Mechanisms of Lipid Droplet Formation

与原始提交相比无变更 项目总结/摘要 脂滴（Lipid drops，LD）是普遍存在的单层结合的细胞器，其在细胞脂质储存（用于代谢）中起作用。 能量或膜合成）。LD从ER形成，但LD是如何形成的仍然未知，这是一个核心问题。 现场提问。目前的模型表明，中性脂质，如三酰甘油（TG），合成 并释放到双层中在临界浓度下，TG从磷脂双层中分层， 形成向胞质溶胶出芽的新生LD的相变。我们假设蛋白质对于 确保这一过程以确定的方式发生，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ER中的脂滴组装复合物（LDAC）中操作以形成LD。两种蛋白质 与seipin形成10-12个亚基的环和相同数量的LDAF 1占据 环的中间。虽然我们已经确定了LD形成机制的组成部分，并获得了一些见解 然而，这些蛋白质如何促进LD形成仍然是一个谜。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？有哪些 Seipin/LDAF 1 LDACs的分子结构？这些低聚复合物是如何组装/分解的？ LDAC在细胞中定位在哪里？它们如何组织LD的形成？我们将解决这些问题 实现四个具体目标。目的1将阐明DGAT 1在内质网中合成TG的机制 酵素我们将扩大我们最近阐明的人DGAT 1的分子结构，结合分子生物学， 动力学和生物化学实验，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。目标2将确定LD组装的方式和地点 复合物在细胞中组装形成LD。我们将确定TG合成与LDAC的关系， Seipin/LDAF 1 LDAC定位于ER小管以及它们如何组装。目标3将集中于阐明分子 Seipin/LDAF 1 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cryo-EM和cryo-ET，以检验seipin和LDAF 1与LDAF 1形成环状结构的假设。 这些LDAC形成于膜弯曲的区域（小管），在那里结构可以采用动态的 配合物的构象和活化。目的4确定seipin/LDAF 1 LDAC的分子功能 体外和分子动力学模拟。我们将重建LD形成来测试假设， seipin/LDAF 1 LDAC催化膜中TG的相变，确保LD在这些指定的温度下形成。 形成地点。这些目标的成功完成将促进对一个基本的分子理解。 能量代谢的核心过程，并提供许多代谢的机制基础信息。 疾病，如肥胖症、动脉粥样硬化和脂肪肝。

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.