Mechanism of cholesterol regulation by the mitochondrial translocator protein (TSPO)

项目摘要 游离胆固醇是膜双层的完整性和流动性所必需的基本结构成分 对细胞信号传导和脂质代谢调节很重要。游离胆固醇也是 类固醇激素和胆汁酸的合成。游离胆固醇与其酯化储存之间的平衡 形式是严格调节的所有细胞在体内，是至关重要的，以防止高胆固醇血症， 相关疾病。直到最近，人们才相信存在于外膜中的转运蛋白（TSPO） 线粒体膜是胆固醇进入线粒体的类固醇激素转运体 生产我们在最近的工作中推翻了这一假设，证明TSPO与此无关。 过程这一空白使人们重新关注TSPO行动的作用。这是极端的 重要性，因为在多种人类病理中观察到TSPO的上调，并且目前有24种 用于治疗或诊断目的的靶向TSPO的人体临床试验。通过重新审视 使用不同的Tspo基因缺失模型，我们已经确定了TSPO和胆固醇之间的新联系， TSPO和胆固醇酯化之间的关系。本申请的目的是阐明 TSPO：（目的1）定义TSPO介导的胆固醇酯化机制，（目的2）了解TSPO在胆固醇酯化中的作用机制。 在Tspo敲除（Tspo-/-）小鼠中脂质稳态的体内变化。该项目的实验计划 从TSPO的分子表征扩展到使用体内代谢研究其功能相关性 模型使用体外模型，我们研究了胆固醇结合和蛋白质相互作用的重要性， TSPO通过评估特定的氨基酸突变推定的结合位点;我们还进行代谢组学， 评估细胞反应并确定与已知胆固醇调节途径的功能整合 在细胞中。使用体内模型，我们研究了TSPO对肝脏和白色脂肪中脂质代谢的影响 组织以及对血浆脂质的相关影响。这些研究的结果有望提供新的见解 TSPO的脂质调节可能解释动脉粥样硬化中观察到的特定病理功能障碍， 非酒精性脂肪性肝病（NAFLD）和非酒精性脂肪性肝炎（NASH）。此外，知识 TSPO功能将与描述其参与病理学的基础和定义TSPO功能高度相关。 TSPO用于推进人类医学的药理学靶向的基本原理。

PROJECT SUMMARY Free cholesterol is an essential structural component required for integrity and fluidity of membrane bilayers important for cell signaling and regulation of lipid metabolism. Free cholesterol is also the precursor for the synthesis of steroid hormones and bile acids. The balance between free cholesterol and its esterified storage form is tightly regulated by all cells in the body, and is crucial for preventing hypercholesterolemia and associated disorders. Until recently, it was believed that the translocator protein (TSPO) present in the outer mitochondrial membrane was a transporter for cholesterol to enter the mitochondria for steroid hormone production. We overturned this assumption in recent work demonstrating that TSPO is not involved in this process. This void has refocused attention on elucidating the role of TSPO action. This is of extreme importance because upregulation of TSPO is seen in multiple human pathologies, and there are currently 24 human clinical trials targeting TSPO for therapeutic or diagnostic purposes. By reexamining the relationship between TSPO and cholesterol using different Tspo gene deleted models we have identified a novel link between TSPO and cholesterol esterification. The objective of this application is to elucidate the precise role of TSPO: (Aim 1) Define the TSPO-mediated mechanism in cholesterol esterification, and (Aim 2) Understand the in vivo changes to lipid homeostasis in Tspo knockout (Tspo-/-) mice. The experimental plans for this project extend from molecular characterization of TSPO to studying its functional relevance using in vivo metabolism models. Using in vitro models, we investigate the importance of cholesterol binding and protein interactions to TSPO by evaluating specific amino acid mutations to putative binding sites; we also perform metabolomics to evaluate cellular responses and determine functional integration with known pathways of cholesterol regulation in cells. Using in vivo models, we examine TSPO effects on lipid metabolism in the liver and white adipose tissue and the associated impact on plasma lipids. Results from these studies are poised to offer novel insights into lipid regulation by TSPO that might explain specific pathological dysfunctions observed in atherosclerosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In addition, knowledge of TSPO function will be highly relevant to describing the basis of its involvement in pathologies and defining the principles underlying pharmacological targeting of TSPO for advancing human medicine.