-centered NADPH oxidase is dependent on the concentration of NoxO1. A surplus of NoxO1 has been observed to exert control over the activity of the complex in accordance with a dose-dependent mechanism. We thus conclude that the ratio of Nox1, NoxA1, and NoxO1 complexes does not adhere to a 1:1 ratio. Conversely, the availability of NoxO1 serves to regulate the formation of ROS by the Nox1-centered NADPH oxidase.

Systematical Evaluation of the Structure-Cardiotoxicity Relationship of 7-Azaindazole-based PI3K Inhibitors Designed by Bioisosteric Approach.

A growing concern of cardiotoxicity induced by PI3K inhibitors has raised the requirements to evaluate the structure-cardiotoxicity relationship (SCR) in the development process of novel inhibitors. Based on three bioisosteric 7-azaindazole-based candidate inhibitors namely FD269, FD268 and FD274 that give same order of inhibitory concentration 50% (IC50) magnitude against PI3Ks, in this work, we proposed to systematically evaluate the SCR of 7-azaindazole-based PI3K inhibitors designed by bioisosteric approach. The 24-h lethal concentrations 50% (LC50) of FD269, FD268 and FD274 against zebrafish embryos were 0.35, 4.82 and above 50 µM (not detected), respectively. Determination of the heart rate, pericardial and yolk-sac areas and vascular malformation confirmed the remarkable reduction in the cardiotoxicity of from FD269 to FD268 and to FD274. The IC50s of all three compounds against the hERG channel were tested on the CHO cell line that constitutively expressing hERG channel, which were all higher than 20 µM. The transcriptomic analysis revealed that FD269 and FD268 induced the up-regulation of noxo1b, which encodes a subunit of an NADPH oxidase evoking the oxidative stress. Furthermore, immunohistochemistry tests confirmed the structure-dependent attenuation of the overproduction of ROS and cardiac apoptosis. Our results verified the feasibility of bioisosteric replacement to attenuate the cardiotoxicity of 7-azaindazole-based PI3K inhibitors, suggesting that the screening for PI3K inhibitors with both high potency and low cardiotoxicity from bioisosteres would be a beneficial trial.

Identification of a Noxo1 inhibitor by addition of a polyethylene glycol chain.

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules derived from molecular oxygen (O2) which can cause DNA damage and lead to skin cancer. NADPH oxidase 1 (Nox1) is a major producer of ROS in the skin upon exposure to ultraviolet light. Functionally, Nox1 forms a holoenzyme complex that generates two superoxide molecules and reduces NADPH. The signaling activation occurs when the organizer subunit Noxo1 translocates to the plasma membrane bringing a cytochrome p450, through interaction with Cyba. We propose to design inhibitors that prevent Cyba-Noxo1 binding as a topical application to reduce UV-generated ROS in human skin cells. Design started from an apocynin backbone structure to generate a small molecule to serve as an anchor point. The initial compound was then modified by addition of a polyethylene glycol linked biotin. Both inhibitors were found to be non-toxic in human keratinocyte cells. Further in vitro experiments using isothermal calorimetric binding quantification showed the modified biotinylated compound bound Noxo1 peptide with a KD of 2 nM. Both using isothermal calorimetric binding and MALDI (TOF) MS showed that binding of a Cyba peptide to Noxo1 was blocked. In vivo experiments were performed using donated skin explants with topical application of the two inhibitors. Experiments show that ultraviolet light exposure of with the lead compound was able to reduce the amount of cyclobutene pyrimidine dimers in DNA, a molecule known to lead to carcinogenesis. Further synthesis showed that the polyethylene glycol but not the biotin was essential for inhibition.

Overexpression of a Novel Noxo1 Mutant Increases Ros Production and Noxo1 Relocalisation.

Noxo1, the organizing element of the Nox1-dependent NADPH oxidase complex responsible for producing reactive oxygen species, has been described to be degraded by the proteasome. We mutated a D-box in Noxo1 to express a protein with limited degradation and capable of maintaining Nox1 activation. Wild-type (wt) and mutated Noxo1 (mut1) proteins were expressed in different cell lines to characterize their phenotype, functionality, and regulation. Mut1 increases ROS production through Nox1 activity affects mitochondrial organization and increases cytotoxicity in colorectal cancer cell lines. Unexpectedly the increased activity of Noxo1 is not related to a blockade of its proteasomal degradation since we were unable in our conditions to see any proteasomal degradation either for wt or mut1 Noxo1. Instead, D-box mutation mut1 leads to an increased translocation from the membrane soluble fraction to a cytoskeletal insoluble fraction compared to wt Noxo1. This mut1 localization is associated in cells with a filamentous phenotype of Noxo1, which is not observed with wt Noxo1. We found that mut1 Noxo1 associates with intermediate filaments such as keratin 18 and vimentin. In addition, Noxo1 D-Box mutation increases Nox1-dependent NADPH oxidase activity. Altogether, Nox1 D-box does not seem to be involved in Noxo1 degradation but rather related to the maintenance of the Noxo1 membrane/cytoskeleton balance.

Deletion of NoxO1 limits atherosclerosis development in female mice.

OBJECTIVE: Oxidative stress is a risk factor for atherosclerosis. NADPH oxidases of the Nox family produce ROS but their contribution to atherosclerosis development is less clear. Nox2 promotes and Nox4 rather limits atherosclerosis. Although Nox1 with its cytosolic co-factors are largely expressed in epithelial cells, a role for Nox1 for atherosclerosis development was suggested. To further define the role of this homologue, the role of its essential cytosolic cofactor, NoxO1, was determined for atherosclerosis development with the aid of knockout mice. METHODS AND RESULTS: Wildtype (WT) and NoxO1 knockout mice were treated with high fat diet and adeno-associated virus (AAV) overexpressing pro-protein convertase subtilisin/kexin type 9 (PCSK9) to induce hepatic low-density lipoprotein (LDL) receptor loss. As a result, massive hypercholesterolemia was induced and spontaneous atherosclerosis developed within three month. Deletion of NoxO1 reduced atherosclerosis formation in brachiocephalic artery and aortic arch in female but not male NoxO1-/- mice as compared to WT littermates. This was associated with a reduced pro-inflammatory cytokine signature in the plasma of female but not male NoxO1-/- mice. MACE-RNAseq of the vessel did not reveal this signature and the expression of the Nox1/NoxO1 system was low to not detectable. CONCLUSIONS: The scaffolding protein NoxO1 plays some role in atherosclerosis development in female mice probably by attenuating the global inflammatory burden.

NoxO1 Knockout Promotes Longevity in Mice.

According to the free radical theory of aging, reactive oxygen species (ROS) have been proposed to be a major cause of aging for a long time. Meanwhile, it became clear that ROS have diverse functions in a healthy organism. They act as second messengers, and as transient inhibitors of phosphatases and others. In fact, their detrimental role is highly dependent on the context of their production. NADPH oxidases (Nox) have been discovered as a controllable source of ROS. NoxO1 enables constitutive ROS formation by Nox1 by acting as a constitutively active cytosolic subunit of the complex. We previously found that both Nox1 and NoxO1 were highly expressed in the colon, and that NoxO1-/- deficiency reduces colon health. We hypothesized that a healthy colon potentially contributes to longevity and NoxO1 deficiency would reduce lifetime, at least in mouse. In contrast, here we provide evidence that the knockout of NoxO1 results in an elongated life expectancy of mice. No better endothelial function, nor an improved expression of genes related to longevity, such as Sirt1, were found, and therefore may not serve as an explanation for a longer life in NoxO1 deficiency. Rather minor systemic differences, such as lower body weight occur. As a potential reason for longer life, we suggest better DNA repair capacity in NoxO1 deficient mice. Although final fatal DNA damage appears similar between wildtype and NoxO1 knockout animals, we identified less intermediate DNA damage in colon cells of NoxO1-/- mice, while the number of cells with intact DNA is elevated in NoxO1-/- colons. We conclude that NoxO1 deficiency prolongs lifetime of mice, which correlates with less intermediate and potentially fixable DNA damage at least in colon cells.

Soluble Regulatory Proteins for Activation of NOX Family NADPH Oxidases.

NOX family NADPH oxidases deliberately produce reactive oxygen species and thus contribute to a variety of biological functions. Of seven members in the human family, the three oxidases NOX2, NOX1, and NOX3 form a heterodimer with p22phox and are regulated by soluble regulatory proteins: p47phox, its related organizer NOXO1; p67phox, its related activator NOXA1; p40phox; and the small GTPase Rac. Activation of the phagocyte oxidase NOX2 requires p47phox, p67phox, and GTP-bound Rac. In addition to these regulators, p40phox plays a crucial role when NOX2 is activated during phagocytosis. On the other hand, NOX1 activation prefers NOXO1 and NOXA1, although Rac is also involved. NOX3 constitutively produces superoxide, which is enhanced by regulatory proteins such as p47phox, NOXO1, and p67phox. Here we describe mechanisms for NOX activation with special attention to the soluble regulatory proteins.

NoxO1 Controls Proliferation of Colon Epithelial Cells.

Aim: Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut. Results: NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells. Conclusion: NoxO1 affects colon epithelium homeostasis and prevents inflammation.

The NADPH organizers NoxO1 and p47phox are both mediators of diabetes-induced vascular dysfunction in mice.

AIM: NADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes. RESULTS: In the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice. INNOVATION AND CONCLUSION: ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response.

C-terminal tail of NADPH oxidase organizer 1 (Noxo1) mediates interaction with NADPH oxidase activator (Noxa1) in the NOX1 complex.

NOX1 (NADPH oxidase) similar to phagocyte NADPH oxidase, is expressed mainly in the colon epithelium and it is responsible for host defense against microbial infections by generating ROS (reactive oxygen species). NOX1 is activated by two regulatory cytosolic proteins that form a hetero-dimer, Noxo1 (NOX organizer 1) and Noxa1 (NOX activator 1). The interaction between Noxa1 and Noxo1 is critical for activating NOX1. However no structural studies for interaction between Noxa1 and Noxo1 has not been reported till date. Here, we studied the inter-molecular interaction between the SH3 domain of Noxa1 and Noxo1 using pull-down assay and NMR spectroscopy. 15N/13C-labeled SH3 domain of Noxa1 has been purified for hetero-nuclear NMR experiments (HNCACB, CBCACONH, HNCA, HNCO, and HSQC). TALOS analysis using backbone assignment data of the Noxa1 SH3 domain showed that the structure primarily consists of ß-sheets. Data from pull-down assay between the Noxo1 and Noxa1 showed that the SH3 domains (Noxa1) is responsible for interaction with Noxo1 C-terminal tail harboring proline rich region (PRR). The concentration-dependent titration of the Noxo1 C-terminal tail to Noxa1 shows that Noxo1 particularly in the RT loop: Q407*, H408, S409, A412*, G414*, E416, D417, L418, and F420; n-Src loop: C430, E431*, V432*, A435, W436, and L437; and terminal region: I447; F448*, F452* and V454 interact with Noxa1. Our results will provide a detailed understanding for interaction between Noxa1 and Noxo1 at the molecular level, providing insights into their cytoplasmic activity-mediated functioning as well as regulatory role of C-terminal tail of Noxo1 in the NOX1 complex.

Organizers and activators: Cytosolic Nox proteins impacting on vascular function.

NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS) in the cardiovascular system. Of the 7 members of the Nox family, at least three depend for their activation on specific cytosolic proteins. These are p47phox and its homologue NoxO1 and p67phox and its homologue NoxA1. Also the Rho-GTPase Rac is important but as this protein has many additional functions, it will not be covered here. The Nox1 enzyme is preferentially activated by the combination of NoxO1 with NoxA1, whereas Nox2 gains highest activity with p47phox together with p67phox. As p47phox, different to NoxO1 contains an auto inhibitory region it has to be phosphorylated prior to complex formation. In the cardio-vascular system, all cytosolic Nox proteins are expressed but the evidence for their contribution to ROS production is not well established. Most data have been collected for p47phox, whereas NoxA1 has basically not yet been studied. In this article the specific aspects of cytosolic Nox proteins in the cardiovascular system with respect to Nox activation, their expression and their importance will be reviewed. Finally, it will be discussed whether cytosolic Nox proteins are suitable pharmacological targets to tamper with vascular ROS production.

The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype.

OBJECTIVE: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. APPROACH AND RESULTS: A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1(-/-) when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1(-/-) lung endothelial cells (LECs) and a more tip-cell-like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1(-/-) LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1(-/-) LECs when compared with wild-type LECs. CONCLUSIONS: NoxO1 stimulates α-secretase activity probably through reactive oxygen species-mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

Nox1 activation by ßPix and the role of Ser-340 phosphorylation.

Rac is an activating factor for Nox1, an O2(-)-generating NADPH oxidase, expressed in the colon and other tissues. Rac requires a GDP-GTP exchange factor for activation. Nox1 activation by ßPix has been demonstrated in cell lines. We examined the effects of ßPix and its phosphomimetic mutant on endogenous Nox1 in Caco-2 cells transfected with Noxo1 and Noxa1. ßPix expression enhanced O2(-) production in resting cells and cells stimulated with EGF or phorbol ester. ßPix(S340E) further enhanced O2(-) production, while ßPix(S340A) eliminated the ßPix effect. ßPix(S340E), but not ßPix(S340A), had higher affinity and GEF activity for Rac than wild-type ßPix. These results suggest that ßPix phosphorylation at Ser-340 upregulates Nox1 through Rac activation, confirming Rac as a trigger for acute Nox1-dependent ROS production.

C-terminal truncation of Noxa1 greatly enhances its ability to activate Nox2 in a pure reconstitution system.

Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67(phox) and p47(phox). In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher Vmax value than full-length Noxa1 with a similar EC50 value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC50 than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67(phox) with either Noxo1 or p47(phox), although the stability was lower than that with p67(phox) and p47(phox). These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production.

Phosphorylation of Noxo1 at threonine 341 regulates its interaction with Noxa1 and the superoxide-producing activity of Nox1.

Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC in vitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A (PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA in vitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners. STRUCTURED DIGITAL ABSTRACT: Noxo1 binds to p22phox by pull down (1, 2, 3) Noxo1 binds to Noxo1 by pull down (View interaction) Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5).