线粒体自噬在类泛素化修饰介导的肝脏脂质代谢紊乱中的价值研究

Post-translational modifications are crucially involved in modulation of life activities and the occurrence of disease，making it a research hotspot recently.Previous studies of applicant indicate that Neddylation is involved in the regulation of lipid metabolism, moreover, high expression of NEDD8, which is a reflection of Neddylation level, is discovered in adipogenesis process and related to the degree of adipose differentiation, and the co-localization of lipid droplet with mitochondria and mitophagy have been significantly suppressed in the process of adipogenesis，which can be reversed by Neddylation inhibitor MLN4924. The overproduction of reactive oxygen species (ROS) in the mitochondria triggered by MLN4924 can initiate collapse of the mitochondrial membrane permeability，which is a prerequisite for mitophagy. .Combined with the report that the inhibition of mitophagy induces accumulation of lipid droplet，it is speculated that Neddylation inhibits mitophagy signaling pathway，which may be the key factor of disordered lipid metabolism. The aim of this project is to comprehensively clarify the role of Neddylation on three levels，including cellular，animal and clinical levels，mainly based on the crosstalk between mitophagy and lipid metabolic pathways. The expected results will shed light on new pathogenesis of NAFLD which is the major cause of global chronic liver disease and provide innovative therapeutic strategies based on the Neddylation intervention.

蛋白质翻译后修饰参与调控重要的生命活动及疾病发生成为研究热点。申请人前期研究发现类泛素化修饰 (Neddylation) 参与调控脂质代谢多个过程，同时发现脂肪形成中反映类泛素化水平的NEDD8高表达，且与脂肪分化程度相关，脂滴与代谢关键细胞器线粒体共定位，MLN4924（Neddylation抑制剂）能逆转此现象。申请人还发现MLN4924激发线粒体ROS产生导致线粒体膜电位丢失形成线粒体自噬的必要条件，结合文献报道抑制线粒体自噬能引起脂滴的堆积，据此推测：Neddylation抑制线粒体自噬信号通路是导致脂质代谢紊乱的重要因素。本项目拟从细胞、动物及NAFLD患者三个层面，以脂质代谢通路与线粒体自噬通路相互调控为主，全面阐明Neddylation在NAFLD的脂质代谢紊乱中的作用。预期结果将阐明NAFLD这一全球主要慢性肝病的发病新机制，并提供基于Neddylation的干预新策略。

近年来研究发现重要的蛋白质翻译后修饰-类泛素化修饰 (Neddylation)参与调控了肿瘤、肥胖、神经退行性病变等多种疾病生理病理过程。类泛素化是将泛素化修饰小分子NEDD8共价结合于底物调节靶蛋白，NEDD8作为类泛素化的重要修饰分子，不仅在前脂肪细胞分化过程中被诱导，亦能在肝细胞中被脂肪酸作用所诱导，在脂质的累积中发挥重要作用。类泛素化抑制剂MLN4924能够显著诱导线粒体自噬影响肝脏脂质代谢：我们在细胞和动物层面均验证了MLN4924显著减少肝细胞内脂肪的合成与累积，从而减缓脂肪肝的发生与发展的重要功能。PINK1和PARKIN是线粒体自噬途径中关键蛋白，我们前期结果发现MLN4924可以通过增加PINK1在线粒体膜外聚集，募集下游PARKIN，从而增强线粒体自噬诱导。阻断PINK1/PARKIN通路可以抑制MLN4924发挥其对脂质代谢的影响作用，提示PINK1和PARKIN是MLN4924在影响肝脏脂质代谢过程中诱导线粒体自噬发生与发展的关键蛋白。同时利用脂质代谢组学的方法比较研究了有无脂肪酸诱导或MLN4924药物作用或有二者共同处理组之间差异代谢物，通过KEGG通路富集分析筛选到了一些显著差异的相关通路，为MLN4924对肝细胞脂质代谢影响的研究提供了研究方向与干预策略。综上所述，该系列研究结果为类泛素化抑制剂MLN4924用于非酒精性脂肪肝病的治疗提供了可靠的科学理论依据，为基于类泛素化在今后全球发病率最高之一的慢性肝病的研究拓展了新思路，同时也为开发非酒精性脂肪肝病治疗提供了潜在用药靶点研究。