hnRNPA1调控肝脏甘油三酯代谢的机制及其作为非酒精性脂肪肝药物靶标的研究

One challenge of NAFLD is that the clinical drugs can not obviously reverse the lipid accumulation in liver. This project focuses on the regulation of triglyceride metabolism in liver as the check point for disease therapy, and will demonstrate the mechanism of hnRNP A1 in pathogenesis of NAFLD. Based on the previous studies, we established hepatocyte conditional knockout mice, Alb-cre: hnRNP A1flox/flox, to verify the important role of hnRNP A1 in NAFLD. RIP-Chip was used to indentify the hnRNP A1-targeting specific genes in dyslipidemia. Furthermore, site mutants are used to study hnRNP A1’s phosphorylation and cellular location in triglyceride metabolism. HnRNP A1 is confirmed to be the drug target for NALFD, and we will setup the drug evaluation system targeting hnRNP A1 and detect the improvement in NAFLD of candidate compounds. By this project, we will evaluate the contribution of hnRNP A1 and its targeting genes in triglyceride metabolism of hepatocyte, which maybe the novel strategies of NAFLD treatment.

NAFLD的主要问题之一是现有临床药物尚不能很好逆转肝脏内的脂质堆积，本课题选择肝脏内甘油三酯代谢的调控为切入点，揭示NAFLD的重要发病机制。我们首次建立了肝实质细胞条件敲除A1的小鼠 ，Alb-cre：hnRNP A1flox/flox，明确A1参与肝细胞甘油三酯代谢以及NAFLD的发病。使用RIP-Chip鉴定脂质代谢障碍时A1的结合基因，基于A1结合基因的多样性，首次提出靶向A1实现多方面网络调控NAFLD的概念。构建系列A1的拟磷酸化和非磷酸化突变体，明确A1蛋白核-浆定位与磷酸化修饰对其结合基因功能的影响。并且确立肝细胞A1作为NAFLD的药物靶标，证实其配体化合物调控甘油三酯代谢的可行性。希望通过本课题的研究，阐明A1及其结合基因对NAFLD病理机制的重要贡献，确立A1作为治疗NAFLD的新靶标，并为相关的药物发现打下基础。

NAFLD的发病环节诸多，其核心病变是肝细胞内甘油三酯的异常积累。本项目首次证实脂肪变性的肝细胞中hnRNP A1会发生核-浆转位。通过比较肝实质细胞特异性缺失hnRNP A1的小鼠与正常鼠，发现敲除hnRNP A1能够显著减轻高脂饲料诱导的肝脏脂质蓄积，降低肝组织和血清的甘油三酯含量。随后证实hnRNP A1泛素化形式在脂代谢中具有重要作用。脂肪肝小鼠的肝实质细胞内hnRNP A1发生明显的泛素化修饰，同样观察到HepG2肝细胞在PA刺激下也出现泛素化修饰。我们进一步揭示，hnRNP A1的泛素化由E3泛素连接酶UBR4所介导，点突变实验证实hnRNP A1的泛素化修饰是由K11连接的泛素化链所致，并且hnRNP A1的泛素化修饰位点主要发生在RRM2区域的赖氨酸残基上。随后，我们探讨了泛素化的hnRNP A1在肝脏脂质代谢中的靶基因，通过RIP-Chip和GO分析证实hnRNP A1的部分靶基因与代谢性疾病密切相关，并且筛选获得其中变化最为显著的结合靶基因serpina1。泛素化hnRNP A1起到增强serpina1 mRNA稳定性的作用，serpina1能够进一步控制肝细胞内的脂代谢过程。最后，我们获得了色酮类化合物S5。S5能够靶向结合hnRNP A1并抑制其泛素化的功能，进而改善NAFLD脂代谢异常和肝脏损伤，可以作为治疗NAFLD的潜在先导化合物。综上所述，本项目首次揭示hnRNP A1参与调控NAFLD的发病机制，明确K11介导的hnRNP A1泛素化修饰在肝细胞内脂质异常积累过程中的作用，即通过调节serpina1 mRNA稳定性控制Srebp-1c的成熟和入核，进而影响下游脂代谢基因转录，并且以hnRNP A1为靶标，获得了潜在的先导化合物S5。本项目为NAFLD提供了新的治疗靶标和研究方向。