Autophagosome closure by the ESCRT machinery

通过 ESRT 机制封闭自噬体

• 批准号: 10132346
• 负责人: HONG-GANG WANG
• 金额: $ 31.22万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-09-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10132346
• 关键词: ATP phosphohydrolase; Aging; Autophagocytosis; Autophagosome; Biogenesis; Biological Assay; Caenorhabditis elegans; Cell Membrane Permeability; Complex; Data; Defect; Drosophila genus; Endosomes; Event; Filament; Goals; Heart Diseases; Homeostasis; Immunity; Left; Libraries; Ligands; Lipids; Liver diseases; Lysosomes; Malignant Neoplasms; Mammals; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Monitor; Neck; Nerve Degeneration; Nuclear Envelope; Nutrient; Pathogenesis; Peptides; Phenotype; Phosphatidylethanolamine; Process; Proteins; Recycling; Reporter; Role; Small Interfering RNA; Sorting - Cell Movement; Starvation; Stress; Structure; Testing; Vesicle; base; human disease; novel; polymerization; recruit; response; screening; seal; tetramethylrhodamine

Project Summary/Abstract During macroautophagy (hereafter referred to as autophagy), a crescent-shaped membrane known as the phagophore elongates to engulf damaged cargo and seals to generate a double-membrane vesicle called the autophagosome. While much of the core machinery regulating autophagy induction and phagophore nucleation and elongation has been identified, the machinery for phagophore closure is largely unknown. The goal of this application is to define the molecular mechanism by which the phagophore seals to generate the completed autophagosome. Phagophore closure is a critical step in autophagy that is required for lysosomal fusion and efficient degradation and recycling of autophagic cargo. The sealing of phagophores has misguidedly been described as a membrane fusion event when the process of generating the inner and outer autophagosomal membrane actually requires membrane scission. Notably, the topology of phagophore closure is similar to ESCRT (endosomal sorting complexes required for transport)-mediated scission, in which the ESCRT machinery mediates membrane fission from within the membrane neck. While ESCRT defects accumulate immature autophagosomal structures in C. elegans, Drosophila and mammals, a molecular understanding for the phenotype remains unclear. Exploiting the topology of LC3 during autophagosome biogenesis, we established a novel HaloTag-LC3 based assay to differentiate phagophores, autophagosomes and autolysosomes. Using this assay, we have identified ESCRT-III as a candidate regulator of phagophore closure. We hypothesize that phagophore-associated factors assemble a novel adaptor for the recruitment and assembly of ESCRT-III filaments for phagophore closure. We will test our hypotheses in the following Specific Aims: (1) to define the core ESCRT machinery required for phagophore closure; (2) to identify the upstream factor(s) that serve as an autophagy-specific adaptor for ESCRT-III assembly during phagophore closure.

项目摘要/摘要 在大型噬菌学（以下称为自噬）中，一种新月形的膜被称为 吞噬孔拉伸以吞噬损坏的货物和密封以产生一个称为的双膜囊泡 自噬体。而大部分核心机械调节自噬诱导和吞噬作用 已经确定了成核和伸长率，吞噬闭合的机械在很大程度上尚不清楚。这 该应用的目的是定义吞噬器密封以生成的分子机制 完成的自噬体。吞噬作用是自噬的关键步骤，这是溶酶体所需的 自噬货物的融合和有效的降解和回收利用。吞噬器的密封具有 当产生内部和外部的过程时，误导性被描述为膜融合事件 自噬体膜实际上需要膜分裂。值得注意的是，关闭吞噬的拓扑 类似于ESCRT（运输所需的内体分类络合物），其中介导的分裂，其中 ESCRT机械从膜颈内介导膜裂变。而ESCRT缺陷 在秀丽隐杆线虫，果蝇和哺乳动物中积累未成熟的自噬体结构，一种分子 对表型的理解尚不清楚。在自噬体期间利用LC3的拓扑 生物发生，我们建立了一种新型的基于Halotag-LC3的测定法，以区分吞噬作用，自噬体 和自溶性。使用该测定法，我们将ESCRT-III确定为吞噬器的候选调节剂 关闭。我们假设与吞噬相关的因素组装了一个新型适配器，用于招募和 用于吞噬闭合的ESCRT-III丝组装。我们将在以下特定中测试我们的假设 目的：（1）定义吞噬闭合所需的核心ESCRT机械； （2）识别上游 在吞噬过程闭合期间用作ESCRT-III组件的自噬特异性适配器的因子。