Discovering the new function of DRP1 on lipid metabolism

摘要 脂滴（LDs）是细胞溶质细胞器，在大多数细胞中充当主要的能量储存器。 细胞类型。动态LD与代谢稳态的调节密切相关。元 LD由内质网（ER）产生。然而， 参与LD的生物发生的机制知之甚少。有趣的是，我们最近发现 动力蛋白相关蛋白1（DRP 1），一种众所周知的线粒体分裂蛋白， 并调节新生LD从ER的解离。在这里，根据我们的 初步观察，我们假设ER上的DRP 1通过其 GT活性。为了验证这一假设，我们提出了三个具体目标。在目标1中，我们定义 DRP 1介导的LD从ER出芽的分子机制。以前我们 发现DRP 1的缺乏导致新生LD在ER中的保留。为了确定直接 为了研究DRP 1在ER结合LD上的裂变功能，我们将应用体外和体内系统， 在组成上模拟ER-LD的管状拓扑结构，以分析DRP 1的收缩和切断 容量我们还将通过以下方法确定LD出芽是否需要DRP 1中的GTdR结构域： 应用DRP 1的GT3突变体（S38 A）进行研究。在目标2中，我们将定义细胞 管理DRP 1招募到ER的机制。我们首先要确定cAMP- PKA信号转导调节DRP 1在ER上的转位。具体来说，我们将分析 通过PKA在DRP 1中的磷酸化位点和其ER位置上的鉴定位点的功能。 然后，我们将研究Seipin，一种已知的ER膜蛋白在ER募集中的分子作用 DRP1。具体来说，我们将确定DRP 1和Seipin之间的相互作用，并检测 在Seipin功能丧失的情况下，DRP 1的ER募集受损。在目标3中，我们 研究DRP 1缺陷脂肪细胞中异常ER-LD表型。首先，我们会调查 通过检查其大小，数量， 形态以及对LD降解和脂质代谢的相应影响。然后， 我们将研究ER形态学变化、ER应激以及其他相关的 病理变化总的来说，这项研究的目标是获得以下基本发现： DRP 1在LD生物发生中的新功能。 了解LD生物成因中的关键因素，如DRP 1和详细事件， 动力学不仅有助于我们剖析LD对脂质代谢的基本细胞功能， 而且还揭示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.