SUMO2/3修饰和SENP3调控肝细胞自噬对代谢和在体肝脏应激应答的影响

Protein sumoylation, in which a small ubiquitin-related modifier (SUMO) is attached to a multitude of proteins, is a lately discovered type of PTM (post translational modification) that affects numerous cellular processes. SUMO2/3 modification specifically occurs under stress, while SUMO2/3-specific protease SENP3 has been reported as a sensing protein via rapid accumulation in response to ROS elevation caused by various stress. Autophagy has emerged as a highly conserved lysosomal pathway that plays a critical role in cell homeostasis maintenance and stress response through degradation of organelles and macromolecules and production of nutrients. However, it remained unclear how cell controls proper autophagic extent when autophagic signaling is initiated. And it has not yet reported whether autophagy related proteins are reversibly modified by SUMO2/3 and consequently modulate autophagy under stress. Recently, we found a novel inhibitive regulatory role of SENP3 in autophagy in not only hepatocytes in vitro but also liver tissues from SENP3+/- mouse. Based on these findings, the present study aims to explore how the autophagic signaling and autophagy extent is negatively regulated by SENP3. We will identify de-SUMO2/3 of Beclin1 and ATG7 and consequent protein interaction and activity alteration. Moreover, the study will screen for other protein substrates to fully understand autophagy modulation by reversible SUMO2/3 modification. Meanwhile the study will demonstrate the regulatory role of SENP3 to maintain hepatocytes metabolism, homeostasis and also organism’s metabolism during the early neonatal starvation period and oncogenic stress response in which massive autophagy has been observed in mice. Therefore the study will expand regulatory mechanisms of autophagy via de-SUMO2/3 modification in vitro and in vivo.

SUMO2/3修饰是对应激敏感的类泛素化修饰，调控蛋白质功能。细胞自噬是细胞对机体应激的一种应答反应，在饥饿和癌基因活化等典型的应激中扮演重要角色。然而，自噬的调控是否发生可逆的蛋白质SUMO2/3修饰尚无报导。我们以往的研究证明了SUMO2/3特异性蛋白酶SENP3是应激敏感蛋白质。最近，我们发现了SENP3能够抑制肝细胞自噬，在基因敲除杂合子小鼠中发现肝细胞自噬的增加和肝脏变小，提示SENP3调控自噬蛋白SUMO化而负性调控自噬水平。我们的发现对回答自噬的关键科学问题如哪些机制避免自噬过度发生和哪些翻译后水平参与可能提供信息。因此，本研究拟全面解析自噬过程中SUMO2/3修饰的蛋白质谱和SENP3对其的调控，分析修饰对核心复合物形成和活性的影响；同时选取新生鼠代谢过渡和成年鼠肝癌发生作为肝脏应激模型，在整体水平探究SENP3负性调控自噬在肝脏代谢、动物存活和肿瘤发生等应激应答中的角色。

本项目聚焦应激敏感的SUMO蛋白酶3（SENP3）, 探讨其抑制自噬的作用、分子机制以及在肝脏应激应答中的生理学意义。项目首先发现在自噬应激时胞质SENP3快速升高，BECN1的SUMO化修饰被去除， VPS34复合物的形成和活性被抑制，自噬水平因而降低，形成了对自噬程度的精细的自我控制。其次，项目也发现了自噬相关重要分子ATG7的SUMO化修饰。结果显示无论是新生小鼠代谢过渡期，还是成年小鼠饥饿应激时，在肝、肺、脑等组织中ATG7均发生SUMO化修饰，该修饰促进了ATG7同源二聚体的形成，也促进了与LC3特异的相互作用。ATG7的SUMO化修饰发生于Lys413（K413）,而K413R突变基因插入小鼠则出现了和多脏器自噬的缺失和新生小鼠快速死亡，高度提示了自噬时SUMO化修饰的调控作用和重要的生理意义。第三，在肝脏致癌剂DEN和化学损伤剂CCl4诱导的小鼠肝癌模型中，肝脏特异敲除SENP3促进了肝癌发生，且早期肝损伤即加重，机制可能与Wnt-β-Catenin信号通路活性增强、肝组织Zone3区细胞的生物转化酶Cyp2E1表达增加有关。研究结果提示了SENP3及SUMO化修饰在肝细胞代谢分区中的重要作用。另外，项目也应用新技术BioID鉴定了肝细胞中SENP3相互作用蛋白，发现静息状态时310种作用蛋白主要与基因表达调控、RNA剪接等相关， 而DEN处理后，在新出现的95种相互作用蛋白中主要与ATP合成、核糖体合成、胆汁酸合成相关，提示了SENP3对应激时代谢的调控作用。项目扩充了肝细胞SENP3及SUMO化修饰的认识，也加深了SENP3调控自噬机制的理解。综上，项目阐明了SENP3通过抑制自噬对应激时肝脏稳态的影响和在新生鼠生存及成年鼠急性肝损伤中的意义。