细胞自噬体，溶酶体及内质网的相互作用及调控

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. Key questions need to be answered that :1)how autophagosome fuses with lysosome with high efficiency and specificity? 2) how autophagasome-autolysosome-ER interplay in this process. We found that autophagic SNAREs Syntaxin17 (STX17)-SNAP29-VAMP8 assemble into a fusion competent four-helices bundle and function as a basal fusion machinery, their assembly on autophagosomes, as well as the fusogenic activity, is promoted by autophagosome membrane binding protein ATG14, as well as other autophagy specific proteins and protein complexes. Besides, we found that a crucial innate immune regulator STING, could sequest STX17 on ER and block autophagy, and our preliminary data also suggested that the retrieval of autophagic SNAREs from autolysosomes is likely mediated by TECPR1 through its interaction with STX17. We therefore hypothesize that autophagic membrane fusion is tightly controlled by the core STX17-SNAP29-VAMP8 SNARE complex and SNARE-binding regulatory proteins. In this study, we aim to determine how STX17-SNAP29-VAMP8 mediated membrane fusion is regulated in our state-of-the-art biochemical and genetic assays. We will also utilize autophagic fusogenic activites as readouts to study STING or TECPR1 involved mechanisms of trafficking to and retrieving away from mature autophagosomes by comprehensive cell biology and biochemical approaches. At last, we will use mice models to define novel functions of STING or TECPR1 in autophagy regulated energy metabolism. These studies will provide new insights into key molecular mechanisms of membrane fusion and regulation in autophagy and help us to design new classes of drugs for the treatment of human diseases caused by autophagy dysfunction.

细胞自噬是细胞中精密调控的代谢途径，由双层膜结构的自噬体包裹胞质，受损细胞器及累积蛋白，并运送至溶酶体降解。自噬功能异常与肿瘤等多种人类重大疾病过程相关。细胞自噬涉及多个细胞器的相互作用，但其机制并不清楚。结合先进的生物化学和生物物理学研究手段，我们发现自噬相关SNAREs STX17-SNAP29-VAMP8构成四束螺旋状结构并以此作为基本功能单元介导膜融合并可能受到一系列蛋白调控。本课题研究中，我们计划使用先进的生化及遗传学研究手段进一步揭示自噬特异蛋白对SNARE介导的膜融合过程的调控机制；在生化水平和细胞水平研究内质网蛋白介导的自噬SNARE由内质网至自噬体的转运机制及对机体能量代谢的影响；并阐明自噬溶酶体蛋白介导的自噬SNARE回收机制。本研究的顺利完成将全面揭示自噬过程中自噬体，溶酶体与内质网的相互作用关系，并提供自噬相关疾病治疗新策略。

自噬体和溶酶体的融合是一个关键的细胞器互作过程，包括了SNAREs融合机器的转运与招募，膜性细胞器的物理拉近和膜脂质层的融合，内容物的交换，以及膜融合后SNARE蛋白复合物的解聚回收。围绕自噬中这一重要的细胞器互作过程，我们开展了系统性的研究，揭示了STING调控STX17由内质网转运至自噬体膜的机制及其对自噬的影响；揭示了C9orf72-Rab39A-HOPS促进自噬体-溶酶体融合的机制；揭示了STXBP5调控SNARE复合物解聚影响自噬流的机理。同时，我们成功生化重建了自噬体和溶酶体融合这一细胞器互作过程，为了解自噬的生理病理功能提供重要基础，并为整个“膜运输”领域提供有意义的借鉴。