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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通过快速膜蛋白热稳定性位移分析鉴定解偶联蛋白 1 的激活配体
DOI：
10.1101/2022.02.03.478984
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Cavalieri R
中科院分区：
文献类型：
--
作者：
Cavalieri R
解偶联蛋白1 （UCP1）在棕色脂肪组织中催化线粒体质子泄漏，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。在褐色脂肪细胞的肾上腺素能刺激过程中，脂肪分解产生的游离脂肪酸通过一种不明确的相互作用激活UCP1。在这里，我们开始表征激活剂与纯化UCP1的结合，以阐明激活过程，识别新的激活剂和靶向UCP1的潜力。方法通过蛋白质热稳定性位移分析来评估配体与纯化UCP1的结合，与许多传统方法不同，该方法可以了解疏水配体与膜蛋白的结合。通过对脂质体、分离的棕色脂肪线粒体和UCP1表达控制细胞系中UCP1活性的研究，开展了详细的激活剂相互作用分析和筛选方法。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UCP1，作为运输底物将蛋白质转移到运输周期中不太稳定的构象。通过检测筛选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UCP1对相互作用的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表达UCP1的HEK293细胞中成功诱导了UCP1活性，但在培养的棕色脂肪细胞中没有，这表明不同细胞类型的药物递送不同。这些发现阐明了激活剂-UCP1相互作用的性质，并证明通过批准的药物靶向细胞中的UCP1原则上是可以作为治疗途径实现的，但需要在棕色脂肪细胞中更有效递送的变体。
ObjectiveUncoupling 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.MethodsWe 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.ResultsWe 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.ConclusionsThese 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.
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DPC 胶束中获得的线粒体解偶联蛋白的结构模型在功能上不相关
DOI：
--
发表时间：
2020
期刊：
The FEBS Journal
影响因子：
--
作者：
Mathilde S. Piel;S. Masscheleyn;F. Bouillaud;K. Moncoq;B. Miroux
通讯作者：
B. Miroux
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
影响因子：
7.7
作者：
Majd H;King MS;Palmer SM;Smith AC;Elbourne LD;Paulsen IT;Sharples D;Henderson PJ;Kunji ER
通讯作者：
Kunji ER
影响因子：
5.7
作者：
Alexandrov, Alexander I.;Mileni, Mauro;Stevens, Raymond C.
通讯作者：
Stevens, Raymond C.
棕色脂肪组织线粒体解偶联蛋白的 Cl-易位途径的新底物和竞争性抑制剂。
DOI：
--
发表时间：
1990
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Jezek,P;Garlid,KD
通讯作者：
Garlid,KD