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NADPH oxidase organizer 1 (NoxO1) is a scaffold cytoplasmic subunit of the reactive oxygen species (ROS) forming Nox1 complex and involved in angiogenesis, differentiation, and atherosclerosis. We found that overexpression of NoxO1 without simultaneous overexpression of any other component of the active Nox1 complex inhibited EGF-induced wound closure and signaling, while NoxO1 KO yielded the opposite effect. Accordingly, we hypothesize NoxO1 to exert Nox1 independent functions. Using the BioID technique, we identified ErbB2 interacting protein (Erbin) as novel interaction partner of NoxO1. Colocalization of NoxO1 with EGFR, as well as with Erbin validated this finding. EGF treatment interrupted colocalization of NoxO1 and EGFR. EGF mediated kinase activation was delayed in NoxO1 overexpressing cells, while knockout of NoxO1 had the opposite effect. In conclusion, Erbin was identified as a novel NoxO1 interacting protein. Through the subsequent interaction of NoxO1 and EGFR, NoxO1 interferes with EGF signaling. The results of this study suggest a potential role of NoxO1 as an adaptor protein with functions beyond the well-established enabling of Nox1 mediated ROS formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces H 2 O 2 which can directly oxidize cysteine residues. With this post-translational modification, activity, stability, localization and protein-protein interactions of the affected protein is altered. Nox4 controls differentiation, cellular homeostasis and prevents inflammation. Therefore, is likely that epigenetic mechanisms contribute to the effects of Nox4. One group of epigenetic modifiers are class IIa histone deacetylases (HDACs). We hypothesize that Nox4-derived H 2 O 2 oxidizes HDACs and analyzed whether HDACs can be differentially oxidized by Nox4. As an artificial system, we utilized HEK293 cells, overexpressing Nox4 in a tetracycline-inducible manner. HDAC4 was oxidized upon Nox4 overexpression. Additionally, Nox4 overexpression increased HDAC4 phosphorylation on Ser632. H 2 O 2 disrupted HDAC4/Mef2A complex, which de-represses Mef2A. In endothelial cells such as HUVECs and HMECs, overexpression of HDAC4 significantly reduced tube formation. Overexpression of a redox insensitive HDAC4 had no effect on endothelial tube formation. Treatment with H 2 O 2 , induction of Nox4 expression by treatment of the cells with TGFβ and co-overexpression of Nox4 not only induced phosphorylation of HDAC4, but also restored the repressive effect of HDAC4 for tube formation, while overexpression of a redox dead mutant of Nox4 did not. Taken together, Nox4 oxidizes HDAC4, increases its phosphorylation, and eventually ensures proper tube formation by endothelial cells., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Cysteinyl leukotriene receptor 1 antagonists (CysLT1RA) are frequently used as add-on medication for the treatment of asthma. Recently, these compounds have shown protective effects in cardiovascular diseases. This prompted us to investigate their influence on soluble epoxide hydrolase (sEH) and peroxisome proliferator activated receptor (PPAR) activities, two targets known to play an important role in CVD and the metabolic syndrome. Montelukast, pranlukast and zafirlukast inhibited human sEH with IC 50 values of 1.9, 14.1, and 0.8 μM, respectively. In contrast, only montelukast and zafirlukast activated PPARγ in the reporter gene assay with EC 50 values of 1.17 μM (21.9% max. activation) and 2.49 μM (148% max. activation), respectively. PPARα and δ were not affected by any of the compounds. The activation of PPARγ was further investigated in 3T3-L1 adipocytes. Analysis of lipid accumulation, mRNA and protein expression of target genes as well as PPARγ phosphorylation revealed that montelukast was not able to induce adipocyte differentiation. In contrast, zafirlukast triggered moderate lipid accumulation compared to rosiglitazone and upregulated PPARγ target genes. In addition, we found that montelukast and zafirlukast display antagonistic activities concerning recruitment of the PPARγ cofactor CBP upon ligand binding suggesting that both compounds act as PPARγ modulators. In addition, zafirlukast impaired the TNFα triggered phosphorylation of PPARγ2 on serine 273. Thus, zafirlukast is a novel dual sEH/PPARγ modulator representing an excellent starting point for the further development of this compound class.

Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC50 = 0.3 μM/PPARγ EC50 = 0.3 μM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.