Mechanisms of Lipid Droplet Formation

脂滴形成机制

• 批准号: 10797435
• 负责人: ROBERT V FARESE
• 金额: $ 4.98万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 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 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797435
• 关键词: Acyltransferase; Address; Adopted; Area; Atherosclerosis; BSCL2 gene; Binding; Binding Proteins; Biochemical; Biochemistry; Biology; Cell physiology; Cells; Cellular biology; Complex; Cryoelectron Microscopy; Cytoplasm; Cytosol; Data; Emulsions; Endoplasmic Reticulum; Energy Metabolism; Ensure; Enzymes; Eukaryotic Cell; Face; Fatty acid glycerol esters; Generations; Glycerol; Human; In Vitro; Investigation; 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; Acyltransferase; Address; Adopted; Area; Atherosclerosis; BSCL2 gene; Binding; Binding Proteins; Biochemical; Biochemistry; Biology; Cell physiology; Cells; Cellular biology; Complex; Cryoelectron Microscopy; Cytoplasm; Cytosol; Data; Emulsions; Endoplasmic Reticulum; Energy Metabolism; Ensure; Enzymes; Eukaryotic Cell; Face; Fatty acid glycerol esters; Generations; Glycerol; Human; In Vitro; Investigation; 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

No changes from original submission Project Summary/Abstract 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.

原始提交没有变化 项目摘要/摘要 脂质液滴（LDS）是无处不在的单层结合细胞器，可在细胞脂质储存中起作用（用于代谢 能量或膜合成）。从急诊室形成LDS，但如何形成LDS仍然未知，是中心 该领域的问题。当前模型表明中性脂质（例如三酰基甘油（TG））已合成 在急诊室并释放到双层。在临界浓度下，TGS从A中的磷脂双层中脱离混合 形成新生LD的相变向细胞质的芽。我们假设蛋白质对于 确保以定义的方式发生此过程，并防止“异位”的形成 功能失调的LD，破坏ER和细胞功能。具体而言，两个ER蛋白 - 塞宾和脂质液滴组件 因子1（LDAF1） - 在ER中的脂质液滴组件复合物（LDAC）中运行以形成LDS。两种蛋白质 形成一个寡聚组件，用塞宾形成一个10-12个亚基的环和相等数量的LDAF1占用的环 戒指的中间。虽然我们已经确定了LD组机械的组件并获得了一些见识 进入它们的结构，这些蛋白质如何促进LD形成仍然是一个谜。我们在这里 利用最新工具和方法的建议，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如何相对于LDAC进行？什么是 Seipin/LDAF1 LDAC的分子结构？这些寡聚复合物如何组装/拆卸？ LDAC在细胞中定位在哪里？它们如何组织LD形成？我们将解决这些问题 通过完成四个具体目标。 AIM 1将解决DGAT1中ER中TG合成的机制 酶。我们将扩展我们最近对人DGAT1分子结构的阐明，结合了分子 动力学和生化实验，以阐明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。 AIM 2将确定LD组装的方式和地点 复合物在细胞中组装以形成LDS。我们将确定TG合成与LDAC的关系，是否是否 Seipin/LDAF1 LDACS定位于ER小管及其组装。 AIM 3将集中于阐明分子 Seipin/LDAF1 LDAC在体外和细胞中的结构。我们将利用细胞和结构生物学方法， 包括冷冻EM和冷冻-ET，以测试Seipin和LDAF1与LDAF1形成环结构的假设 中心，这些LDAC在膜曲率（管）的区域形成，该区域可能采用动态 复合物的构象和激活。 AIM 4将确定Seipin/LDAF1 LDAC的分子功能 体外和分子动力学模拟。我们将重新建立LD形成，以检验以下假设。 Seipin/LDAF1 LDAC催化Tg在膜中的相变，确保在这些指定的LDS形成 地层地点。这些目标的成功完成将提高对基本的分子理解 能源代谢的中心过程，并提供有关许多代谢基础的机械基础的信息 疾病，例如肥胖，动脉粥样硬化和脂肪肝病。