Structural and Functional Investigations on Cholesterol Signaling and Metabolism

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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.