16 Vascular redox signalling and protein oxidation: focus on palmitoylation of NADPH oxidase 5 (Nox5) as a potential posttranslational regulatory process. (22nd March 2023)

• Record Type: Journal Article
• Authors: Adu, Samuel; Delles, Christian; Touz, Rhian; Fuller, Will
• Is Part Of: Heart. Volume 109(2023)Supplement 2
• Page Start: A5
• Page End: A6
• Publication Date: 2023-03-22
• Subjects: Heart -- Diseases -- Treatment -- Periodicals; Cardiology -- Periodicals; 616.12
• DOI: 10.1136/heartjnl-SCF-2023.16
• Languages: English
• ISSNs: 1355-6037

Abstract

Abstract : NOX5 is a significant source of reactive oxygen species (ROS), which controls vascular tone and blood pressure, making it an interesting drug target for hypertension. Understanding the regulatory mechanisms of Nox5 is a high priority. We, therefore, investigate how palmitoylation as a posttranslational modification regulates Nox5 trafficking, localization and function. We engineered HEK cells stably expressing tetracycline-inducible wild-type and mutant (C338/342A) YFP-tagged Nox5. The substantial level of palmitoylation observed in the WT was abolished in the mutant Nox5. Confocal microscopy analysis indicated that Nox5 was predominantly localised in the ER, as previously reported. A small population of Nox5 also localises at the cell surface membrane. Membrane labelling and purification with biotinylation reagents revealed no significant difference in surface abundance between the WT and mutant Nox5. We evaluated Nox5 activity using the ROS sensor L-012 and detected no ROS in the absence of tetracycline. However, in tetracycline-treated cells, ROS were only detected following stimulation of cells with Nox5 agonists (400nM PMA and 1uM Ionomycin). The greater ROS generation observed in WT than in mutant cells indicates that palmitoylation enhances Nox5 activity. Next, using TurboID-mediated proximity labelling and mass spectroscopy, we identified Nox5 interaction partners, including palmitoylating (zDHHC5), depalmitoylating (APT2) enzymes and previously proven interactors. Evidence herein suggests that palmitoylation controls Nox5 activity but not its subcellular distribution. Further experiments will investigate the importance of identified palmitoylation enzymes, the effect of palmitoylation on Nox5 activity (and localization), and the downstream functional consequence of Nox5 palmitoylation in VSMC growth, migration, contraction, and inflammation.