Vici综合症蛋白EPG5协同调控内吞运输和自噬通路的机制研究

The Vici syndrome protein EPG5 functions in both the autophagy and endocytic trafficking pathways. Autophagy involves the enclosure of cytoplasmic material in the autophagosome and its subsequent delivery to the lysosome for degradation. Endocytic trafficking internalizes the extracellular materials and membrane proteins on cell surface within endosomes, which are subsequently transported between the membrane organelles in cytosol. Recent studies indicate a complex interplay between autophagy and endocytic trafficking. Endosomes contribute to membrane source for autophagosome formation, and are also involved in autophagosome maturation. Autophagy affects the efficiency of endocytic trafficking and some autophagy genes participate in the regulation of endocytic trafficking. The coordinate regulation of the autophagy and endocytic trafficking pathways are essential for the development and health of organisms. Dysfunction of both pathways causes a series of metabolic diseases. EPG5, depletion of which causes defects in autophagy and endocytic trafficking, couples the two pathways. The function of EPG5 in autophagy has been extensively studied. However, little is known about the function of EPG5 in the endocytic trafficking pathway. In this proposal, we will investigate the role of EPG5 in endocytic trafficking using C. elegans and mammalian cells. The molecular mechanism by which EPG5 coordinately regulates autophagy and endocytic trafficking will be investigated. These studies will help us to understand the physiological function of EPG5 in the autophagy and endocytic trafficking pathways and also provide insights into how dysfunction of EPG5 leads to the development of related metabolic diseases.

Vici综合症蛋白EPG5在自噬和内吞运输中同时发挥重要作用。自噬是通过形成自噬小体包裹底物运送到溶酶体降解的过程。内吞运输是将胞外物质和胞膜蛋白转入细胞质并在膜细胞器间转运的过程。自噬和内吞运输之间密切相关：一方面内体为自噬小体形成提供膜来源，并帮助自噬小体成熟；另一方面自噬影响内吞运输效率，并且一些自噬基因在内吞运输中发挥功能。自噬和内吞运输的协同调控对于机体发育和健康至关重要，两条通路的异常都会导致多种疾病的发生。EPG5对于维持两条通路的有序运行必不可少。目前对EPG5的功能和机制研究主要集中在自噬方面，而在内吞运输中的作用完全不清楚。本项目中，我们将利用线虫遗传学、细胞生物学、生物化学等手段，研究EPG5在内吞运输中的作用，并阐述EPG5协同调控内吞运输和自噬通路的分子机制。这些研究将加深我们对EPG5在生理和疾病中功能的理解，并为相关代谢疾病的发生发展及治疗提供重要的理论依据。

EPG5基因突变或功能缺失导致多系统紊乱疾病Vici综合征，前期研究表明EPG5以tether蛋白的方式介导自噬小体与晚期内体/溶酶体的特异性融合，以调控自噬通路活性。EPG5同样在内吞运输通路中发挥重要功能，但具体的作用方式和分子机制尚不清楚。本项目研究了EPG5在内吞运输中的作用，发现EPG5直接参与逆行性内吞运输和基底外侧内吞循环过程，其功能缺失将导致内吞运输相关的机体发育异常。内吞运输通路的正常运行和功能维持依赖于多种RAB蛋白的转化过程。线虫中的EPG-5通过直接结合RAB-5的GAP蛋白TBC-2，调节RAB-5/RAB-10等内吞运输相关RAB蛋白的转化过程。EPG5功能缺失引起内吞运输通路中的RAB蛋白转化异常，进而导致各种内体结构形成异常嵌合的结构，并抑制多种内吞运输底物的转运过程。EPG5对内吞运输通路的调控独立于其在自噬中的tether功能，其对内吞运输和自噬通路的协同调控是通过不同的分子机制所介导的，EPG5是一个多功能的蛋白。此外，通过线虫遗传筛选，发现IPMK功能缺失可以抑制EPG5的自噬缺陷表型。IPMK通过调节转录因子TFEB的相分离，负调控自噬-溶酶体通路活性。进一步研究发现MLX、IPMK和MYC以协同表面活性剂的方式调控TFEB相分离凝聚体的界面张力，进而调控自噬-溶酶体通路活性。通过小分子化合物筛选研究，发现Ro-3306等小分子药物，可以通过改变TFEB凝聚体的界面张力等物理性质，调节其转录活性，进而改变自噬-溶酶体通路活性。Ro-3306处理可以逆转细胞系中EPG5缺失导致的自噬缺陷表型。本项目研究揭示了EPG5在内吞运输中的作用机制，有助于进一步理解Vici综合征的致病机理；表明IPMK和MLX及TFEB凝聚体的物理性质是潜在的Vici综合征等神经退行性疾病的治疗靶点，Ro-3306是潜在的用于Vici综合征等神经退行性疾病治疗的先导药物，相关药物筛选方法可以广泛适用于其他基于生物相分离凝聚体的生物学通路的人工干预及相关疾病的治疗。