肿柄菊内酯C诱导溶酶体生成分子探针的构建及分子作用机制研究

Lysosomes are single-membrane organelles that serve as major sites for degradation and cell signaling transduction within the cells. Appropriate biogenesis and homeostasis of lysosomes are critical to a variety of cellular activities. Ample studies have focused on the function and degradation mechanisms of lysosomes. However, the mechanisms of biogenesis and homeostasis of lysosomes and lysosomal response to diverse cellular signals are far from being elucidated. Chemical biology provides unique approaches to investigate the lysosome-mediated signaling pathways and cellular responses. Based on our screening assay to identify lysosome-inducing small molecular chemicals from a pool of natural compounds purified from plants, we found that tagitinin C promoted the biogenesis of lysosomes. What’s more, tagitinin C promoted nuclear translocation of both TFEB and TFE3, two critical transcriptional factors in the lysosome biogenesis pathway, and further promoted the autophagy by upregulating autophagy related genes. In this project, we aim at 1)to design the molecular probe of tagitinin C, which help bait the candidate interacting protein by immunoprecipitation, to investigate the underlying mechanisms of how tagitinin C interfere with lysosome-mediated cellular activities, such as degradation of autophagosomes; 2) to optimize the chemical by structure modification to improve their effectiveness, based on which we aim to develop new drugs that can be applied to treat lysosome-related diseases including lysosomal storage disease, neuron degenerative disorders. For aim 1, I plan to identify target proteins and/or signaling pathways of tagitinin C to elucidate how tagitinin C functions to promote the lysosome biogenesis within cells, using a combination of approaches including the established lysosome-based cell system, protein-protein interaction, live-cell imaging, protein modifications. For aim 2, I plan to further investigate the functional groups of tagitinin C based on its chemical structure, and using chemical modifications to optimize the chemical for improved specificity and efficacy. These biological and chemical studies will provide important insights into lysosome biogenesis and homeostasis, as well as novel therapy target for lysosome-related pathogenesis.

溶酶体是细胞内物质降解的主要细胞器，在细胞的物质降解和代谢信号控制方面具有举足轻重的地位。尽管人们对溶酶体功能和降解作用机制的认识在不断加强，但溶酶体的生物发生对细胞响应不同外界刺激的机制还远未阐明。基于前期促进溶酶体生成活性筛选，发现倍半萜类化合物肿柄菊内酯C能显著的增加细胞溶酶体的数量，以及促进转录因子TFEB 和TFE3入核起始溶酶体及自噬相关基因的表达。本项目旨在通过结构修饰深入探讨肿柄菊内酯C化学结构与促进溶酶体生成之间的构效关系，构建肿柄菊内酯C的小分子探针，以化学生物学的研究方法解析肿柄菊内酯C的靶蛋白以及溶酶体的调控机制；另一方面利用活细胞成像、蛋白亚细胞定位、蛋白质修饰、蛋白质互作等多种手段，研究促进溶酶体介导的细胞自噬等过程，通过促进溶酶体的生物发生的调控，为溶酶体贮积症及神经退行性疾病等相关疾病的提供新的治疗靶点。

溶酶体（lysosome）是细胞内物质降解的主要场所，在细胞的多种生命活动包括生长、代谢、分化、稳态维持及功能执行等方面起着至关重要的作用。针对溶酶体生物发生、抑制溶酶体酸化及维持溶酶体稳态调控的小分子化合物的筛选和机制研究具有重要的意义。结合前期建立的线虫和细胞筛选模型，共筛选天然小分子化合物1000余个，包括生物碱、萜类、甾体类、黄酮类、蒽醌类等多种骨架化合物。筛选出抑制溶酶体酸化化合物1类共4个活性化合物；促进溶酶体生物发生化合物9类68个活性化合物：其中包括7类化合物激活转录因子TFEB和TFE3；2类化合物促进溶酶体生成但不依赖转录因子和转录抑制因子。从化学生物学研究策略入手，选择活性强、靶向性好的活性化合物，构建了Htg-3活性小分子的光亲和标记物，并经实验证实其与母核化合物具有相似的生物学功能，并检测了潜在靶蛋白。Hdj-15可以显著诱导内源性ROS在细胞内的积累、细胞凋亡、自噬发生以及溶酶体生物发生。ROS是Hdj-15下游表型（凋亡、自噬及TFEB表达）调控的关键点，当清除掉ROS后，下游表型会极大的回复。Hdj-15可激活IRE1-XBP1-TFEB/TFE3转录激活转录因子TFEB和TFE3；另外还激活ROS-TRPML1-Calcineurin去磷酸化TFEB/TFE3，从而两条通路的激活，促进了溶酶体生成和细胞自噬。通过系统的研究揭示了溶酶体生物发生和功能维持的调控新机制，同时利用肿瘤细胞毒性活性化合物影响溶酶体功能的筛选及其作用机制研究，探索了溶酶体稳态调控机制与细胞毒性之间的相关性及其规律性，发现了具有新颖作用机制的溶酶体相关疾病和抗肿瘤的先导化合物。