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.

项目总结/摘要 巨自噬（Macroautophagy）（下文称为自噬）是与细胞内的细胞因子密切相关的细胞降解过程。 老化的过程。自噬通过递送潜在的细胞毒性物质来维持细胞健康和稳态。 通过重新形成双膜自噬体将货物运送到溶酶体。 这个过程随着年龄的增长而减弱。此外，核心自噬基因的缺失已经被证实是一种重要的基因突变。 在多种生物体中显示，拯救实验会缩短寿命，而拯救实验则相反地恢复完整的寿命。 自噬在维持长寿神经元的健康方面尤其重要， 在各种神经退行性疾病中，包括帕金森病、阿尔茨海默病和肌萎缩性疾病， 侧索硬化自噬蛋白ATG 2是这一过程和该蛋白的组织特异性消融所必需的 导致寿命、运动性和与年龄相关的组织退化降低。我们最近证明， ATG 2是一种脂质转移蛋白，具有允许大量脂质递送的新结构。这项活动至关重要 自噬体的生物合成。我预测ATG 2将脂质直接递送到新生的自噬体中， 扩大生长膜。到目前为止，脂质捐赠细胞器的身份尚不清楚。有趣的是， 在哺乳动物生物学中，在一个方向上传递脂质净转移的机制没有明确的先例。 由于这种机制可能取决于两种膜的生物物理性质，我建议正式 鉴定ATG 2从中提取脂质的供体膜。该项目旨在提供培训 在未来的职业生涯中实现独立研究是必要的。此外，正如本项目将阐明的那样， 这一关键的延缓衰老细胞生物过程的膜源，这一研究机构将确定 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。 在这个提议中，我试图了解ATG 2将脂质净转移到细胞中的机制。 通过解决三个主要问题来研究自噬体。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， 活细胞荧光显微镜以正式识别供体膜。引人注目的是，初步数据显示， ATG 2在自噬体生物发生过程中位于ER出口位点（ERES），这是自噬体的一个高度特化的亚结构域。 急诊室第二，ATG 2脂质转移是否具有内在的方向性，还是依赖于局部膜 能量学？我已经开发了两种体外脂质转移试验来模拟ERES-自噬体接触位点， 通过它我可以评估脂质通过整体集合转移的方向性和特异性 测量和目视检查。第三，ATG 2是如何被募集到供体膜上的？以下 通过鉴定供体膜，我现在可以系统地评估ATG 2的募集， 生物化学和细胞生物学技术的结合。这项拟议的研究将进一步阐明 通过识别自噬的关键参与者来研究健康衰老的分子机制。