Regulation of Autophagosome Membrane Dynamics by the Atg8 Family of Proteins

Atg8 蛋白家族对自噬体膜动力学的调节

• 批准号: 9239658
• 负责人: Thomas James Melia
• 金额: $ 49.93万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9239658
• 关键词: ATG3 gene; Affinity; Area; Automobile Driving; Autophagocytosis; Autophagosome; Binding; Biological; Biological Models; Cell Line; Cell model; Cells; Chronic; Complex; Cytosol; Dimensions; Encapsulated; Exhibits; Family; Goals; Growth; Image; In Vitro; Infection; Invaded; Knock-out; Lipids; Liposomes; Lumen of the Lysosome; Lysosomes; Maps; Membrane; Membrane Proteins; Microscopy; Modeling; Morphology; Nerve Degeneration; Nutrient; Organelles; Pathology; Pathway interactions; Peptide Hydrolases; Phosphatidylethanolamine; Process; Protein Family; Protein Isoforms; Proteins; Proteome; Publications; Radial; Reaction; Recruitment Activity; Regulation; Resolution; Role; Shapes; Starvation; Structure; System; Tertiary Protein Structure; Testing; Time; Toxin; Ubiquitin Like Proteins; Vesicle; Yeasts; base; biological adaptation to stress; design; flexibility; in vitro Assay; in vivo; interfacial; microorganism; pathogen; protein aggregate; protein protein interaction; reconstitution; response; scaffold; stressor; unilamellar vesicle

Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This “packaging” is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane coordinates the capture of cytosolic toxins with its own expansion and closure is an area of intense study. One factor implicated in both cargo-capture and autophagosome dynamics is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the preautophagosomal membrane and remains bound through the maturation process of the autophagosome. Our preliminary results suggest that Atg8-PE can directly deform the membrane perhaps contributing to the unique cup-like morphology of the immature autophagosome. Further, we show that several proteins driving Atg8 recruitment are designed to recognize unique features of the autophagosome including curvature. By combining these low affinity interactions across multiple proteins in a complex, these proteins would achieve dramatic targeting selectivity for only the transient intermediate in the autophagosome growth. Once cargo-capture is complete and the autophagosome closes, curvature- sensitive components are released. Atg8-PE must also eventually be recycled and we describe how the proteases responsible for Atg8-PE release are also sensitive to the membrane structure and composition. Our discoveries are made possible by two important technological advances. First we have developed a variety of in vitro reconstitution approaches to study how Atg8-PE and other autophagy proteins influence membrane deformation and structure. In particular, we have now reconstituted Atg8-PE formation on Giant Unilamellar Vesicles that comprise both a highly tractable membrane manipulation model and also are large enough to support fluorescent-microscopy based interrogation of protein-membrane organization. Second, we can now image autophagosome intermediate structures at super resolution in three dimensions so that we can now visualize both the cup-like intermediate and its eventual resolution following fission. With this proposal, we expect to demonstrate exactly how Atg8-PE proteins coordinate the dual responsibilities of protein-protein interaction supporting cargo encapsulation with the protein- membrane complexes that shape and close the autophagosome.

宏大学是细胞内应力响应途径，细胞包装部分的部分 用于输送到溶酶体的细胞质。这种“包装”是由从头形成进行的 一种称为自噬体的新细胞器，它生长并封装了胞质材料 最终溶酶体降解。自噬体如何形成，尤其是如何 膜协调捕获胞质毒素与自身膨胀和闭合是一个区域 激烈的研究。在货运和自噬体动力学中实施的一个因素是 泛素样蛋白，ATG8。在自噬期间，ATG8与 磷脂酰乙醇胺（PE）在牙胶膜上，并保持通过 自噬体的成熟过程。 我们的初步结果表明，ATG8-PE可以直接变形膜也许可以造成 未成熟自噬体的独特杯状形态。此外，我们证明了几个 驱动ATG8募集的蛋白质旨在识别自噬体的独特功能 包括曲率。通过将这些低亲和力相互作用结合在复合物中的多种蛋白质中 这些蛋白质将仅对瞬态中间体实现戏剧性的靶向选择性 自噬体增长。一旦货物捕获完成并且自噬体关闭，曲率 - 敏感组件已释放。 ATG8-PE有时还必须回收利用，我们描述了如何 负责ATG8-PE释放的蛋白酶也对膜结构和 作品。通过两个重要的技术进步使我们的发现成为可能。首先 已经开发了各种体外重构方法来研究ATG8-PE和其他 自噬蛋白会影响膜的变形和结构。特别是，我们现在有 在巨型Unilamellar囊泡上重构的ATG8-PE形成，这两个都可以完成高度拖动 膜操纵模型，并且足够大，可以支持基于荧光的微观镜检查 蛋白质膜组织的询问。第二，我们现在可以图像自噬体 超级分辨率的中间结构在三个维度上，因此我们现在可以看到两个 裂变后杯状中间体及其最终分辨率。 通过此提案，我们希望准确地证明ATG8-PE蛋白如何协调双重 蛋白质 - 蛋白质相互作用的责任支持货物封装与蛋白质 膜复合体形成并关闭自噬体。