Mechanisms of Lipid Droplet Formation

项目总结 脂滴(LDs)是一种普遍存在的单层结合细胞器，具有细胞脂存储(代谢能量)的功能 或膜合成)。内质网形成LDS，但LDS是如何形成的仍是未知的，这是一个中心问题 菲尔德。目前的模型表明，中性脂质，如三酰甘油(TG)，在内质网中合成和释放 进入双层。在临界浓度下，TGS在形成的相变中从磷脂双层中解混 向胞质溶质萌发的初生芽。我们假设蛋白质是确保这一过程发生在 明确的方式，并防止形成“异位”和潜在功能障碍的LD，扰乱内质网和细胞 功能。具体地说，两种内质网蛋白质--seipin和脂滴组装因子1(LDAF1)--在脂滴中工作 内质网中的组装复合体(LDAC)形成LDS。这两种蛋白质都与Seipin形成一个环，形成一个寡聚体 10-12个亚基和占据环中央的相同数量的LDAF1。虽然我们已经确定了组件 了解了LD的形成机制，并深入了解了它们的结构，以及这些蛋白质如何发挥促进LD的作用 在很大程度上，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：甘油三酯是如何以及在哪里产生的 相对于LDAC？Seipin/LDAF1 LDAC的分子结构是什么？这些低聚物是如何 组装/拆卸？LDAC在细胞中定位在哪里？它们如何在组织LD形成中发挥作用？我们会 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 DGAT1酶。我们将对我们最近阐明的人类DGAT1的分子结构进行扩展，结合 分子动力学和生化实验，以阐明甘油三酯的精确生成机制，并确定如何 TG被释放到ER膜形成LD。目标2将确定LD组装复合体的方式和地点 在细胞中组装形成LDS。我们将确定甘油三酯的合成与LDAC的关系，是否Seipin/LDAF1 LDAC定位于内质网小管，以及它们如何组装。目标3将集中于阐明分子的分子结构 Seipin/LDAF1 LDAC的体外和细胞内表达。我们将利用细胞和结构生物学方法，包括冷冻-EM和 Cryo-et来验证这样的假设，即Seipin和LDAF1形成以LDAF1为中心的环结构，并且这些LDAC 在膜曲率(小管)区域形成，在那里结构可以采用动态构象并激活 很复杂。目的4确定Seipin/LDAF1 LDAC的体外分子功能和分子动力学 模拟。我们将重建LD形成以验证Seipin/LDAF1 LDAC催化相的假设 Tg在膜中的过渡，确保在这些指定的形成位置形成LDS。成功完成这些任务 AIMS将促进对能量代谢中心的基本过程的分子理解，并提供 关于许多代谢性疾病的机制基础的信息，如肥胖、动脉粥样硬化和脂肪肝 疾病。

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.