Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis.
Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis.
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DOI：
10.1016/j.molmet.2022.101526
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发表时间：
2022-08
影响因子：
8.1
通讯作者：
Crichton, Paul G.
中科院分区：
文献类型：
--
作者：
Cavalieri, Riccardo;Hazebroek, Marlou Klein;Cotrim, Camila A.;Lee, Yang;Kunji, Edmund R. S.;Jastroch, Martin;Keipert, Susanne;Crichton, Paul G.
关键词：
解偶联蛋白1（UCP 1）催化棕色脂肪组织中的线粒体质子泄漏，以促进营养氧化产生热量，如果在人类中被激活，可能会对抗代谢疾病。在棕色脂肪细胞的肾上腺素能刺激过程中，脂解产生的游离脂肪酸通过不清楚的相互作用激活UCP 1。在这里，我们开始研究激活剂与纯化的UCP 1的结合，以阐明激活过程，识别新的激活剂和靶向UCP 1的潜力。我们通过蛋白质热稳定性变化分析评估了配体与纯化的UCP 1的结合，与许多常规方法不同，该方法可以告知疏水配体与膜蛋白的结合。进行了详细的激活剂相互作用分析和筛选方法，通过研究脂质体中的UCP 1活性，分离的棕色脂肪线粒体和UCP 1表达控制的细胞系得到支持。我们发现，脂肪酸和其他激活剂通过特定的不稳定相互作用影响UCP 1，作为转运底物，将蛋白质转移到转运周期的不太稳定构象。通过检测屏幕中的特定稳定性变化，我们确定了新的激活剂，包括非处方药布洛芬，其中配体分析表明UCP 1对相互作用的分子具有相对广泛的结构特异性。Iceland在脂质体、分离的棕色脂肪线粒体和表达UCP 1的HEK 293细胞中成功地诱导了UCP 1活性，但在培养的棕色脂肪细胞中没有，这表明药物递送在每种细胞类型中不同。这些发现澄清了激活剂-UCP 1相互作用的性质，并证明了通过批准的药物靶向细胞中的UCP 1原则上可以作为治疗途径实现，但需要在棕色脂肪细胞中更有效递送的变体。UCP 1热稳定性变化可以识别脂肪酸激活剂结合。激活剂使UCP 1不稳定，在该机制中作为转运底物结合。UCP 1对激活剂具有广泛的配体特异性。筛选确定新的激活剂，包括获得许可的药物布洛芬。细胞中靶向UCP 1的药物可以增加能量消耗。
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. UCP1 thermostability shifts can identify fatty acid activator binding. Activators destabilise UCP1, binding as transport substrates in the mechanism. UCP1 has a wide ligand specificity for activators. Screens identify new activators, including the licenced drug ibuprofen. Drug-targeting UCP1 in cells is viable to increase energy expenditure.
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影响因子：
64.5
作者：
Fedorenko A;Lishko PV;Kirichok Y
通讯作者：
Kirichok Y
影响因子：
3.4
作者：
Dehez, Francois;Schanda, Paul;Chipot, Christophe
通讯作者：
Chipot, Christophe
DOI：
10.1016/j.bbabio.2015.09.013
发表时间：
2016-01
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
King MS;Kerr M;Crichton PG;Springett R;Kunji ERS
通讯作者：
Kunji ERS
影响因子：
3.9
作者：
Crichton PG;Lee Y;Kunji ER
通讯作者：
Kunji ER
影响因子：
29
作者：
Cypess AM;Weiner LS;Roberts-Toler C;Franquet Elía E;Kessler SH;Kahn PA;English J;Chatman K;Trauger SA;Doria A;Kolodny GM
通讯作者：
Kolodny GM