Autophagosome closure by the ESCRT machinery

通过 ESRT 机制封闭自噬体

• 批准号: 10383918
• 负责人: HONG-GANG WANG
• 金额: $ 7.5万
• 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/10383918
• 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 在巨型自噬(以下称为自噬)过程中，一个新月形的膜被称为 噬菌体伸长，吞噬受损的货物并密封，从而产生一种双层膜小泡，称为 自噬小体。而调控自噬诱导和吞噬载体的许多核心机制 成核和伸长已经确定，吞噬菌体关闭的机制在很大程度上是未知的。这个 本应用程序的目标是定义吞噬菌体海豹产生 完整的自噬小体。噬菌体关闭是自噬的关键步骤，自噬是溶酶体所必需的 自食性货物的融合和高效降解和回收。对吞噬细胞的封存被误导了 被描述为膜融合事件的时候，内部和外部产生的过程 自噬体膜实际上需要膜的断裂。值得注意的是，噬菌体闭合的拓扑结构 类似于ESCRT(运输所需的内体分选复合体)介导的分裂，在这种情况下 ESCRT机器从膜颈部内介导膜分裂。而ESCRT缺陷 在线虫、果蝇和哺乳动物中积累未成熟的自噬结构，分子 对这种表型的理解还不清楚。利用自噬小体中的LC3的拓扑结构 生物发生学，我们建立了一种新的基于HaloTag-LC3的方法来区分吞噬体、自噬小体 和自溶酶体。利用这一检测方法，我们已经确定ESCRT-III是吞噬细胞的候选调节因子 结案了。我们假设吞噬相关因子组装了一种新的适配器用于招募和 用于噬菌体关闭的ESCRT-III细丝的组装。我们将在以下具体实例中测试我们的假设 目标：(1)定义吞噬菌体关闭所需的核心ESCRT机制；(2)确定上游 在噬菌体关闭过程中，作为ESCRT-III组装的自噬特异性接头的因子(S)。