Biochemical Dissection and Reconstitution of Autophagic Membrane Fusion

自噬膜融合的生化解剖与重建

• 批准号: 9004982
• 负责人: Qing Zhong
• 金额: $ 31.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9004982
• 关键词: Aging; Autophagocytosis; Autophagosome; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biogenesis; Biological Assay; Cellular biology; Chemicals; Chimeric Proteins; Communicable Diseases; Complex; Cytosol; Data; Disease; Dissection; Foundations; Functional disorder; Genes; Genetic; Homo; In Vitro; Invaded; Knowledge; Lysosomes; Malignant Neoplasms; Mediating; Membrane; Membrane Fusion; Membrane Protein Traffic; Metabolic Diseases; Molecular; Nerve Degeneration; Organelles; Paper; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Play; Post-Translational Protein Processing; Process; Proteins; Publishing; Recruitment Activity; Recycling; Retrieval; Role; SNAP receptor; Staging; Stress; Structure; System; Vertebrates; Vesicle; Yeasts; design; drug discovery; genetic analysis; human disease; in vivo; insight; microorganism; novel; protein aggregate; protein complex; protein protein interaction; public health relevance; reconstitution; structural biology; trafficking

 DESCRIPTION (provided by applicant): Autophagy is a highly regulated cellular degradation system that engulfs cytosol, damaged organelles, protein aggregates and invading microorganisms into a double-membrane vesicle termed autophagosome that delivers cargoes to endolysosomes for degradation. Dysfunction of autophagy has been implicated in a broad spectrum of human diseases including cancers. It is still largely unknown how this process is regulated biochemically. One unsolved question in autophagy is how autophagosome fuses with lysosome. Genetic analysis suggests important roles of multiple SNARE proteins in the autophagic membrane fusion, however, whether these SNAREs function as fusogens and how their fusogenic activities are regulated in an autophagy specific manner is unknown. We found that autophagic SNAREs Syntaxin17 (STX17)-SNAP29-Vamp8 assemble into a fusion competent four-helices bundle and functions as a basal fusion machinery, their assembly on autophagosomes, as well as the fusogenic activity, is promoted by autophagosome membrane binding protein ATG14. ATG14 colocalizes with STX17 on the complete autophagosome where the oligomerized ATG14 physically interacts with STX17-SNAP29 SNARE binary complex to promote autophagosome fusion with lysosome. This regulated membrane fusion in vitro recapitulates stress induced autophagosome fusion with lysosome in vivo. Our preliminary data also suggest that the retrieval of autophagic SNAREs from autolysosomes is likely mediated by TECPR1 through its interaction with STX17. We therefore hypothesize that the autophagic membrane fusion is tightly controlled by the core STX17-SNAP29-VAMP8 SNARE complex and two SNARE-binding proteins ATG14 and TECPR1. In this study, we aim to determine how ATG14 regulates STX17-SNAP29-VAMP8 mediated membrane fusion in our state-of-the-art biochemical and genetic assays (Aim 1). The recruitment and retrieval of autophagic proteins on mature autophagosomes (complete autophagosome and autolysosome) is scarcely studied. We will utilize the autophagic fusogenic proteins as readouts to study the mechanism of the trafficking to and the retrieving away from the mature autophagosomes by comprehensive cell biology and biochemical approaches (Aim 2). At last, our study suggests that autophagic membrane fusion is tightly regulated. ATG14 homo-oligomerization plays a crucial role in mediating in its binding to autophagic SNAREs. We will utilize biochemical and structural biology approaches to investigate the regulatory mechanism in autophagic membrane tethering/fusion (Aim 3). These studies will provide new insights into the molecular mechanism of membrane fusion in autophagy and help us to design new classes of drugs for the treatment of human diseases caused by autophagy dysfunction.

 描述(申请人提供)：自噬是一种高度调控的细胞降解系统，它将细胞质、受损的细胞器、蛋白质聚集体和入侵的微生物吞噬到称为自噬小体的双层膜泡中，将货物运送到内溶体进行降解。自噬功能障碍与包括癌症在内的多种人类疾病有关。在很大程度上，人们仍然不知道这一过程是如何通过生物化学来调节的。自噬中一个悬而未决的问题是自噬小体与溶酶体是如何融合的。遗传分析表明，多个SNARs蛋白在自噬膜融合中发挥重要作用，然而，这些SNARs是否作为融合原发挥作用，以及它们的融合活性如何以自噬特异的方式调节尚不清楚。我们发现，自噬SNARs Synaxin17(STX17)-SNAP29-Vamp8组装成一个可融合的四螺旋束，并作为基础融合机制发挥作用，它们在自噬体膜结合蛋白ATG14的促进下在自噬体上的组装以及融合活性。ATG14与STX17共定位在完整的自噬小体上，寡聚化的ATG14与STX17-SNAP29 SNARE二元复合体物理作用，促进自噬小体与溶酶体的融合。这种体外调节的膜融合概括了应激诱导的自噬小体与体内溶酶体的融合。我们的初步数据还表明，自溶酶体中自噬陷阱的恢复可能是由TECPR1通过其与STX17的相互作用来调节的。因此，我们假设自噬膜融合受到核心STX17-SNAP29-VAMP8 SNARE复合体和两个SNARE结合蛋白ATG14和TECPR1的严格控制。在这项研究中，我们旨在确定ATG14如何在我们最先进的生化和遗传检测中调节STX17-SNAP29-VAMP8介导膜融合(目标1)。自噬蛋白在成熟的自噬小体(完整的自噬小体和自溶酶体)上的募集和回收几乎没有研究。我们将利用自噬融合蛋白作为读数，通过综合的细胞生物学和生物化学方法来研究成熟自噬小体的转运和回收机制(目标2)。最后，我们的研究表明，自噬膜融合受到严格的调控。ATG14同源寡聚在介导ATG14与自噬陷阱结合过程中起着至关重要的作用。我们将利用生化和结构生物学的方法来研究自噬膜系留/融合的调控机制(目标3)。这些研究将为自噬中膜融合的分子机制提供新的见解，并帮助我们设计治疗自噬功能障碍引起的人类疾病的新型药物。