ATG9 scrambles lipids in concert with ATG2 lipid delivery to directly grow the autophagosome

ATG9 与 ATG2 脂质输送协同扰乱脂质，以直接生长自噬体

• 批准号: 10391326
• 负责人: Taryn Olivas
• 金额: $ 4.02万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391326
• 关键词: Aging; Alanine; Area; Autophagocytosis; Autophagosome; Biochemistry; Biological Process; Biology; Cardiovascular Diseases; Cell Line; Cells; Cellular Membrane; Collaborations; Data; Data Reporting; Density Gradient Centrifugation; Disease; Disputes; Encapsulated; Foundations; Future; Gene Expression; Genes; Golgi Apparatus; Growth; Homeostasis; Immunoprecipitation; In Vitro; Integral Membrane Protein; Knock-out; Libraries; Lipid A; Lipids; Literature; Lysosomes; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Methods; Microscopy; Modeling; Morphology; Mutagenesis; Mutate; Nature; Nerve Degeneration; Organelles; Population; Protein Isoforms; Proteins; Protocols documentation; Reporting; Research; Research Project Grants; Resolution; Seeds; Source; Structure; Styrenes; System; Techniques; Testing; Therapeutic; Time; Training; Vesicle; career; cell type; copolymer; design; in vitro Assay; in vivo; insight; lipid I; lipid transport; lipidomics; maleic acid; mutant; nanodisk; screening; trafficking

Project Summary/Abstract The autophagosome encapsulates cellular debris into double-membrane vesicles responsible for trafficking this cargo to the lysosome for degradation, thereby maintaining cellular homeostasis. How the autophagosome grows and particularly the method by which lipid is delivered to the growing structure has been a key area of dispute. The model favored by our lab is direct delivery of lipid from a source membrane to the expanding phagophore by the early-acting autophagy-related protein ATG2 that we have defined as a bulk lipid transporter. If ATG2 is growing the phagophore through direct lipid transport, then there must be lipid flipping activity that allows for the population of the inner leaflet of the bilayer. Due to the nature of lipid-flipping proteins, they must span the membrane at least once to facilitate lipid crossover. ATG9 is the only transmembrane protein required for autophagy, and its function remains to be determined. Preliminary data from our collaborator shows that ATG9 is capable of flipping lipids in vitro without the presence of ATP, making it a potential scramblase. This data, the reported direct interaction with ATG2, and the fact that ATG9 accumulates with ATG2 genes knocked out led to the hypothesis that ATG9 is a lipid scramblase on the seed membrane that facilitates membrane expansion of the phagophore through direct lipid delivery by ATG2. This project is designed to provide the training necessary to achieve a future career in independent research. Further, this project will increase the understanding of a crucial cell biological process and in turn lay the foundation for future therapeutic lines of inquiry. This proposal outlines two aims – Aim 1: Determine whether ATG9 functions as a scramblase in vivo, and if so, whether it is embedded in the donor or acceptor membrane during autophagosome expansion; Aim 2: Evaluate the lipid composition of the autophagosomal membrane and ATG9 vesicles to trace the origin of the lipids required for autophagosome formation. Aim 1 will assess changes in the biology correlating to manipulation of the ATG9 protein by a powerful combination of biochemistry and microscopy techniques. Disruptions in autophagy will be evaluated by several classic readouts of autophagy progression such as LC3 puncta formation and autophagy factor turnover (i.e. p62), and will allow for efficient screening of ATG9 mutants before submitting them to in vitro scramblase activity assessment. Aim 2 will leverage high resolution quantitative lipidomics and several knockout cell lines that trap autophagosome morphological intermediates to determine the membrane composition of the mammalian autophagosome for the first time. The composition of the autophagosome is expected to approximate the composition of the source membrane. Finally, to separate the autophagosomal membrane from contaminating cargo, styrene maleic acid (SMA) nanodiscs will be employed to rigorously assess the lipids that comprise the autophagosome. This proposed study will contribute new insight into the mechanism of autophagosome formation and answer a decades-long question in the field.

项目总结/摘要 自噬体将细胞碎片包裹在双膜囊泡中， 这种货物的溶酶体降解，从而维持细胞内稳态。自噬体是如何 生长，特别是将脂质输送到生长结构的方法一直是 争端我们实验室所青睐的模型是将脂质从源膜直接递送到扩张的膜。 吞噬细胞的早期作用的自噬相关蛋白ATG 2，我们已经定义为散装脂质 传送器如果ATG 2通过直接的脂质运输来生长吞噬细胞，那么一定有脂质翻转 允许双层的内小叶的群体的活性。由于脂质翻转的性质 当脂质与蛋白质结合时，它们必须跨越膜至少一次以促进脂质交叉。ATG 9是唯一 自噬所需的跨膜蛋白，其功能仍有待确定。初步数据 我们的合作者的研究表明，ATG 9能够在不存在ATP的情况下在体外翻转脂质，使 这是一个潜在的混乱。该数据，报告的与ATG 2的直接相互作用，以及ATG 9 随着ATG 2基因的敲除而积累，导致假设ATG 9是种子上的脂质乱序酶 膜，其通过ATG 2的直接脂质递送促进吞噬细胞的膜扩张。这 该项目旨在提供必要的培训，以实现未来的职业生涯中的独立研究。 此外，该项目将增加对关键细胞生物学过程的理解，并反过来奠定 是未来治疗探索的基础 该提案概述了两个目标-目标1：确定ATG 9是否在体内起扰频酶的作用， 因此，在自噬体扩增过程中，它是否包埋在供体或受体膜中;目的2： 评估自噬体膜和ATG 9囊泡的脂质组成，以追踪自噬体的起源。 自噬体形成所需的脂质。目标1将评估与以下因素相关的生物学变化： 通过生物化学和显微镜技术的强大组合来操纵ATG 9蛋白。 自噬的破坏将通过自噬进展的几个经典读数来评估，如LC 3 斑点形成和自噬因子周转（即p62），并将允许有效筛选ATG 9 突变体，然后将它们提交给体外乱序酶活性评估。Aim 2将利用高分辨率 定量脂质组学和几个敲除细胞系，捕获自噬体形态中间体， 首次确定了哺乳动物自噬体的膜组成。的组合物 预期自噬体接近源膜的组成。最后，为了分离 来自污染货物苯乙烯马来酸（SMA）纳米盘的自噬体膜将被 用于严格评估构成自噬体的脂质。这项研究将有助于 这是对自噬体形成机制的新见解，并回答了该领域长达数十年的问题。