Biochemical Dissection and Reconstitution of Autophagic Membrane Fusion

 描述（由申请人提供）：自噬是一种高度调节的细胞降解系统，将细胞溶质、受损细胞器、蛋白质聚集体和入侵微生物吞噬到称为自噬体的双膜囊泡中，自噬体将货物递送至内溶酶体进行降解。自噬功能障碍与包括癌症在内的多种人类疾病有关。目前还不清楚这一过程是如何在生物化学上进行调节的。自噬中一个尚未解决的问题是自噬体如何与溶酶体融合。遗传分析表明，多个SNARE蛋白在自噬膜融合中的重要作用，然而，这些SNARE是否作为融合因子以及它们的融合活性如何以自噬特异性方式调节尚不清楚。我们发现自噬SNARE Syntaxin 17（STX 17）-SNAP 29-Vamp 8组装成一个具有融合能力的四螺旋束，并作为一个基础融合机制发挥作用，它们在自噬体上的组装以及融合活性都受到自噬体膜结合蛋白ATG 14的促进。ATG 14与STX 17共定位在完整的自噬体上，其中寡聚化的ATG 14与STX 17-SNAP 29 SNARE二元复合物物理相互作用以促进自噬体与溶酶体融合。这种体外调节的膜融合重现了体内应激诱导的自噬体与溶酶体的融合。我们的初步数据还表明，自噬SNARE从自体溶酶体的检索可能是由TECPR 1通过其与STX 17的相互作用介导的。因此，我们假设自噬膜融合是由核心STX 17-SNAP 29-VAMP 8 SNARE复合物和两个SNARE结合蛋白ATG 14和TECPR 1严格控制的。在这项研究中，我们的目的是确定如何ATG 14调节STX 17-SNAP 29-VAMP 8介导的膜融合在我们的国家的最先进的生化和遗传测定（目的1）。自噬蛋白在成熟的自噬体（完全自噬体和自噬溶酶体）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研究自噬体的运输和回收机制（目的2）。最后，我们的研究表明，自噬膜融合受到严格的调控。ATG 14同源寡聚化在介导其与自噬SNARE的结合中起关键作用。我们将利用生物化学和结构生物学方法研究自噬膜束缚/融合的调控机制（目的3）。这些研究将为自噬中膜融合的分子机制提供新的见解，并帮助我们设计新的药物用于治疗自噬功能障碍引起的人类疾病。

 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.