Structural and Functional Investigations on Cholesterol Signaling and Metabolism

总结 胆固醇是一种必需的脂质，在维持膜刚性方面起着重要作用， 它具有良好的渗透性，并且对于哺乳动物细胞的生长和活力是必需的。胆固醇也是一种 类固醇激素、胆汁酸、维生素D生物合成的前体，并且是刺猬信号传导器。 在这个项目中，我们将采用细胞生物学和结构的方法来研究所涉及的膜蛋白 在胆固醇信号传导（刺猬信号通路），生物合成（胆固醇合成酶），和 胆固醇酯化酶（cholesterol esterification enzymes）1)Hedgehog（HH）信号转导失调，这是 正常的胚胎发育和成体组织的体内平衡，导致肿瘤发生。在过去的五年里， 我们已经确定了单独的人PTCH 1、人PTCH 1-HH复合物、SMO-Gi复合物的结构， 在不同的状态下，DISP 1单独和与HH复合。这些结构，沿着我们的功能研究， 提供HH信号转导的分子见解。在这个项目中，我们将继续在这条道路上努力。 具体来说，我们将集中在运输和信号调节的HH配体通过HH-PTCH 1轴。（二） 十八种酶将乙酰辅酶A转化为羊毛甾醇，羊毛甾醇是一系列反应中第一个类似甾醇的中间体 在内质网（ER）中合成胆固醇。一个关键反应是由HMGCR（3- 羟基-3-甲基戊二酰辅酶A还原酶），催化乙酰辅酶A转化为甲羟戊酸 酸HMGCR是降胆固醇药物他汀类药物的靶点，因为它在体内起限速酶的作用。 胆固醇合成。UBIAD 1稳定HMGCR并防止其降解，而E3连接酶的结合 gp 78和Insig触发HMGCR的降解。最近，我们确定了HMGCR的冷冻电镜结构， 与UBIAD 1绑定。这个项目的成功完成将有助于我们调查HMGCR是如何 通过Insig被gp 78泛素化。此外，我们还将研究膜包埋胆固醇合成酶 进行胆固醇的生物合成。3)在ER中，ACAT酶（ACAT-1和ACAT-2）催化 长链脂肪酰基转移到胆固醇，产生胆固醇酯， 脂滴中分泌或储存的脂蛋白。这种反应导致在一个低胆固醇浓度， 它是维持细胞内胆固醇稳态的关键。ACAT-1广泛表达于 ACAT-2的表达仅限于肝脏和肠道。ACAT的药理学抑制作用- 2降低血液胆固醇水平和动脉粥样硬化。使用结构方法，我们计划调查 脂质介导的ACATs调节机制，以深入了解新的抑制模式， 这些信息有助于开发特异性抑制ACAT-2和降低血浆LDL胆固醇的小分子。

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.