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: 10051183
• 负责人: Thomas James Melia
• 金额: $ 51.75万
• 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/10051183
• 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; established cell line; falls; flexibility; microorganism; pathogen; progenitor; protein aggregation; reconstitution; recruit; response; stressor; unilamellar vesicle; vector

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.

宏自噬是细胞内应激反应途径，细胞通过该途径包装部分 胞质溶胶输送到溶酶体中，这种“包装”是通过从头形成的。 一种称为自噬体的新细胞器，它生长并封装胞质物质，以最终实现 溶酶体降解。自噬体如何形成，特别是膜如何形成。 协调捕获胞质毒素与其自身的扩张和闭合是一个强烈的区域 研究中涉及货物捕获和自噬体动力学的一个因素是泛素样。 蛋白质 Atg8 在自噬过程中，Atg8 与磷脂酰乙醇胺 (PE) 共价结合。 位于自噬体前膜上，并在自噬体的成熟过程中保持结合 自噬体。 我们的初步结果表明 Atg8-PE 装饰了最早的自噬体膜 祖结构并在组织膜的积累中发挥着不可或缺的作用 预示着自噬体本身的最终生长。 Atg8-PE 将单个囊泡束缚到底层支持物中，至关重要的是，Atg8 必须保持关联。 我们的结果现在也描述了自噬体的整个生长过程。 该 Atg8-PE 库受到保护，免受回收蛋白酶的影响，否则这些蛋白酶将发挥组成型作用 两项重要的技术使我们的发现成为可能。 首先，我们开发了多种体外重构方法来研究 Atg8- 的作用。 PE 和其他自噬蛋白影响膜变形和结构。 能够在三个维度上以超分辨率对自噬体中间结构进行成像 识别微米深电子断层扫描中的相同结构，揭示最早的关键特征 Atg8 修饰的膜。 通过这个提议，我们希望准确地证明 Atg8-PE 蛋白如何组织蛋白质和 支持自噬体膜扩张的膜。