Structural and Functional Investigations on Cholesterol Signaling and Metabolism

摘要 胆固醇是一种必需的脂质，在维持细胞膜的刚性和 它具有通透性，对哺乳动物细胞的生长和存活至关重要。胆固醇也是一种 是类固醇激素、胆汁酸、维生素D生物合成的前体，是刺猬信号转导。 在这个项目中，我们将使用细胞生物学和结构方法来研究所涉及的膜蛋白。 在胆固醇信号(Hedgehog信号通路)、生物合成(胆固醇合成酶)和 储存(胆固醇酯化酶)。1)Hedgehog(HH)信号调节异常，这是 正常的胚胎发育和成人组织的动态平衡会导致肿瘤的发生。五年来， 我们已经确定了单独的人ptch1、人ptch1-HH复合物、SMO-GI复合物的结构 在不同的状态下，DISP1单独和与HH形成络合物。这些结构，连同我们的功能研究， 提供对HH信号转导的分子洞察。在这个项目中，我们将继续在这条道路上工作。 具体地说，我们将重点关注HH配体通过HH-ptch1轴的运输和信号调节。2) 18种酶将乙酰辅酶A转化为羊毛甾醇，这是一系列反应中的第一个类固醇中间体 在内质网(ER)中合成胆固醇。一个关键的反应是由HMGCR(3- 羟基-3-甲基戊二酰辅酶A还原酶)，催化乙酰辅酶A转化为甲伐他隆 酸。HMGCR是降胆固醇药物他汀类药物的靶点，因为它是 胆固醇合成。UBIAD1稳定HMGCR并阻止其降解，而E3连接酶的结合 Gp78和Insig触发HMGCR的降解。最近，我们确定了HMGCR的低温EM结构 绑定到UBIAD1。该项目的成功完成将有助于我们对HMGCR是如何 通过Insig被gp78泛素化。此外，我们将研究嵌入膜的胆固醇合成酶是如何 进行胆固醇的生物合成。3)在内质网中，ACAT酶(ACAT-1和ACAT-2)催化 将长链脂肪酰基转移到胆固醇上，生成胆固醇酯，并整合到 用于分泌或储存在脂滴中的脂蛋白。这一反应导致低胆固醇浓度。 内质网是维持细胞内胆固醇动态平衡的关键。ACAT-1普遍表达在 而ACAT-2的表达仅限于肝脏和肠道。ACAT-的药理抑制作用 2可降低血胆固醇水平和动脉粥样硬化。使用结构方法，我们计划调查 脂类调节ACAT的机制以深入了解新的抑制模式并利用这一点 开发专门抑制ACAT-2和降低血浆低密度脂蛋白胆固醇的小分子的信息。

Summary Cholesterol is an essential lipid that plays an important role in the maintenance of membrane rigidity and permeability and is essential for the growth and viability of mammalian cells. Cholesterol also functions as a precursor in the biosynthesis of steroid hormones, bile acids, vitamin D, and is a Hedgehog signaling transducer. In this project, we will employ cell biological and structural approaches to study the membrane proteins involved in cholesterol signaling (Hedgehog signaling pathway), biosynthesis (cholesterol synthetic enzymes), and storage (cholesterol esterification enzymes). 1) Dysregulation of Hedgehog (HH) signaling, which is required for proper embryonic development and adult tissue homeostasis, leads to tumorigenesis. Over the past five years, we have determined the structures of human PTCH1 alone, human PTCH1-HH complexes, SMO-Gi complexes in distinct states, and DISP1 alone and in complex with HH. These structures, along with our functional studies, provide molecular insights into HH signal transduction. In this project, we will continue to work on this pathway. Specifically, we will focus on the trafficking and signal regulation of the HH ligand via the HH-PTCH1 axis. 2) Eighteen enzymes convert acetyl-CoA into lanosterol, the first sterol-like intermediate in a series of reactions that synthesize cholesterol in the endoplasmic reticulum (ER). One key reaction is mediated by HMGCR (3- Hydroxy-3-methylglutaryl coenzyme A reductase), which catalyzes the conversion of acetyl-CoA to mevalonic acid. HMGCR is a target of the cholesterol-lowering drugs statins due to its role as the rate-limiting enzyme in cholesterol synthesis. UBIAD1 stabilizes HMGCR and prevents its degradation, while the binding of the E3 ligase gp78 and Insig trigger the degradation of HMGCR. Recently, we determined the cryo-EM structures of HMGCR bound to UBIAD1. The successful completion of this project will aid our investigation into how HMGCR is ubiquitinated by gp78 via Insig. In addition, we will study how the membrane-embedded cholesterol synthetases carry out the cholesterol biosynthesis. 3) In the ER, the ACAT enzymes (ACAT-1 and ACAT-2) catalyze the transfer of long-chain fatty acyl groups to cholesterol, generating cholesterol esters that are integrated into lipoproteins for secretion or storage in lipid droplets. This reaction results in a low cholesterol concentration in the ER and is crucial for maintaining intracellular cholesterol homeostasis. ACAT-1 is ubiquitously expressed in tissues, whereas ACAT-2 expression is restricted to the liver and intestine. Pharmacological inhibition of ACAT- 2 reduces blood cholesterol levels and atherosclerosis. Using structural methods, we plan to investigate mechanisms for lipid-mediated regulation of ACATs to gain insight into novel modes of inhibition and use this information to develop small molecules that specifically inhibit ACAT-2 and lower plasma LDL cholesterol.