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: 10536404
• 负责人: Devin Fuller
• 金额: $ 4.68万
• 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/10536404
• 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; Light; Lipids; Longevity; Lysosomes; Maintenance; Maps; Measurement; Mediating; Melia; Membrane; Membrane Biology; Mentorship; Methods; Microscopy; Modeling; Molecular; Mutagenesis; Neurodegenerative Disorders; Neurons; Organelles; Organism; Parkinson Disease; Problem Solving; Process; 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; base; 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; skills; 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.

项目摘要/摘要 宏观噬菌（以下称自噬）是一个密切相关的细胞降解过程 衰老过程。自噬通过潜在的细胞毒性递送维持细胞健康和稳态 通过双膜自噬体的从头形成，货物到达溶酶体。 随着年龄的增长，此过程受到损害。此外，核心自噬基因的缺失已经 在多种生物体中显示以降低寿命，而救援实验则恢复了完整的寿命。 自噬对于维持长寿神经元的健康和自噬结果缺陷特别重要 在各种神经退行性疾病中，包括帕金森氏病，阿尔茨海默氏病和肌萎缩症 侧硬化。自噬蛋白ATG2对于此过程和组织特异性消融至关重要 结果改善了寿命，运动性和与年龄相关的组织定义。我们最近证明了 ATG2是一种具有新型结构的脂质转移蛋白，可实现大量脂质递送。这项活动是必不可少的 用于自噬体生物发生。我预测ATG2将脂质直接传递到新生的自噬体中 扩展生长的膜。尚不清楚尚不清楚脂质细胞器的身份。有趣的是， 在哺乳动物的生物学中，脂质净转移净转移的机制在一个方向上没有明确的先例。 由于这种机制可能取决于两种机制的生物物理特性，因此我建议正式 确定供体膜，从中提取脂质的供体膜。该项目旨在提供培训 对于实现独立研究的未来职业所必需的。此外，该项目将阐明 这种关键的延迟年龄细胞生物学过程的膜来源，这一研究将确定 自噬的主要调节剂，代表与年龄相关的治疗干预的其他方法。 在此提案中，我试图了解ATG2将脂质净传递到该的机制 通过解决三个主要问题，自噬体。首先，什么是自噬体的供体膜 生物发生？我建议利用与邻近性标签的无偏见和高分辨率的先端策略 活细胞荧光显微镜以正式识别供体膜。令人惊讶的是，初步数据表明 自噬体生物发生期间，ATG2位于ER出口位点（ER），这是一个高度专业的子域 急诊室。其次，ATG2脂质转移具有内在的方向性，还是依赖于局部膜 能量学？我已经开发了两个体外脂质转移测定法，以模仿ERES-Autophagosome接触位点， 我可以通过大量回合评估脂质转移的方向性和特异性 测量和通过视觉检查。第三，如何将ATG2招募到供体膜？下列的 识别供体膜，我现在可以通过A系统地评估ATG2招募 生化和细胞生物学技术的组合。这项提出的研究将进一步阐明 通过鉴定自噬中的关键参与者的健康衰老的分子机制。