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.

大自噬是细胞内应激反应途径，细胞通过该途径包装部分 细胞溶质用于递送到溶酶体中。这种“包装”是通过重新形成 一种叫做自噬体的新细胞器， 最终的溶酶体降解。自噬体是如何形成的，尤其是 细胞膜通过其自身的扩张和闭合来协调细胞溶质毒素的捕获， 紧张的学习。与货物捕获和自噬体动力学有关的一个因素是 泛素样蛋白Atg 8。在自噬过程中，Atg 8与 磷脂酰乙醇胺（PE）的前自噬体膜上，并保持结合，通过 自噬体的成熟过程。 我们的初步结果表明，Atg 8-PE可以直接使膜变形，这可能有助于 未成熟自噬体独特的杯状形态。此外，我们还发现， 驱动Atg 8募集的蛋白质被设计成识别自噬体的独特特征 包括曲率。通过将这些低亲和力的相互作用结合在复合物中的多个蛋白质上， 这些蛋白质将仅对细胞中的瞬时中间体实现显著的靶向选择性， 自噬体生长一旦货物捕获完成，自噬体关闭，曲率- 释放敏感成分。Atg 8-PE也必须最终回收，我们描述如何 负责Atg 8-PE释放的蛋白酶也对膜结构敏感， 混合物.两项重要的技术进步使我们的发现成为可能。首先我们 已经开发了多种体外重建方法来研究Atg 8-PE和其他细胞如何在体外重建。 自噬蛋白影响膜的变形和结构。特别是，我们现在 在巨大单层囊泡上重建的Atg 8-PE形成，其包含高度易处理的 膜操作模型，也足够大，以支持基于荧光显微镜 蛋白质膜组织的询问。第二，我们现在可以成像自噬体， 在三维空间中以超分辨率显示中间结构，这样我们现在就可以看到 杯状中间体及其在裂变后的最终分解。 有了这个提议，我们希望能确切地证明Atg 8-PE蛋白如何协调双链蛋白。 蛋白质-蛋白质相互作用的责任，支持货物与蛋白质的包封- 形成并关闭自噬体的膜复合物。