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; 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 或膜合成）。从急诊室形成LDS，但是如何形成LDS仍然未知，这是一个核心问题 场地。当前模型表明中性脂质（例如三酰基甘油（TG））在ER中合成并释放 在临界浓度下，TGS在形成的相变中从磷脂双层中脱离混合物 向细胞质芽发芽的新生LD。我们假设蛋白质对于确保此过程发生至关重要 定义的方式并防止形成“异位”和可能功能障碍的LDS，破坏ER和细胞 功能。具体而言，两种ER蛋白 - 塞宾和脂质液滴组装因子1（LDAF1） - 在脂质液滴中起作用 ER中的组装复合物（LDAC）形成LDS。两种蛋白质都形成一个寡聚组件，塞宾形成一个环 10-12个亚基和相等数量的LDAF1占据了环的中间。虽然我们已经确定了组件 LD形成机械的构造，并获得了对其结构的深入了解，这些蛋白质如何促进LD 形成主要是一个谜。在这里，我们建议利用包括生物化学在内的最新工具和方法 结构生物学，分子模拟和细胞生物学，以解决以下问题：如何以及在何处进行TG 相对于LDACS？ Seipin/LDAF1 LDAC的分子结构是什么？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 通过完成四个具体目标来解决这些问题。 AIM 1将解决ER中TG合成的机制 DGAT1酶。我们将扩展我们最近阐明人DGAT1分子结构的结合 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组装综合体的方法和地点 在细胞中组装以形成LDS。我们将确定TG合成与LDAC的关系，无论SEIPIN/LDAF1是否 LDACS本地化为ER小管及其组装。 AIM 3将集中于阐明的分子结构 Seipin/LDAF1 LDAC体外和细胞中。我们将利用细胞和结构生物学方法，包括冷冻EM和 冷冻-ET检验了塞宾和LDAF1在中心中形成带有LDAF1的环结构的假设，并且这些LDACS 在结构可能采用动态构象并激活结构的膜曲率区域的形式 复杂的。 AIM 4将在体外和分子动力学中确定Seipin/LDAF1 LDAC的分子功能 模拟。我们将重建LD形成，以检验Seipin/LDAF1 LDAC催化相的假设 TG在膜上的过渡，确保在这些指定形式的位点上LDS形式。这些成功完成 目标将提高对能量代谢中心的基本过程的分子理解，并提供 有关许多代谢疾病的机械基础的信息，例如肥胖，动脉粥样硬化和脂肪肝 疾病。