The interaction of mitochondrial Uncoupling Protein-1 with regulatory ligands

Cavalieri, Riccardo (2021) The interaction of mitochondrial Uncoupling Protein-1 with regulatory ligands. Doctoral thesis, University of East Anglia.

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Brown adipose tissue of mammals possesses the specialised ability to oxidise nutrients to generate heat for thermoregulation. In adult humans, the thermogenic capacity of the tissue has attracted much interest for its potential to help combat obesity and metabolic disease. Brown fat thermogenesis relies on the Uncoupling protein 1 (UCP1), a member of the mitochondrial carrier family of metabolite transporters. When activated, UCP1 catalyses proton leak across the mitochondrial inner membrane, uncoupling mitochondrial nutrient oxidation from ATP production, releasing energy as heat. The protein is inhibited by cytosolic purine nucleotides and activated by free fatty acids, generated in brown adipocytes when stimulated. Through biochemical analysis of purified UCP1, these studies investigated the interaction of UCP1 with regulatory ligands to gain insight into the activation mechanism.

Assessment of ligand binding to native UCP1 by protein thermostability shift analysis, indicated that fatty acid activators likely interact as transport substrates of UCP1. Specific UCP1 thermostability shifts were used to identify ligands in screens, which revealed novel activators of UCP1, including the drug ibuprofen. The analysis also identified an interaction of UCP1 with the metabolite acyl-Coenzyme A, which could compete off the GDP inhibitor from the protein, representing a possible regulatory mechanism.

Yeast-expressed isoforms of human, mouse, and ovine UCP1 were generated, to show similar nucleotide binding and fatty acid-dependency for proton leak, suggesting that past claims of an inherent proton leak activity in only rodent UCP1 might be unlikely.

Finally, the generation of homology models of UCP1 isoforms, utilising recently available crystal structures of the related ADP/ATP carrier, suggests that UCP1 retains key structural features consistent with a conventional carrier transport mechanism. Novel state-specific bonding networks were also identified, potentially linking transport state shifts with changes in nucleotide affinity, leading to a structural hypothesis of how di- and tri-phosphopurine nucleotides interact with UCP1.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: James Tweddle
Date Deposited: 09 Dec 2022 14:36
Last Modified: 31 Dec 2023 01:38


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