Mechanisms of Lipid Droplet Formation

项目摘要 脂滴（Lipid drops，LD）是细胞内普遍存在的单层细胞器，其功能是储存细胞内的脂质（代谢能量 或膜合成）。LD从ER形成，但LD如何形成仍然未知，并且是研究的中心问题。 领域目前的模型表明，中性脂质，如三酰甘油（TG），是在ER合成和释放 进入双层。在临界浓度下，TG在相变中从磷脂双层中分层， 向胞质溶胶出芽的新生LD。我们假设，蛋白质是必不可少的，以确保这一过程发生在一个 以明确的方式，并防止形成“异位”和潜在的功能失调的LD，破坏ER和细胞 功能具体而言，两种ER蛋白- seipin和脂滴组装因子1（LDAF 1）-在脂滴中起作用。 组装复合物（LDAC）在ER中形成LD。这两种蛋白质形成一个寡聚体组装与seipin形成一个环 10-12个亚基，相同数目的LDAF 1占据环的中间。虽然我们已经确定了 的LD形成机制，并获得了一些深入了解他们的结构，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：如何和在哪里是TG 相对于LDAC？Seipin/LDAF 1 LDAC的分子结构是什么？这些低聚复合物 组装/拆卸？LDAC在细胞中定位在哪里？它们如何组织LD的形成？我们将 通过完成四个具体目标来解决这些问题。目的1将通过以下途径阐明内质网中TG合成的机制： DGAT 1酶。我们将扩大我们最近阐明的人DGAT 1的分子结构，结合 分子动力学和生物化学实验来阐明TG产生的精确机制，并确定如何 TG被释放到ER膜中以形成LD。目标2将确定如何以及在哪里LD组装复杂 在细胞中组装形成LD。我们将确定TG合成与LDACs的关系，无论seipin/LDAF 1 LDAC定位于ER小管以及它们如何组装。目标3将集中在阐明的分子结构的 Seipin/LDAF 1 LDAC在体外和细胞中的表达。我们将利用细胞和结构生物学方法，包括冷冻EM和 cryo-ET来检验seipin和LDAF 1形成环状结构，LDAF 1位于中心，并且这些LDAC 在膜弯曲区域（小管）形成，在那里结构可以采用动态构象并激活细胞膜。 复杂.目的4将在体外和分子动力学中确定seipin/LDAF 1 LDAC的分子功能 仿真。我们将重构LD形成以检验seipin/LDAF 1 LDAC催化相 TG在膜中的过渡，确保LD在这些指定的形成位点形成。成功完成这些 目标将推进对能量代谢的基本过程的分子理解，并提供 关于许多代谢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.