Ambra1调控细胞自噬的分子机制研究

Autophagy is a physiological process that cells degrade organelles and redundant proteins during stimulation in order to maintain their normal metabolism. During autophagy induction, double-membrane vesicles called autophagosomes are produced to sequester intracellular cargos and fused with lysosomes to form autolysosomes for subsequent degradation. Because of its essential roles in embryonic development and disease process, autophagy becomes an important issue these years. Beclin1 is an essential protein for autophagy. Deficiency of Beclin1 leads to abrogation of autophagy. Beclin1 is also a tumor suppressor by increasing autophagy level. Low Beclin1 activity is responsible for tumorigenesis. Ambra1 is one of the most important proteins for autophagy. Ambra1 is a WD repeat domain-containing protein that activates Beclin1-dependent autophagy. Ambra1 deficiency leads to early embryonic lethality, and autophagy defects. Ambra1 interacts with Beclin1 and promotes the association of Beclin1 with Vps34, which is essential for autophagy initiation. However, the molecular mechanism of Ambra1-regulated autophagy is unknown. In our study, we found that Beclin1 is ubiquitinated during autophagy. Ubiquitination of Beclin1 promotes the interaction between Beclin1 and Vps34, and also increases the activity of Vps34. We found that knockdown of Ambra1 reduces ubiquitination of Beclin1, indicating Ambra1 takes part in Beclin1 ubiquitination. According to our results, in this project, we want to verify the mechanism of Ambra1 regulating Beclin1 ubiquitination, find out the Ambra1 complex which ubiquitinates Beclin1, and reveal the molecular mechanism of Ambra1-regulated autophagy.

自噬是细胞在受到外界刺激或是某些特定生理过程中，细胞降解自身的细胞器和冗余的蛋白质，从而维持细胞正常代谢的一种细胞生物学过程。自噬因其在胚胎发育和疾病发生等过程中发挥着重要作用，近几年来成为研究热点。Beclin1-Vps34复合物对于自噬的进行非常重要，Beclin1活性的丧失会抑制自噬，并且促进肿瘤的发生。Ambra1是自噬调控过程中的重要分子之一，但Ambra1调控自噬的分子机理还不清楚。我们在研究中发现在自噬过程中，Beclin1能进行的K63位修饰的泛素化，而这种类型的泛素化修饰能够增强Beclin1-Vps34复合物的活性。同时也我们发现Ambra1敲低以后，Belcin1的泛素化水平受到抑制。本课题希望进一步证实Ambra1动态调控Beclin1泛素化的分子机制，寻找Ambra1调控Beclin1泛素化的复合物，揭示Ambra1调控自噬的分子机制。

本项目主要研究了Ambra1蛋白介导细胞自噬的分子机制。通过研究发现Ambra1蛋白能够招募DDB1-Culin4复合物形成E3连接酶，形成的Ambra1-DDB1 E3连接酶复合物能够将Beclin1泛素化。泛素化的Beclin1与Vps34结合增强，促进了Vps34的活性，促进了自噬的发生。我们在研究中证明了Ambra1形成的E3复合物成分，确定了Ambra1介导的泛素化对活化Beclin1-Vps34的重要作用。我们确定了Ambra1对于Beclin1的泛素化位点为K437位。将此泛素化位点突变以后，Beclin1蛋白不能泛素化，并且Vps34复合物的活性降低，自噬活性受到抑制。我们还发现Ambra1蛋白在自噬过程的后期会被泛素化降解，通过免疫共沉淀实验，我们鉴定了RNF2为Ambra1蛋白的E3连接酶。RNF2蛋白的缺失会导致Ambra1蛋白降解受阻，自噬过度发生。我们系统研究了Ambra1蛋白在自噬过程中调控和被调控的过程，相关研究成果发表在Cell Research杂志上面。本项目支持的研究还发表在Immunity, Nat Commun, Nat. Immunol, 和Cell. Mol. Life Sci.等杂志上面。