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

项目摘要/摘要 巨噬细胞自噬(以下简称自噬)是一种细胞降解过程，与 衰老的过程。自噬通过传递潜在的细胞毒性来维持细胞健康和动态平衡 货物通过双膜自噬小体的从头形成到达溶酶体。 随着年龄的增长，这一过程会受到损害。此外，核心自噬基因的缺失已经被 在多个生物体中显示出减少寿命，而救援实验相反地恢复了完整的寿命。 自噬对于维持长期存活的神经元的健康和自噬结果的缺陷尤为关键 在各种神经退行性疾病中，包括帕金森氏病、阿尔茨海默病和肌营养不良 侧索硬化症。自噬蛋白ATG2对这一过程和对该蛋白的组织特异性消融是必不可少的 导致寿命、活力和与年龄相关的组织退化。我们最近证明了 ATG2是一种具有新型结构的脂类转移蛋白，可实现大批量脂类转运。这项活动是必不可少的 用于自噬生物发生。我预测ATG2将脂类直接输送到新生的自噬小体中 展开生长的膜。到目前为止，供脂细胞器的身份尚不清楚。有趣的是， 在哺乳动物生物学中，一种在一个方向上传递脂类净转移的机制是没有明确先例的。 由于这一机制可能取决于两种膜的生物物理性质，我建议正式 确定ATG2从中提取脂质的供体膜。该项目旨在提供培训 为了在未来的独立研究中实现职业生涯所必需的。此外，正如本项目将阐明的 这一关键的延缓衰老的细胞生物学过程的膜来源，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。 在这项提议中，我试图了解ATG2将脂质净转移到 通过解决三个主要问题来实现自噬。首先，自噬的供体膜是什么？ 生物起源？我建议利用无偏见和高分辨率的邻近标记APEX策略 活细胞荧光显微镜正式鉴定供体膜。引人注目的是，初步数据显示， ATG2在自噬小体生物发生过程中驻留在内质网出口部位(ERE)，自噬小体生物发生是 急诊室。第二，ATG2脂转移是具有固有的方向性，还是依赖于局部膜 精力充沛？我已经开发了两种体外脂转移试验来模拟ERES-自噬小体接触部位， 通过它，我可以评估通过整体集成进行脂质转移的方向性和特异性。 测量和目视检查。第三，ATG2是如何被招募到供体膜上的？跟随 对供体膜的鉴定，我现在可以系统地评估ATG2的招募 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 通过确定自噬中的一个关键角色来研究健康衰老的分子机制。

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.