Discovering the new function of DRP1 on lipid metabolism

发现DRP1对脂质代谢的新功能

• 批准号: 10618321
• 负责人: Kai Sun
• 金额: $ 39万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-06 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618321
• 关键词: Ablation; Adipocytes; Adrenergic Agents; Adverse effects; Affect; Apoptosis; Autophagocytosis; BSCL2 gene; Binding; Biogenesis; Cell physiology; Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Data; Disease; Dissociation; Dynamin; Endoplasmic Reticulum; Energy Intake; Energy Metabolism; Event; Goals; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Impairment; In Vitro; Inflammation; Isoproterenol; Knowledge; Link; Lipid Binding; Lipids; Location; Mass Spectrum Analysis; Measures; Mediating; Membrane Proteins; Metabolic; Metabolic Diseases; Mitochondria; Molecular; Morphology; Names; Obesity; Organelles; Pathologic; Phenotype; Phosphorylation; Phosphorylation Site; Process; Proteins; Regulation; Role; Signal Transduction; Site; System; Testing; Transmission Electron Microscopy; Tubular formation; Vesicle; cell type; constriction; endoplasmic reticulum stress; experimental study; in vivo; insight; lipid disorder; lipid metabolism; loss of function; membrane reconstitution; mutant; novel; novel strategies; recruit; response; spatiotemporal; superresolution microscopy

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.

抽象的 脂质液滴（LDS）是胞质细胞器，在大多数人中都是主要能量储层 细胞类型。 Dynamics LD与代谢稳态的调节密切相关。新生 LDS是由内质网（ER）产生的。但是，关键因素是 参与LD的生物发生知之甚少。有趣的是，我们最近发现 该动力蛋白相关的蛋白1（DRP1）是一种众所周知的线粒体裂变蛋白，易位 上ER并调节新生LDS与ER的解离。在这里，根据我们的 初步观察，我们假设drp1在ER上直接裂解微地位通过其裂痕 GTPase活性。为了检验假设，我们提出了三个具体目标。在AIM 1中，我们将定义 DRP1介导的LD从ER萌芽的分子机制。以前我们 发现缺乏DRP1会导致新生LDS在ER中的保留。确定直接 DRP1在ER结合的LDS上的裂变函数，我们将在体外和体内系统应用， 构图模仿ER-LD的管状拓扑，以分析DRP1收缩和切断 容量。我们还将确定是否需要DRP1中的GTPase域才能通过 在研究中应用DRP1的GTPase突变体（S38a）。在AIM 2中，我们将定义细胞 管理DRP1招募到ER的机制。我们将首先确定如何营地 PKA信号传导调节DRP1易位到ER。具体来说，我们将分析 PKA中DRP1中的磷酸化位点以及已确定位置在其ER位置的功能。 然后，我们将研究Seipin的分子作用，Seipin是一种已知的ER膜蛋白在ER渗透中的作用 DRP1。具体而言，我们将确定DRP1和Seipin之间的相互作用，并检测到 DRP1的ER恢复受损，代替了Seipin的功能丧失。在AIM 3中，我们将 研究DRP1缺陷脂肪细胞中的异常ER-LD表型。首先，我们将调查 通过检查其大小，数量， 形态以及对LD降解和脂质代谢的结果影响。然后， 我们将检查ER的形态变化，ER压力，因此 病变。总的来说，该研究的目的是实现有关的基本发现 DRP1在LD生物发生上的新功能。 了解关键因素，例如DRP1和LD生物发生和 动力学不仅会帮助我们剖析LDS在脂质代谢上的基本细胞功能，还可以 而且还阐明了治疗与肥胖相关脂质疾病的新型策略。