Mechanisms of Lipid Droplet Formation

脂滴形成机制

• 批准号: 10475248
• 负责人: ROBERT V FARESE
• 金额: $ 6.16万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475248
• 关键词: Acyltransferase; Address; Adopted; Area; Atherosclerosis; BSCL2 gene; Binding Proteins; Biochemical; Biochemistry; Biology; Cell physiology; Cells; Cellular biology; Complex; Cryoelectron Microscopy; Cytosol; Data; Diffuse; Emulsions; Endoplasmic Reticulum; Energy Metabolism; Ensure; Enzymes; Eukaryotic Cell; Face; Fatty acid glycerol esters; Generations; Glycerol; Human; In Vitro; Investigation; Lead; Lipid Bilayers; Lipid Inclusion; Lipids; Membrane; Membrane Lipids; Metabolic; Metabolic Diseases; Modeling; Molecular; Molecular Conformation; Molecular Structure; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organelles; Phase Transition; Phospholipids; Process; Proteins; Research; Set protein; Site; Structure; Testing; Triglycerides; Work; experimental study; fatty liver disease; in vitro testing; insight; membrane synthesis; molecular dynamics; monolayer; monomer; novel therapeutics; particle; prevent; protein complex; protein function; reconstitution; simulation; structural biology; tomography; tool

PROJECT SUMMARY Lipid droplets (LDs) are ubiquitous monolayer-bound organelles that function in cellular lipid storage (for metabolic energy or membrane synthesis). LDs form from the ER, but how LDs are formed remains unknown and is a central question for the field. The current model indicates that neutral lipids, such as triacylglycerols (TG), are synthesized in the ER and released into the bilayer. At a critical concentration, TGs de-mix from the phospholipid bilayer in a phase transition that forms nascent LDs that bud toward the cytosol. We hypothesize that proteins are essential to ensure this process occurs in a defined manner and to prevent the formation of “ectopic” and potentially dysfunctional LDs, disrupting ER and cell function. Specifically, two ER proteins – seipin and lipid droplet assembly factor 1 (LDAF1) – operate in the lipid droplet assembly complex (LDACs) in the ER to form LDs. Both proteins form an oligomeric assembly with seipin forming a ring of 10-12 subunits and an equal number of LDAF1 occupying the middle of the ring. While we have identified components of the LD formation machinery and gained some insight into their structures, how these proteins function to facilitate LD formation remains mostly a mystery. Here we propose to utilize the latest tools and approaches, including biochemistry, structural biology, molecular simulations, and cell biology, to address the following questions: How and where is TG made relative to LDACs? What are the molecular structures of the seipin/LDAF1 LDACs? How do these oligomeric complexes assemble/disassemble? Where do LDACs localize in cells? How do they function to organize LD formation? We will address these questions by completing four specific aims. Aim 1 will address the mechanism of TG synthesis in the ER by the DGAT1 enzyme. We will expand on our recent elucidation of the molecular structure of human DGAT1, combining molecular dynamics and biochemical experiments to elucidate the precise mechanism of TG generation and determine how TG is released into the ER membrane for LD formation. Aim 2 will determine how and where LD assembly complexes assemble in cells to form LDs. We will determine the relationship of TG synthesis to LDACs, whether seipin/LDAF1 LDACs localize to ER tubules and how they assemble. Aim 3 will focus on elucidating the molecular structure of the seipin/LDAF1 LDAC in vitro and in cells. We will utilize cell and structural biology approaches, including cryo-EM and cryo-ET to test the hypothesis that seipin and LDAF1 form a ring structure with LDAF1 in center and that these LDACs form at areas of membrane curvature (tubules) where the structure may adopt dynamic conformations and activate of the complex. Aim 4 will determine the molecular function of the seipin/LDAF1 LDAC in vitro and in molecular dynamics simulations. We will reconstitute LD formation to test the hypothesis that the seipin/LDAF1 LDAC catalyzes phase transition of TG in the membrane, ensuring LDs form at these designated formation sites. Successful completion of these aims will advance the molecular understanding of a fundamental process central to energy metabolism and provide information on the mechanistic underpinning of many metabolic diseases, such as obesity, atherosclerosis, and fatty liver disease.

项目概要 脂滴 (LD) 是普遍存在的单层结合细胞器，在细胞脂质储存中发挥作用（用于代谢能量） LD 是由 ER 形成的，但 LD 是如何形成的仍然未知，也是当前研究的核心问题。 目前的模型表明，中性脂质，例如三酰甘油（TG），是在内质网中合成并释放的。 在临界浓度下，TG 在相变中从磷脂双层中分离出来，形成相变。 我们发现蛋白质对于确保这一过程在细胞质中发生至关重要。 定义的方式并防止形成“异位”和潜在功能失调的 LD，破坏 ER 和细胞 具体而言，两种 ER 蛋白——seipin 和脂滴组装因子 1 (LDAF1)——在脂滴中发挥作用。 ER 中的组装复合物 (LDAC) 形成 LD，两种蛋白与 seipin 形成寡聚组装体，形成环。 10-12 个亚基和相同数量的 LDAF1 占据环的中间，同时我们已经确定了组件。 了解 LD 形成机制，并深入了解其结构、这些蛋白质如何发挥作用以促进 LD 形成仍然是一个谜，在这里我们建议利用最新的工具和方法，包括生物化学， 结构生物学、分子模拟和细胞生物学，以解决以下问题：TG 是如何以及在哪里制造的 相对于 LDAC，seipin/LDAF1 LDAC 的分子结构如何？ LDAC 在细胞中定位于何处？它们如何发挥作用来组织 LD 形成？ 通过完成四个具体目标来解决这些问题，目标 1 将通过以下方式解决 ER 中 TG 合成的机制。 我们将结合最近对人类 DGAT1 分子结构的阐明进行扩展。 分子动力学和生化实验阐明 TG 生成的精确机制并确定如何 TG 被释放到 ER 膜中以形成 LD，目标 2 将决定 LD 组装复合物的方式和位置。 在细胞中组装形成LD，我们将确定TG合成与LDAC的关系，无论是seipin/LDAF1。 LDAC 定位于 ER 小管及其组装方式，目标 3 将重点阐明其分子结构。 我们将利用细胞和结构生物学方法，包括冷冻电镜和细胞内的 seipin/LDAF1 LDAC。 冷冻电子断层扫描 (cryo-ET) 检验 seipin 和 LDAF1 形成以 LDAF1 为中心的环状结构以及这些 LDAC 的假设 在膜曲率（管）区域形成，结构可以采用动态构象并激活 目标 4 将确定 seipin/LDAF1 LDAC 的体外和分子动力学分子功能。 我们将重构 LD 形成来测试 seipin/LDAF1 LDAC 催化相的假设。 TG 在膜中的转变，确保 LD 在这些指定的形成位点成功完成。 目标将促进对能量代谢核心基本过程的分子理解，并提供 有关许多代谢疾病（例如肥胖、动脉粥样硬化和脂肪肝）的机制基础的信息 疾病。