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: 10312028
• 负责人: Thomas James Melia
• 金额: $ 49.75万
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312028
• 关键词: 3-Dimensional; Architecture; Area; Autophagocytosis; Autophagosome; Binding; Biogenesis; Cell Line; Cells; Characteristics; Complement; Cytosol; Electrons; Encapsulated; Event; Excision; Exhibits; Family; Family member; Goals; Growth; Image; In Vitro; Individual; Invaded; Knock-out; Lipids; Lumen of the Lysosome; Lysosomes; Mediating; Membrane; Morphology; Nerve Degeneration; Nutrient; Organelles; Pathology; Pathway interactions; Peptide Hydrolases; Phase; Phosphatidylethanolamine; Phosphorylation; Phosphoserine; Physiologic pulse; Play; Process; Protein Family; Proteins; Recycling; Regulation; Resistance; Resolution; Role; Seeds; Site; Source; Starvation; Structure; System; Tomogram; Toxin; Ubiquitin Like Proteins; Vesicle; Yeasts; biological adaptation to stress; chronic infection; delivery vehicle; established cell line; falls; flexibility; microorganism; pathogen; progenitor; protein aggregation; reconstitution; recruit; response; 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 decorates the earliest autophagosome membrane progenitor structure and plays an integral role in organizing the accumulation of membranes that presages the eventual growth of the autophagosome itself. These activities depend upon the ability of Atg8-PE to tether individual vesicles into an underlying support. Critically, Atg8 must remain associated with the membranes throughout the growth of the autophagosome. Our results now also describe how this pool of Atg8-PE is protected from recycling proteases that otherwise function to constitutively remove Atg8 at other sites. 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. Second, we are now able to image autophagosome intermediate structures at super resolution in three dimensions and identify the same structures in micron deep electron tomograms revealing key features of the earliest Atg8-decorated membranes. With this proposal, we expect to demonstrate exactly how Atg8-PE proteins organize the proteins and membranes that support autophagosome membrane expansion.

宏自噬是细胞内的应激反应途径，细胞通过它包装部分 胞浆输送到溶酶体中的胞浆。这种“包装”是由从头形成的 一种名为自噬小体的新细胞器，它生长并包裹胞液物质，最终 溶酶体降解。自噬小体是如何形成的，尤其是膜如何形成 协调捕获胞质毒素与其自身的扩展和关闭是一个强烈的领域 学习。在货物捕获和自噬小体动力学中涉及的一个因素是泛素样蛋白 蛋白质，Atg8。在自噬过程中，ATG8与磷脂酰乙醇胺(PE)共价结合 在自噬前膜上，并在成熟过程中保持结合 自噬小体。 我们的初步结果表明，Atg8-PE修饰了最早的自噬体膜 祖细胞结构，并在组织膜的积累方面起着不可或缺的作用， 预示着自噬小体本身的最终生长。这些活动取决于 ATG8-PE将单个囊泡拴在下面的支撑物上。至关重要的是，Atg8必须保持关联 在自噬小体的整个生长过程中都有膜。我们的结果现在也描述了 这个Atg8-PE池受到保护，不能回收本来起到结构性作用的蛋白酶 移除其他站点的Atg8。我们的发现是通过两项重要的技术实现的 预付款。首先，我们开发了各种体外重建方法来研究Atg8是如何- PE和其他自噬蛋白影响膜的变形和结构。第二，我们现在 能够以超分辨率对自噬中间结构进行三维成像 在微米深电子断层图像中发现相同的结构，揭示了最早的 ATG8修饰的膜。 通过这项提议，我们希望确切地展示Atg8-PE蛋白质是如何组织蛋白质和 支持自噬体膜扩张的膜。