ATG2 mediated lipid transport is essential for lipid droplet homeostasis

ATG2 介导的脂质转运对于脂滴稳态至关重要

• 批准号: 10679372
• 负责人: Justin Korfhage
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679372
• 关键词: Adipose tissue; Amphipathic Alpha Helix; Autophagosome; Binding; Biochemistry; Biogenesis; Biological; Biological Assay; Biological Process; Biology; Biotinylation; C-terminal; Carrier Proteins; Cell physiology; Cells; Cellular biology; Characteristics; Co-Immunoprecipitations; Complex; Consumption; Data; Defect; Development; Diabetes Mellitus; Engineering; Esters; Family; Fatty Acids; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Goals; Homeostasis; Hydrophobicity; Image Analysis; In Vitro; Knock-out; Label; Lead; Link; Lipid Mobilization; Lipids; Lipoproteins; Liposomes; Maintenance; Mediating; Melia; Membrane; Mentorship; Metabolic; Metabolic Diseases; Metabolic syndrome; Mitochondria; Movement; Obesity; Organelles; Organism; Phenocopy; Phospholipids; Physiologic pulse; Process; Protein Family; Proteins; Publishing; Regulation; Role; Site; Small Interfering RNA; Structure; Testing; Training; Triglycerides; Validation; Work; automated image analysis; design; fatty acid analog; fatty acid oxidation; hydrophilicity; in vivo; knock-down; lipid metabolism; lipid transport; member; mutant; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; novel strategies; quantitative imaging; skills; structural biology; trafficking

Project Summary/Abstract Lipid droplets (LDs) are cellular energy reservoirs in the form of triglycerides and steryl esters and are central to the maintenance of membrane structure and energy homeostasis. They serve as the primary organelle for energy storage both in cells and in organisms (as adipose tissue). To regulate cellular and organismal energetics, lipids must be trafficked to other organelles where they are consumed by fatty acid oxidation or enzymatically altered to maintain membrane structure. Dysregulation of cellular lipid metabolism is a common feature of metabolic disorders such as obesity, diabetes, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). The VPS13 or repeating beta groove (RBG) family of proteins- containing ATG2A/B- comprises proteins thought to partake in pipe-like, bulk lipid transport between two membranes. Deletion of ATG2 and two of its known binding partners lead to massive accumulation of LDs, defects in lipoprotein biogenesis, and full-blown NASH. ATG2 localizes primarily to LDs, and our group has recently demonstrated that the protein can facilitate bulk lipid transfer in vitro. Loss of ATG2 blocks fluorescent fatty acid movement from the LD to mitochondria through yet undefined mechanisms. However, whether ATG2 directly participates in lipid transport at the LD and how other organelles and protein machinery partake in this process is not known. In this proposal, I outline a strategy to directly test ATG2 mediated lipid transport at the LD, identify the membranes that participate in lipid transport at the LD, and mechanistically define the proteins that cooperate in lipid transport. This project is structured to maximize progress toward my training goals in in vitro biochemistry, assay design, and quantitative image analysis, thereby equipping me with a full set of technical and intellectual skills to generate mechanistic explanations to complex biological questions. Accordingly, I place a strong focus on in vitro biochemistry, cell-based assays, and automated image analysis. In aim 1, I draw heavily on the expertise of the Melia and Reinisch labs to directly test for lipid transport activity of ATG2 at the LD using a combination of newly published and newly engineered in vitro lipid transport assays and cell-based assays of LD accumulation. In aim 2, I build on an APEX proximity labeling approach validated in the Melia lab to identify proteins and membranes that ATG2 links to the LD. In aim 3, I employ a wide range of cell-based assays, binding assays, and automated image analysis to determine which proteins mechanistically cooperate with ATG2 to move lipids at the LD. This project will pioneer new approaches to test lipid transport in VPS13/RBG family proteins and it will elucidate novel mechanisms of lipid transfer at the LD.

项目概要/摘要 脂滴（LD）是甘油三酯和甾醇酯形式的细胞能量储存库，是细胞的核心 维持膜结构和能量稳态。它们作为主要细胞器 细胞和生物体（如脂肪组织）中的能量储存。调节细胞和有机体 从能量学角度来看，脂质必须被运输到其他细胞器，在那里它们被脂肪酸氧化或 酶促改变以维持膜结构。细胞脂质代谢失调是一种常见的现象 肥胖、糖尿病、非酒精性脂肪肝 (NAFLD) 等代谢性疾病的特征 非酒精性脂肪性肝炎（NASH）。 VPS13 或重复 β 沟 (RBG) 蛋白质家族 ATG2A/B- 包含被认为参与两个膜之间的管道状大量脂质运输的蛋白质。 ATG2 及其两个已知结合伴侣的缺失会导致 LD 的大量积累， 脂蛋白生物合成和全面的 NASH。 ATG2 主要定位于 LD，我们小组最近 证明该蛋白质可以促进体外大量脂质转移。 ATG2 的缺失会阻断荧光脂肪 酸通过尚未明确的机制从 LD 转移到线粒体。然而，ATG2是否 直接参与 LD 的脂质运输以及其他细胞器和蛋白质机器如何参与这一过程 过程未知。在本提案中，我概述了一种直接测试 ATG2 介导的脂质转运的策略 LD，识别在 LD 处参与脂质转运的膜，并机械地定义蛋白质 共同参与脂质运输。 该项目旨在最大限度地实现我在体外生物化学、分析设计、 和定量图像分析，从而使我具备了全套的技术和智力技能 对复杂的生物学问题产生机械解释。因此，我非常关注 体外生物化学、基于细胞的测定和自动图像分析。在目标 1 中，我大量利用专业知识 Melia 和 Reinisch 实验室使用组合直接测试 LD 处 ATG2 的脂质转运活性 新发表和新设计的体外脂质转运测定和基于细胞的 LD 测定 积累。在目标 2 中，我基于 Melia 实验室验证的 APEX 邻近标记方法来识别 ATG2 与 LD 连接的蛋白质和膜。在目标 3 中，我采用了多种基于细胞的检测方法， 结合测定和自动图像分析以确定哪些蛋白质与 ATG2 在 LD 处移动脂质。该项目将开创测试 VPS13/RBG 中脂质运输的新方法 家族蛋白，它将阐明 LD 脂质转移的新机制。