靶向蛋白磷酸酶2A信号调控是AFB1和HBx协同诱导肝毒性的一条新通路

Protein phosphatase 2A (PP2A) regulates the cellular function and outcomes by dephosphorylating the target molecules in toxicity pathway. Our previous studies demonstrated that the regulation of PP2A subunits expression was involved in the hepatotoxicity induced by xenobiotics. Our recent study suggested that the transcription profile of PP2A subunits gene was altered in HBV x protein (HBx) transfected hepatocytes and regulated by aflatoxin B1 (AFB1) exposure which resulted in the hepatotoxicity, but the interactions and regulation mechanisms are still obscure. We firstly propose that the dephosphorylation of substrates in signaling pathway regulated by PP2A is the target of their synergic toxicity, and try to prove this hypothesis. In the current project, firstly, HepaRG cell model will be constructed to screen the certain PP2A subunits targeting by HBx using structure biology methods. Next, we will verify the interaction between HBx and PP2A subunits in overexpression and knockdown cell models. Then, using AFB1 synergic exposure cell models, the dephosphorylating regulation pattern of HBx targeting PP2A subunits will be investigated, and their functions on the hepatotoxicity and malignant transformation will be figured out. The results will clarify the molecular mechanisms of targeting to the dephosphorylation of signal cascades mediated by PP2A is a novel pathway for synergistic hepatotoxicity and outcomes induced by AFB1 and HBx, and provide basis for exploring new intervening models with PP2A as the regulation core and targets in xenobiotics exposure.

蛋白磷酸酶2A(PP2A)通过对毒性通路靶分子的去磷酸化参与调节细胞转归；我们研究表明PP2A亚基调控表达可介导外源物诱导的肝细胞毒性。我们新近发现乙肝病毒X蛋白(HBx)使肝细胞PP2A亚基表达谱改变、并受黄曲霉毒素B1(AFB1)暴露的调节，影响了肝细胞毒性反应；但它们靶向PP2A的交互作用和调控机制尚不明确。我们首次提出"PP2A调控信号通路去磷酸化作为毒作用协同靶点"的假设并拟验证。本项目在HepaRG细胞建立模型，首先结构生物学筛查HBx对PP2A特定亚基的靶向性；并在PP2A调控表达细胞中功能试验确证与HBx的交互作用；再采用AFB1协同染毒细胞模型探讨HBx靶向PP2A介导的信号通路调控模式，探明诱导肝细胞毒性和转化的特征。结果阐明靶向PP2A介导的信号级联去磷酸化调控作为AFB1和HBx协同诱导肝毒性转归新通路的分子机制；为探索以PP2A调控为靶点的干预新模式提供依据。

研究表明蛋白磷酸酶2A(PP2A)亚基调控表达可介导外源物诱导的肝细胞毒性。本研究探讨了PP2A亚基调控HBx蛋白和AFB1联合暴露协同靶向诱导肝细胞毒性转归的机制；开展了4个方面内容的研究：包括(1)HBx、(2)黄曲霉毒素B1(AFB1)、(3)HBx联合AFB1暴露、(4)不同外源物介导线粒体-内质网(ER)质量控制的PP2A调控机制调节性细胞死亡(RCD)、毒性评价与靶向干预应用。.主要结果发现：(1)证实HBx表达介导肝细胞PP2A-B56γ表达，经由p53磷酸化调控，影响细胞ER应激、凋亡等转归；在HBx转基因小鼠体内试验中得到证实。(2)AFB1暴露人肝HepaRG细胞中PP2A-B55δ、-B56γ等改变，经由氧化应激诱导线粒体自噬，介导坏死性凋亡，并可被靶向干预抑制或基因干预；GEO数据分析表明AFB1暴露的肝细胞毒性损伤与PP2A表达关联。(3)模拟HBV感染的HBx表达肝细胞中，AFB1暴露可诱导环氧合酶-2(COX-2)在线粒体转位分布，介导线粒体自噬导致线粒体动态平衡失调，导致肝细胞脂质蓄积；证实肝脏局部免疫微环境介导的枯否细胞中NLRP3依赖性炎症反应与肝细胞焦亡有关。(4) 选择代表性肝毒性受试物，开展体内外试验和靶向干预评价。镉和纳米颗粒等经由B56γ或B55δ表达诱导ER应激、自噬、坏死性凋亡、焦亡等介导毒性损伤的不同转归结局；诱导人肝L02细胞发生Drp1依赖性线粒体质量控制(MQC)，经由p53和/或RB的转录非依赖性调控机制介导肝细胞毒性。.本项目探明了外源物诱导线粒体-ER通路轴关联性RCD介导的肝细胞毒性；阐明了“靶向PP2A介导的信号级联去磷酸化调控作为AFB1和HBx协同诱导肝毒性转归新通路”的分子机制。结果为探索外源物暴露肝毒作用的早期筛查评价和PP2A调控信号通路靶点有效干预的新模式提供了依据。