假性蛋白激酶TRB3通过促进肝细胞FOXA2降解调控肝纤维化进展

Hepatic fibrosis is the common pathological mechanism of various chronic liver diseases. Pseudokinase TRB3 is an important effective protein for CHOP/ATF4 pathway in the endoplasmic reticulum stress (ERS), and participates in the regulation of hepatocyte function, which is dominated by ERS under glycolipid metabolism and acute liver injury. However, the role of TRB3 in the process of hepatic fibrosis has not been studied. Our previous study indicated that the expression of TRB3 was up-regulated in fibrotic liver tissues. Forced hepatocyte-specific expression of TRB3 in vivo markedly aggravated hepatic fibrosis and inhibited the expression of liver functional genes. It was more important that TRB3 could decrease the level of FOXA2 protein in hepatocytes. FOXA2 was proved to alleviate hepatocytes injury and fibrous deposition in liver. Based on these findings, we will investigate the effect of TRB3 on the liver function and hepatic fibrogenesis. We will explore the underlining mechanism of FOXA2 ubiquitination and degradation by TRB3 using lysine motif enrichment, mass spectrometry and immunoprecipitation analysis. Moreover, we will discover the molecular and cellular mechanisms of TRB3 affecting liver function and hepatic fibrosis through hepatocyte-specific FOXA2 conditional knockout mice. This study will clarify the role and mechanism of TRB3 in the progression of liver fibrosis, preliminarily ascertain the efficacy of suppression of hepatocytes ERS in lhepatic fibrosis and provide a novel method for the treatment of chronic liver diseases.

肝纤维化是各种慢性肝病的共同病理机制。假性蛋白激酶TRB3是内质网应激（ERS）通路CHOP/ATF4的重要靶蛋白，参与糖脂代谢及急性肝损伤下ERS主导的肝细胞功能调控，然而TRB3在肝纤维化进程中的作用未见研究。我们前期研究发现，TRB3在肝纤维化进展时表达上调；特异性上调肝细胞TRB3表达可加重肝纤维化，抑制肝脏功能基因表达，更为重要的是降低肝细胞FOXA2蛋白水平。FOXA2被证实可减轻肝细胞损害及肝脏纤维沉积。本课题拟以TRB3为靶点，探讨肝细胞TRB3表达变化对肝脏功能及纤维沉积的影响；利用赖氨酸基序富集、质谱分析及免疫共沉淀等方法观察TRB3对FOXA2蛋白的泛素化降解作用，并通过模式动物探索TRB3影响肝细胞功能及肝纤维化进展的分子及细胞机制。本课题将阐明TRB3在肝纤维化进程中的作用及机制，初步明确抑制肝细胞ERS对肝纤维化的疗效，有望为临床治疗慢性肝病提供新的策略与方法。

非酒精性脂肪性肝病（NAFLD）是一种进展性肝病, 是代谢综合征（胰岛素抵抗、肥胖和高脂血症）的肝脏表现。随着生活水平的提升，能量消耗及摄入之间的逐渐失衡，我国普通人群中NAFLD的发病率显著提高。以肝细胞损伤、炎症和纤维化发展为特征的非酒精性脂肪性肝炎（NASH）可演变为肝硬化及肝细胞癌。NASH与内质网应激关系密切，TRB3是内质网应激驱动的重要效应分子。我们首先分析了NASH进展过程中TRB3表达的变化规律及细胞定位；随后通过结合质谱分析以及RNA-Seq分析发现TRB3与HNF4α存在相互作用，并通过体内外结合实验加以验证。更为重要的是，HNF4α蛋白水平受TRB3的负向调控。此外，体外结果证实TRB3作为衔接因子，介导TRIM8催化HNF4α发生K48依赖的泛素化降解，并明确了HNF4α第470位赖氨酸是影响TRIB3-TRIM8-HNF4α泛素化降解轴的关键位点，随后体内回补K470R突变型HNF4α可显著抑制TRB3促进NASH进展的作用，在此基础上，我们通过分子对接及突变实验进一步明确了影响TRB3与HNF4α结合的关键氨基酸位点，并通过I-TASSER服务器筛选TRB3潜在的α-螺旋结构，将穿膜肽与模拟α-螺旋的序列肽组装，形成竞争性的野生多肽，并将多肽上影响结合的关键氨基酸位点进行突变产生对照多肽。通过体内外一系列实验，证明野生多肽通过阻遏TRB3与HNF4α结合显著抑制HNF4α的泛素化降解，进而缓解NASH进展。本课题主要阐明了TRB3促进NASH进展的作用及主要分子机制，揭示了TRIB3-TRIM8-HNF4α-K470泛素化降解轴是驱动NASH代谢紊乱的关键一环，并通过一系列蛋白功能实验筛选出可抑制TRB3与HNF4α结合的竞争性多肽，明确其对NASH的治疗作用，为临床NASH的诊治提供新的策略及方法。