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

基本信息

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
项目类别：
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

项目摘要

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最终溶酶体降解。自噬体如何形成，特别是如何形成。膜通过其自身的扩张和闭合来协调胞质毒素的捕获，是一个区域与货物捕获和自噬体动力学有关的一个因素是深入研究的。泛素样蛋白 Atg8 在自噬过程中，Atg8 与它共价结合。磷脂酰乙醇胺（PE）位于自噬体前膜上，并通过自噬体的成熟过程。我们的初步结果表明 Atg8-PE 可以直接使膜变形，这可能有助于未成熟自噬体独特的杯状形态进一步，我们展示了一些。驱动 Atg8 募集的蛋白质旨在识别自噬体的独特特征通过将多个蛋白质之间的这些低亲和力相互作用结合在一个复合物中，这些蛋白质将仅对瞬时中间体实现显着的靶向选择性一旦货物捕获完成并且自噬体关闭，曲率- Atg8-PE 最终也必须被释放，我们描述了如何回收。负责 Atg8-PE 释放的蛋白酶也对膜结构敏感，并且我们的发现得益于两项重要的技术进步。开发了多种体外重构方法来研究 Atg8-PE 和其他自噬蛋白尤其影响膜的变形和结构。在巨型单层囊泡上重建 Atg8-PE 结构，该囊泡包含高度易处理的膜操纵模型也足够大以支持基于荧光显微镜的其次，我们现在可以对自噬体进行成像。三维超分辨率的中间结构，以便我们现在可以可视化杯状中间体及其裂变后的最终分解。通过这个提议，我们期望准确地证明 Atg8-PE 蛋白如何协调双蛋白质-蛋白质相互作用的责任支持货物封装与蛋白质- 形成和关闭自噬体的膜复合物。