ATG2 transfers lipids from ER exit site membranes to directly expand the growing autophagosome

ATG2 从 ER 出口位点膜转移脂质以直接扩展生长中的自噬体

• 批准号: 10707025
• 负责人: Devin Fuller
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707025
• 关键词: Ablation; Address; Age; Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Architecture; Artificial Membranes; Automobile Driving; Autophagocytosis; Autophagosome; Binding Sites; Biochemical; Biogenesis; Biological; Biological Assay; Biological Process; Biology; Carrier Proteins; Cell physiology; Cells; Cellular Stress; Co-Immunoprecipitations; Collaborations; Cytoplasm; Data; Defect; Deterioration; Engineering; Environment; Funding; Future; Genes; Goals; Growth; Health; Homeostasis; Hydrophobicity; Impairment; In Vitro; Kinetics; Label; Length; Lipids; Longevity; Lysosomes; Maintenance; Maps; Measurement; Mediating; Melia; Membrane; Membrane Biology; Mentorship; Methods; Microscopy; Modeling; Molecular; Mutagenesis; Neurodegenerative Disorders; Neurons; Organelles; Organism; Parkinson Disease; Process; Property; Proteins; Proteome; Proteomics; Publishing; Reaction; Research; Resolution; Rod; Side; Site; Small Interfering RNA; Source; Specificity; Structure; System; Techniques; Technology; Testing; Therapeutic Intervention; Tissues; Training; Visual; age related; biophysical properties; career; cell motility; cytotoxic; design; experimental study; healing; healthy aging; in silico; lipid transfer protein; lipid transport; live cell imaging; novel; prevent; reconstitution; recruit; skill acquisition; success

Project Summary/Abstract Macroautophagy (hereafter autophagy) is a cellular degradative process that is intimately connected to the process of aging. Autophagy maintains cell health and homeostasis through the delivery of potentially cytotoxic cargo to the lysosome through the de novo formation of the double membrane autophagosome. This process is impaired with increased age. Furthermore, deletion of the core autophagy genes has been shown in multiple organisms to decrease lifespan, while rescue experiments conversely restore a full lifespan. Autophagy is especially critical in maintaining the health of long-lived neurons, and defects in autophagy result in various neurodegenerative diseases, including Parkinson’s Disease, Alzheimer’s Disease, and amyotrophic lateral sclerosis. Autophagy protein ATG2 is essential to this process and tissue-specific ablation of this protein results in decreased lifetime, motility, and age-related tissue deterioration. We recently demonstrated that ATG2 is a lipid transfer protein with a novel structure that allows for bulk lipid delivery. This activity is essential for autophagosome biogenesis. I predict that ATG2 delivers lipids directly into the nascent autophagosome to expand the growing membrane. As-of-yet, the identity of the lipid-donating organelle is not known. Intriguingly, a mechanism to deliver a net transfer of lipids in one direction is without clear precedent in mammalian biology. As this mechanism likely depends on the biophysical properties of both membranes, I propose to formally identify the donor membrane from which ATG2 extracts lipids. This project is designed to provide the training necessary to achieve a future career in independent research. Furthermore, as this project will elucidate the membrane source for this critical age-delaying cell biological process, this body of research will identify key regulators of autophagy that represent additional methods of age-related therapeutic intervention. In this proposal, I seek to understand the mechanism by which ATG2 delivers a net transfer of lipids into the autophagosome by addressing three main questions. First, what is the donor membrane for autophagosome biogenesis? I propose to leverage the non-biased and high resolution APEX strategy of proximity labeling with live cell fluorescent microscopy to formally identify the donor membrane. Strikingly, preliminary data reveal that ATG2 resides at ER exit sites (ERES) during autophagosome biogenesis, a highly specialized subdomain of the ER. Second, does ATG2 lipid transfer possess intrinsic directionality, or does it rely upon local membrane energetics? I have developed two in vitro lipid transfer assays to mimic the ERES-autophagosome contact site, through which I can assess the directionality and specificity of lipid transfer through bulk-ensemble measurement and by visual examination. Third, how is ATG2 recruited to the donor membrane? Following identification of the donor membrane, I can now systematically assess ATG2 recruitment through a combination of biochemical and cell biological techniques. This proposed study will further elucidate the molecular mechanisms of healthy aging through the identification of a key player in autophagy.

项目概要/摘要 巨自噬（以下简称自噬）是一种细胞降解过程，与 自噬通过提供潜在的细胞毒性来维持细胞健康和体内平衡。 通过双膜自噬体的从头形成将货物运送到溶酶体。 此外，核心自噬基因的缺失也会随着年龄的增长而受到损害。 在多种生物体中显示会缩短寿命，而救援实验相反会恢复完整的寿命。 自噬对于维持长寿神经元的健康尤其重要，自噬缺陷会导致 各种神经退行性疾病，包括帕金森病、阿尔茨海默病和肌萎缩症 自噬蛋白 ATG2 对该过程和该蛋白的组织特异性消融至关重要。 我们最近证明，这会导致寿命缩短、活动能力和与年龄相关的组织退化。 ATG2 是一种脂质转移蛋白，具有新颖的结构，可实现大量脂质递送。 对于自噬体的生物发生，我预测 ATG2 将脂质直接输送到新生的自噬体中。 有趣的是，目前尚不清楚提供脂质的细胞器的身份。 在哺乳动物生物学中，向一个方向传递脂质净转移的机制尚无明确的先例。 由于这种机制取决于两种膜的生物物理特性，我可能建议正式 识别 ATG2 提取脂质的供体膜 该项目旨在提供培训。 此外，正如该项目将阐明的那样，这是实现未来职业生涯所必需的。 该研究机构将确定这一关键的延缓细胞生物过程的膜来源 自噬的关键调节因子代表了与年龄相关的治疗干预的其他方法。 在本提案中，我试图了解 ATG2 将脂质净转移到细胞中的机制。 自噬体通过解决三个主要问题首先，什么是自噬体的供体膜。 生物起源？我建议利用邻近标记的无偏见和高分辨率 APEX 策略 令人惊讶的是，初步数据显示，活细胞荧光显微镜正式鉴定供体膜。 ATG2 在自噬体生物发生过程中驻留在 ER 出口位点 (ERES)，这是自噬体的一个高度专业化的子域 其次，ATG2 脂质转移是否具有内在的方向性，还是依赖于局部膜？ 我开发了两种体外脂质转移测定法来模拟 ERES-自噬体接触位点， 通过它我可以通过整体集成评估脂质转移的方向性和特异性 第三，ATG2 是如何被招募到供体膜上的？ 识别供体膜后，我现在可以通过 这项拟议的研究将生物化学和细胞生物学技术相结合，进一步阐明这一点。 通过识别自噬中的关键参与者来了解健康衰老的分子机制。