蛋白磷酸酶2A调控外源化学物代谢酶表达的分子通路及其功能研究

Metabolism activation is required for most xenobiotics to exert toxicity effect. However, the regulatory mechanism of metabolic enzymes expression still remains elusive. In this project, we intend to take dephosphorylation mediated by protein phosphatase 2A (PP2A) as a clue to define the key pathways of regulating metabolic enzymes expression by using gene knockout mouse and human cell mdels in vitro. Igbp1 knockout mouse have been successfully created and applied as an animal model with PP2A activity suppression. Firstly, the metabolic enzymes regulated by PP2A were screened under the condition of animal and cell level，PP2A activity suppression with different degree or induced by chemicals. Meanwhile, the variant proteins regulated by PP2A were identified by the phosphorylation analysis of known transcription factors regulating metabolic enzymes expression, and spectrometry analysis of serine/threonine protein enriched by immunoprecipitation. Through further function verification, we will define the key targets and signaling pathways involved in metabolic enzymes expression regulated by PP2A, and compare the differences of regulatory pathway in hepatocytes from human and mouse. In addition, to validate the response of pathways underlying metabolic enzymes expressions regulated by PP2A, we will measure the toxicity effect induced by chemicals including hepatorenal function, DNA damage, oxidative stress, and metabolites of chemicals. We speculate that our findings will reveal the regulatory mechanism underlying metabolic enzymes expressions and provide the targets for the prevention of toxicity effect induced by chemicals.

大部分外源化学物需要经过代谢酶的代谢活化才能引起生物学效应，然而目前代谢酶表达的调控机制仍未阐明。本研究以蛋白磷酸酶2A (PP2A)介导的去磷酸化作为切入点，利用基因敲除动物和体外细胞模型筛查调控代谢酶表达的关键通路。已经获得的Igbp1基因敲除小鼠可作为PP2A活性下降的动物模型。首先在动物和细胞水平、不同程度的PP2A活性降低、化学物诱导和非诱导的状态下筛查受PP2A调控的代谢酶，并通过已知调控代谢酶的转录因子的磷酸化检测以及免疫沉淀富集的丝苏氨酸磷酸化蛋白的质谱分析，筛查受PP2A调控的差异磷酸化蛋白。通过功能验证，阐明PP2A调控代谢酶表达的关键靶点以及信号通路，并比较人和小鼠细胞中调控通路的差异。此外，通过观察化学物诱导的毒效应包括肝肾功能损害、DNA损伤、氧化应激等，验证PP2A调控代谢酶表达的关键通路的应答。本课题将阐明代谢酶表达的调控机制，为预防化学物毒效应提供干预靶点。

大部分外源化学物需要经过代谢酶的代谢活化才能引起生物学效应，然而目前代谢酶表达的调控机制仍未阐明。该项目利用Aalpha基因敲除小鼠和细胞模型，筛查PP2A介导调控的代谢酶以及关键的信号通路，通过观察调控通路对化学物诱导的毒性效应的影响，阐述代谢酶表达调控的分子机制。通过3年的努力，完成了计划书的研究任务。已发表科研论文6篇，其中SCI 论著3篇。主要成果包括以下几个方面：(1)筛查PP2A调控外源化学物诱导代谢酶表达的信号通路 构建了肝特异性的PP2A活性下调的小鼠模型，利用蛋白组学技术筛查PP2A调控的代谢酶，体外实验证实PP2A的激活剂可逆转体外肝细胞中多个代谢酶表达下调，提示PP2A在维持代谢酶表达中起着关键作用。进一步机制揭示PP2A通过调控HNF4alpha的磷酸化水平来影响代谢酶表达。(2)PP2A在苯诱导血液毒性中的作用研究 利用PP2A Aalpha肝特异性敲除的小鼠模型，采用动式吸入苯的染毒方式，探讨PP2A在苯诱导的血液毒性中的作用以及机制，发现肝脏敲除PP2A Aalpha缓解了苯诱导的血液毒性，进一步机制揭示PP2A通过调控苯的关键代谢酶cyp2e1的表达来影响苯诱导的血液毒性。(3) 肝特异性敲除PP2A Aalpha小鼠的表型观察 成功构建了肝特异性敲除PP2A Aalpha的小鼠模型，并进行表型观察到18个月。观察到9个月时，雌性HO小鼠的肝脏出现纤维化，12个月时更加显著。当用乙醇处理PP2A Aalpha缺失小鼠，发现alphaα缺失显著促进肝纤维化的进程。进一步利用蛋白组学揭示相关的信号通路。此外，发现两个lncRNA TUG1和HOTAIR可能在Aalpha缺失诱导的肝纤维化的发生过程中起作用。(4) PP2A参与表观遗传调控外源化学物诱导DNA损伤修复和细胞转化 证实组蛋白H3Ser10磷酸化修饰是调控化学致癌物诱导细胞转化及肿瘤的发生发展中的重要靶点。进一步揭示PP2A通过直接调控组蛋白H3Ser10磷酸化水平影响致癌物AFB1诱导的DNA损伤修复以及细胞转化的机制。此外，在AFB1诱导的细胞恶性转化模型中筛查发现RUNX3高甲基化，与细胞恶性转化密切相关密切。所阐述的靶点被国际权威毒性与基因组比较数据库（CTD）收录。