Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, United Kingdom.
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91, Stockholm, Sweden.
Mol Metab. 2022 Aug;62:101526. doi: 10.1016/j.molmet.2022.101526. Epub 2022 Jun 9.
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.
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.
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.
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.
解偶联蛋白 1（UCP1）可催化棕色脂肪组织中线粒体质子泄漏，促进营养物质氧化产热，如果在人体中被激活，则可能有助于治疗代谢疾病。在棕色脂肪细胞的肾上腺素刺激过程中，脂解产生的游离脂肪酸通过不明确的相互作用激活 UCP1。在此，我们旨在对纯化的 UCP1 与激活剂的结合进行特征描述，以阐明激活过程、识别新的激活剂以及靶向 UCP1 的潜力。
我们通过蛋白热稳定性转移分析评估了配体与纯化的 UCP1 的结合情况，与许多传统方法不同，该方法可以提供有关疏水性配体与膜蛋白结合的信息。我们进行了详细的激活剂相互作用分析和筛选方法，同时还对脂质体、分离的棕色脂肪线粒体和 UCP1 表达控制的细胞系中的 UCP1 活性进行了研究。
我们揭示了脂肪酸和其他激活剂通过特定的不稳定相互作用影响 UCP1，它们作为转运底物，将蛋白质转移到转运循环中不太稳定的构象。通过在筛选中检测到特定的稳定性变化，我们鉴定出了包括非甾体抗炎药布洛芬在内的新型激活剂，配体分析表明 UCP1 对相互作用分子具有相对广泛的结构特异性。布洛芬在脂质体、分离的棕色脂肪线粒体和 UCP1 表达的 HEK293 细胞中成功诱导了 UCP1 的活性，但在培养的棕色脂肪细胞中没有，这表明在每种细胞类型中药物的递送方式都不同。
这些发现阐明了激活剂-UCP1 相互作用的性质，并表明通过已批准的药物靶向细胞中的 UCP1 在原则上是可行的，作为一种治疗途径，但需要在棕色脂肪细胞中具有更有效递送的变体。
