角蛋白8/18磷酸化修饰在非酒精性脂肪性肝病糖脂代谢异常的作用及机制研究

Non-alcoholic fatty liver disease (NAFLD) is the most common causes of chronic liver disease in the worldwide, and hepatic insulin resistance plays a major role in determining the severity of metabolism abnormalities in NAFLD. Keratin8/18 function as the largest member of cytoskeleton protein that have been reported to be closely associated with glycometabolism and insulin signal pathway. Our previous study have found that keratin mutations which disrupt the structure of protein lead or predispose to the development of insulin resistance. Phosphorylation modification of keratins have been demonstrated as a progression marker of chronic liver disease. So we hypothesis that phosphorylation modification of keratins has a important conserved role in the development of insulin resistance. In this study, we plan to analyze the change of phosphorylation modification of keratins in NAFLD models in vitro and vivo by means of mass spectrum and Western blot. We will identify those specific phosphorylation sites with phosphorylation levels change significantly, and further explore its influence in site-directed mutation cell model and transgenic animal. Immunofluorescence staining ,euzymelinked immunosorbent assay, Western blot and co-immunoprecipitation will be performed to discern the relationship between keratin phosphorylation sites, keratin protein structure, glycometabolism and insulin signal pathway, by which we can further elucidate the mechanism of protein post-translational modification and constitution of keratin to cause insulin resistance. We aim to find out a new therapeutic target and provide theoretical basis for it.

非洒精性脂肪性肝病(NAFLD)是全球首位慢性肝病，肝脏胰岛素抵抗（IR）介导的全身代谢异常加剧是其预后不良的重要原因。角蛋白作为肝脏细胞骨架中间丝的最主要成分，与糖代谢及胰岛素信号转导密切相关。我们前期研究表明角蛋白基因遗传变异引起的蛋白结构异常可能参与IR形成，而病理刺激下角蛋白磷酸化修饰改变可导致蛋白结构异常并与多种肝病的进展相关，因此NAFLD中角蛋白的磷酸化修饰改变可能是IR发生的新机制。本项目拟运用质谱技术及蛋白印迹法在细胞、动物组织和临床肝穿标本分析NAFLD中角蛋白磷酸化修饰的改变，针对磷酸化水平显著改变的位点通过CRISPR-Cas9系统构建定点突变细胞模型和转基因动物，利用免疫荧光染色、酶联免疫法检测糖代谢产物、蛋白印迹法和免疫共沉淀等方法检测磷酸化位点对角蛋白结构、肝细胞糖代谢及胰岛素信号通路转导的影响，从而阐明角蛋白结构异常引起IR的机制，为IR的治疗提供新靶点。

中间丝细胞骨架是胞内物质运输、代谢的重要结构，可其在疾病、应激下异常的磷酸化改变并参与疾病发生。角蛋白（Keratin, K）8(K8)和K18是肝脏中间丝家族的最主要成员，其与非酒精性脂肪性肝病（NAFLD）糖脂代谢紊乱的关系仍不明确。本项目首先通过病例对照研究结合基因外显子测序明确NAFLD患者角蛋白变异率显著高于对照组(5.0% vs. 0.58%,P=0.015)，并发现1例全新的K18外显子错义突变(N193S)，NAFLD合并胰岛素抵抗(IR)组的突变率高于不合并IR组(8.86% vs. 2.5%P=0.043)，多因素回归表明K8/K18基因变异（OR=0.110，95%置信区间：0.014-0.872，P=0.012）是NAFLD发病及胰岛素抵抗发生的危险因素。进一步运用定量磷酸化蛋白组学结合高盐富集骨架蛋白全面鉴定NAFLD中包括K8/K18在内的细胞骨架蛋白家族磷酸化修饰特征变化，在NAFLD小鼠模型中鉴定细胞骨架蛋白鉴定出27个细胞骨架及70个骨架结合蛋白在NAFLD模型中磷酸化修饰显著变化，其中K8的24位、28位、46位、103位、110位、280位、316位丝氨酸位点、97位酪氨酸磷酸化水平降低，而K18的384位和413位磷酸化位点磷酸化水平显著降低。相关性分析及临床标本Western blot验证显示细胞骨架蛋白磷酸化位点与胰岛素通路分子的磷酸化修饰水平具有高度相关。经过筛选发现角蛋白8的280位丝氨酸（S-280）磷酸化下调在NAFLD模型小鼠中作为显著，因此以其为靶点研究对肝脏糖代谢的影响及可能参与胰岛素抵抗发生的机制。NAFLD小鼠及患者肝脏活检组织肝脏脂肪变程度越高，s280磷酸化信号越弱，且体外模型抑制其磷酸化抑制肝细胞线粒体氧化磷酸化水平及胰岛素介导的糖原生成及糖酵解。蛋白组学鉴定体外模型中持续K8-S280磷酸化激活和失活状态下KEGG通路和GSEA分析显示相关蛋白与糖代谢和AKT通路密切相关，并通过构建注射腺病毒AAV-K8 S280持续磷酸化（S280D）及去磷酸化（S280A）突变的小鼠模型，证实K8 S280磷酸化持续激活状态下可减轻肝脏脂肪沉积，增强肝脏糖代谢活跃度，及引起AKT通路激活。本研究全面分析K8/18磷酸化在NAFLD中的改变并发现K8-S280磷酸化降低可能是介导NAFLD胰岛素抵抗的分子事件。