Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis

Cavalieri, Riccardo, Hazebroek, Marlou Klein, Cotrim, Camila A., Lee, Yang, Kunji, Edmund R. S., Jastroch, Martin, Keipert, Susanne and Crichton, Paul G. ORCID: (2022) Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis. Molecular Metabolism, 62. ISSN 2212-8778

[thumbnail of Cavalieri_etal_2022_MolecularMetabolism]
PDF (Cavalieri_etal_2022_MolecularMetabolism) - Accepted Version
Available under License Creative Commons Attribution Non-commercial No Derivatives.

Download (2MB) | Preview


Objective: Uncoupling protein 1 (UCP1) catalyses mitochondrial proton leak in brown adipose tissue to facilitate nutrient oxidation for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we set out to characterise activator binding to purified UCP1 to clarify the activation process, discern novel activators and the potential to target UCP1. Methods: We assessed ligand binding to purified UCP1 by protein thermostability shift analysis, which unlike many conventional approaches can inform on the binding of hydrophobic ligands to membrane proteins. A detailed activator interaction analysis and screening approach was carried out, supported by investigations of UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1 expression-controlled cell lines. Results: We reveal that fatty acids and other activators influence UCP1 through a specific destabilising interaction, behaving as transport substrates that shift the protein to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the over-the-counter drug ibuprofen, where ligand analysis indicates that UCP1 has a relatively wide structural specificity for interacting molecules. Ibuprofen successfully induced UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1-expressing HEK293 cells but not in cultured brown adipocytes, suggesting drug delivery differs in each cell type. Conclusions: These findings clarify the nature of the activator-UCP1 interaction and demonstrate that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.

Item Type: Article
Additional Information: Acknowledgements: The authors thank local Norfolk and Suffolk farms for access to new-born lambs that died of natural causes during the lambing season. This work was supported a PhD studentship award (R.C.) from Norwich Medical School and the Faculty of Medicine and Health, University of East Anglia, UK, and by funding from the UK Biological and Biotechnological Sciences Research Council (BB/S00940X/1), Novo Nordisk Research Foundation (0059646 to M.J.) and Swedish Research Council (2018-02150 to S.K).
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
UEA Research Groups: Faculty of Medicine and Health Sciences > Research Groups > Cardiovascular and Metabolic Health
Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health
Related URLs:
Depositing User: LivePure Connector
Date Deposited: 13 Jun 2022 15:30
Last Modified: 19 Oct 2023 03:21
DOI: 10.1016/j.molmet.2022.101526


Downloads per month over past year

Actions (login required)

View Item View Item