Discovering the new function of DRP1 on lipid metabolism

摘要 脂滴是一种胞质细胞器，在大多数情况下是主要的能量储存库。 单元类型。动力学LDS与代谢动态平衡的调节密切相关。新生的 内质网是由内质网产生的。然而，关键因素是 关于腰椎发育迟缓的生物发生，人们知之甚少。有趣的是，我们最近发现 动力蛋白相关蛋白1(Drp1)，一种众所周知的线粒体分裂蛋白，易位 在内质网上，并调节新生LDS从内质网的解离。在这里，基于我们的 初步观察，我们假设内质网上的Drp1通过ITS直接裂变微LD GTP酶活性。为了检验这一假设，我们提出了三个具体目标。在目标1中，我们将定义 调控DRP1介导的LD从内质网萌发的分子机制(S)。以前我们 研究发现，缺乏Drp1导致内质网中新生LDS的滞留。要确定直接的 Drp1在内质网结合的LDS上的裂变功能，我们将在体外和体内应用 成分模拟ER-LD的管状拓扑结构来分析Drp1的收缩和切断 容量。我们还将通过以下方法确定Drp1中的GTPase结构域是否是LD萌发所必需的 将Drp1的GTP酶突变体(S38A)应用于本研究。在目标2中，我们将定义细胞 (S)我们将首先确定夏令营如何- PKA信号调节Drp1在内质网上的转位。具体地说，我们将分析 PKA在Drp1中的磷酸化位点及其在内质网位置上的功能。 然后，我们将研究已知的ER膜蛋白Seipin在ER募集中的分子作用 Drp1的基因序列。具体地说，我们将确定Drp1和Seipin之间的交互并检测 内质网受损-DRp1的招募取代了Seipin功能的丧失。在《目标3》中，我们将 研究Drp1基因缺陷脂肪细胞ER-LD的异常表型。首先，我们将调查 通过检查其大小，数量， 形态以及由此对LD降解和脂代谢的影响。然后, 我们将研究内质网的形态变化、内质网应激以及因此与之相关的其他 病理变化。总而言之，这项研究的目标是获得关于以下方面的基本发现 Drp1在LD生物发生中的新功能。 了解关键因素，如Drp1和LD生物发生和 动力学不仅将帮助我们剖析LDS在脂肪代谢方面的基本细胞功能， 但也阐明了治疗肥胖相关脂质紊乱的新策略。

ABSTRACT Lipid droplets (LDs) are cytosolic organelles that serve as the major energy reservoir in most cell types. Dynamics LDs are tightly linked to the regulation of metabolic homeostasis. Nascent LDs are generated from the endoplasmic reticulum (ER). However, the key factors that are involved in the biogenesis of the LDs are poorly understood. Interestingly, we recently discovered that dynamin-related-protein1 (DRP1), a well-known mitochondrial fission protein, translocates onto ER and regulates the dissociation of the nascent LDs from the ER. Here, based on our preliminary observations, we hypothesize that DRP1 on ER directly fissions micro-LDs via its GTPase activity. To test the hypothesis, we propose three Specific Aims. In Aim 1, we will define the molecular mechanism(s) governing DRP1-mediated LD budding off from ER. Previously we found that lack of DRP1 leads to the retention of the nascent LDs in ER. To determine the direct fission function of DRP1 on the ER-bound LDs, we will apply both in vitro and in vivo systems that compositionally mimic the tubular topology of ER-LD to analyze DRP1 constricting and severing capacity. We will also determine whether GTPase domain in DRP1 is required for LD budding by applying a GTPase mutant (S38A) of DRP1 in the study. In Aim 2, we will define the cellular mechanism(s) governing the recruitment of DRP1 onto the ER. We will first determine how cAMP- PKA signaling regulates translocalization of DRP1 onto the ER. Specifically, we will analyze the phosphorylation sites in DRP1 by PKA and the function of the identified sites on its ER location. Then, we will study the molecular role of Seipin, a known ER membrane protein in ER-recruitment of DRP1. Specifically, we will determine the interaction between DRP1 and Seipin and detect the impaired ER-recruitment of DRP1 in the lieu of the loss-of-function of Seipin. In Aim 3, we will study the abnormal ER-LD phenotype in the DRP1-deficient adipocytes. First, we will investigate the abnormal changes on the pre-existing cytosol LDs by examining their size, number, morphology as well as the consequential effects on LD degradation and lipid metabolism. Then, we will examine the ER morphological changes, ER stress and hence other associated pathological changes. Collectively, the goal of the study is to achieve fundamental findings about DRP1 novel function on LD biogenesis. Understanding the critical factors, such as DRP1 and the detailed events in LD biogenesis and dynamics will not only help us to dissect the basic cellular function of LDs on lipid metabolism, but also shed light on the novel strategies to treat obesity-related lipid disorders.