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.

项目总结/摘要 脂滴（LD）是甘油三酯和甾醇酯形式的细胞能量储存库， 维持细胞膜结构和能量平衡它们作为主要的细胞器， 能量储存在细胞和生物体中（如脂肪组织）。调节细胞和器官 从能量学上讲，脂质必须被运输到其他细胞器，在那里它们被脂肪酸氧化消耗， 酶促改变以维持膜结构。细胞脂质代谢失调是一种常见的 代谢紊乱的特征，如肥胖症、糖尿病、非酒精性脂肪性肝病（NAFLD），以及 非酒精性脂肪性肝炎（NASH）。含有VPS 13或重复β沟（RBG）的蛋白质家族 ATG 2A/B-包含被认为参与两个膜之间的管状、大量脂质转运的蛋白质。 缺失ATG 2及其两个已知的结合伴侣导致LD的大量积累， 脂蛋白生物合成和全面NASH。ATG 2主要定位于LD，我们的团队最近 证明该蛋白质可以促进体外大量脂质转移。ATG 2的缺失阻断了荧光脂肪 通过尚未确定的机制从LD到线粒体的酸运动。无论是ATG 2 直接参与LD的脂质转运，以及其他细胞器和蛋白质机器如何参与其中 过程是未知的。在这个建议中，我概述了一个策略，直接测试ATG 2介导的脂质转运在 LD，识别参与LD处脂质转运的膜，并机械地定义蛋白质 在脂质运输中起协同作用。 这个项目的结构是为了最大限度地提高我在体外生物化学，分析设计， 和定量图像分析，从而使我具备了一整套技术和智力技能， 为复杂的生物学问题提供机械解释。因此，我将重点放在 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，我大量借鉴了 Melia和Reinisch实验室使用组合直接测试在LD处的ATG 2的脂质转运活性 新发表的和新设计的体外脂质转运试验和基于细胞的LD试验 积累在目标2中，我建立在一个APEX接近标记的方法，在Melia实验室验证，以确定 蛋白质和膜，ATG 2连接到LD。在目标3中，我采用了广泛的基于细胞的测定， 结合分析和自动图像分析，以确定哪些蛋白质与 ATG 2在LD处移动脂质。该项目将开拓新的方法来测试VPS 13/RBG中的脂质转运 家族蛋白质，它将阐明新的机制，脂质转移在LD。