真兽哺乳动物线粒体解偶联蛋白1二聚体调控产热的质子渗漏

Eutherians have developed a crucial function of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) to prevent energy storage as fat. The thermogenic ability of BAT is conferred by a BAT-specific mitochondrial UCP1. UCP1 increases inner mitochondrial membrane conductance, which allows H+ to dissipate the mitochondrial H+ gradient and convert the energy of substrate oxidation into heat. Free fatty acids (FFA) and purine nucleotides are known to be potent regulators of UCP1, but the interaction of UCP1 and inhibitors as well as the mechanism of FFA activation of UCP1 are not completely understood. In 2012, Andriy Fedorenko et al. carried out direct patch-clamp measurements of UCP1 currents from the inner mitochondrial membrane of BAT mitochondria and showed that UCP1 is a long-chain fatty acid (LCFA) anion/H+ symporter. However, a high-resolution 3-dimensional structure of UCP1 remains unsolved, and an in-depth explanation for the mechanism is not available. In addition, the aggregation state of functional UCP1 is controversial. Interestingly, UCP1 orthologs have been identified in adipose tissue in the interscapular region of the Australian fat-tailed dunnartand gray short-tailed opossum, which are dasyurid marsupial species that separated from eutherians approximately 150 million year ago. UCP1 in the fat-tailed dunnart is induced by cold and norepinephrine. However, there is currently no evidence for a thermogenic role of UCP1 in the fat-tailed dunnart. Its regulation and expression is suggestive of a thermogenic role, but no evidence of non-shivering thermogenesis or "adaptive-NST", i.e., an adaptive organismal response to cold, has been demonstrated. A significant, five-fold increase in the rate of UCP1 evolution in the eutherian lineage was observed, and a relaxed constraint on UCP1 is responsible. In this research area, several hypotheses have been suggested, but more investigations are needed to clarify and confirm these hypotheses. In this project, biochemistry, molecular biology, biophysics and bioinformatics-related techniques will be applied to identify the key amino acid residues during the evolution of metatheria to eutheria and the relationship between these residues and UCP1 aggregation state, and build a homology model of UCP1 homodimer complex and a docking model of GDP and UCP1 to elucidate its function and the mechanism of inhibiting UCP1 by GDP. Furthermore, the marsupial UCP1 proton leak and NST function were further determined and the role of these evolution-related amino acid residues in NST were studied by lentivirus and UCP1-KO (or trangenic)mice experiments to demonstrate that mitochondrial UCP1 is a monophyletic trait of eutherians and dimerized eutherian UCP1 regulates thermogenic proton leaks, and only eutherians have real non-shivering thermogenesis.

真兽哺乳动物的褐色脂肪组织的非震颤性产热（NST）能力来自于组织中特异表达的解偶联蛋白1（UCP1），对其深入研究将推动肥胖等疾病的治疗。有趣的是，后兽有袋目有UCP1的存在，并且UCP1进化分析展示它在真兽哺乳动物进化的速度有5倍增加。自UCP1在1978年发现，它的结构和聚集状态亦尚未阐释。本项目拟从进化角度揭示UCP1结构和功能。具体来讲，确定哺乳动物后兽到真兽UCP1进化的关系及关键位点，这些位点对UCP1聚集状态的影响并构建UCP1二聚体的三维结构模型阐释UCP1结构和功能的联系及抑制剂阻碍UCP1行使功能的可能机制。深入鉴定后兽有袋目哺乳动物UCP1是否具有质子渗漏功能和二聚体存在。并利用UCP1敲除或转基因小鼠等，证明有袋目哺乳动物是否存在非震颤性产热功能以及分析进化关键位点在该功能出现或行使过程中的作用。最终提出只有真兽哺乳动物才具有真正NST，并受UCP1二聚体调控。

解偶联蛋白1（uncoupling protein 1，UCP1）是线粒体内膜上膜蛋白，介导质子渗漏使氧化磷酸化解偶联并产热，而且参与肥胖等代谢疾病。UCP1介导的产热方式是一种UCP1依赖的适应性非震颤性产热（non-shivering thermogenesis，NST），对哺乳动物体温维持具有重要作用。线粒体膜蛋白的聚集状态是其功能重要参数，前人亦采用多种技术手段对其鉴定分析，但结论尚不明确，尤其是UCP1，它的单体和二聚体形式仍须确认。哺乳动物可分为两个亚纲，原兽亚纲和兽亚纲（真兽次亚纲和后兽次亚纲）。真兽通常具有真正的UCP1依赖的NST功能，但前期有文章指出UCP1表达于后兽灰色短尾负鼠和脂尾袋鼩，并应答去甲肾上腺素和冷刺激。对于这种类似真兽UCP1的特征，后兽物种还未系统诠释，至少在功能方面。.本项目研究内容主要包括以下几个部分：UCP1 系统发生树以及UCP1关键功能位点，真兽UCP1 聚集形式和功能，UCP1相关的三维结构模型，后兽有袋目UCP1的质子渗漏和NST生物学功能，比较基因组学的简单分析。为了获得重要结果和关键数据，本项目采用生物化学、分子生物学、生物信息学等技术和化学交联等方法围绕上述研究内容，详细阐释真兽/后兽UCP1相关的科学问题。数据分析揭示真兽UCP1与后兽UCP1处于不同进化分支，后者更接近于鱼纲/两栖纲UCP1和UCP1旁系同源基因UCP2/3。交联实验鉴定UCP1的单体↔二聚体动态转换聚集形式，且二聚体与其功能紧密相关，但未在UCP2/3中发现。同源建模和对接方法构建多个物种的UCPs和真兽UCP1二聚体以及UCP1-GDP结构模型。化学交联实验进一步验证UCP1二聚体模型的合理性。在结构方面，有袋目UCP1与真兽UCP1/2相似。在功能方面，酵母/哺乳动物细胞体系和动物实验结果指出有袋目UCP1并不具有真兽UCP1的质子渗漏活性和NST生物学功能。.总之，本项目提出真兽UCP1的单体↔二聚体动态转换模型，确定UCP1的特征性生物学功能，即质子渗漏和NST，而后兽UCP1则无此类功能。这为哺乳动物UCP1的进化和功能划分等提供更多参数。由于UCP1与疾病的关系，本项目的研究为肥胖等代谢疾病的药物开发和临床治疗提供潜在的理论支持。