PP2A介导抑癌蛋白转录非依赖性通路对外源物诱导线粒体质量控制的调控

Cytoplasmic tumor suppressor p53 and Rb, located at mitochondria, play anti-tumor function through transcription-independent pathway. Our research showed that the regulation on the expression of protein phosphatase 2A (PP2A) subunits affect the damage and repair induced by xenobiotics in hepatocytes. Recently, we found that gene overexpression and/or knockdown of PP2A subunits will change the phosphorylation profile of p53 and affect endoplasmic reticulum (ER) stress and autophagy in hepatocytes. However, the molecular mechanism of PP2A subunits targeting on cytoplasmic tumor suppressors is still unclear. We firstly proposed the hypothesis that PP2A dephosphorylate cytoplasmic tumor suppressors which mediates them targeting mitochondria and affecting their function. Basing on of different hepatocytes and their overexpression and knockdown models, we will firstly screen which subunits of PP2A target at p53/Rb. Next, we will carry function tests to verify the regulation of PP2A on p53/Rb to locate at mitochondria and PP2A interaction with them in transcription-independent signaling pathway. Then, xenobiotics exposure to hepatocytes to find out the effects of PP2A subunits regulation on mitochondrial quality controls and cell functions and outcomes. Futher, we can validate the hypothesis on primary human hepatocyte (PHH) chimeric FRG mice. Through above steps, we aim to clarify the molecular mechanism of the regulation of PP2A on tumor suppressors in the transcription-independent pathway and outcomes induced by hepatotoxicants targeted mitochondrial damage and repair. The aim of the project is to explore a new dephosphorylation regulation mechanism and markers in mitochondria and to provide new effective targets for early evaluation and intervention of toxic effects caused by environmental factors exposure.

抑癌蛋白可定位到线粒体上经由转录非依赖性调控影响其功能。我们研究表明蛋白磷酸酶2A(PP2A)亚基调控表达影响外源物诱导肝细胞损伤；新近又发现不同亚基差异可改变p53磷酸化谱、内质网应激和自噬等的诱导。但PP2A对胞质抑癌蛋白的靶向调控及其作用尚不明。本项目首次提出“PP2A调控胞质抑癌蛋白去磷酸化介导靶向线粒体和功能”假设。拟基于肝细胞调控模型，首先筛证特定PP2A亚基对胞质p53和Rb的靶向性，功能试验确证PP2A调控表达对p53和Rb定位于线粒体及与靶蛋白交互作用的转录非依赖性调控特征；再给外源物暴露探讨靶向调控对线粒体质量控制及诱导细胞损伤和转归的影响；进而在人肝细胞嵌合小鼠体内试验验证。阐明PP2A介导抑癌蛋白转录非依赖性调控新通路及其影响外源物靶向线粒体质量控制诱导肝细胞毒性的分子机制。探索以抑癌蛋白为核心的新调控模式和信号轴标志，为早期快速应答的评价和毒作用干预提供新思路。

外源物暴露时抑癌蛋白p53/RB作为细胞应激关键调节子，可定位于线粒体经由转录非依赖机制参与调控细胞内稳态；我们证实磷酸酶2A(PP2A)不同亚基靶向特异蛋白去磷酸化调控外源物诱导肝细胞毒性。本项目完成了鉴定不同PP2A-B亚基、靶向调控p53和RB蛋白去磷酸化、调节其转位于线粒体和与线粒体质量控制(MQC)信号分子互作、介导代表性外源物诱导肝毒性转归的体内外试验等研究内容。.主要结果发现：(1)探讨PP2A亚基介导外源物诱导肝毒性的调控作用；包括B56γ和B55δ亚基调控HBx表达和AFB1及其联合暴露等诱导肝细胞毒性转归；在转基因(Tg)和人肝嵌合(HLC)小鼠体内试验证实。(2)探明PP2A介导p53定位线粒体的机制；包括B56γ亚基调控p-p53Thr55和p53S20去磷酸化、转位线粒体，参与HBx和AFB1诱导肝细胞线粒体依赖性凋亡的调控；线粒体p53介导与溶酶体和脂滴相关蛋白互作，以及B55β调控线粒体p53质核逆转信号，介导AFB1等诱导肝细胞线粒体自噬、脂质代谢动态、调节性细胞死亡等肝细胞毒性；并可被靶向干预抑制或基因干预拯救。(3)探究PP2A介导线粒体RB蛋白调节肝毒性的机制；包括RB蛋白转位线粒体膜、与Drp1互作，介导CdCl2诱导肝细胞坏死性凋亡和肝毒性损伤，采用Met干预的体内外试验验证。(4)探索PP2A介导MQC调节肝毒性的转归评价；包括PP2A调节MQC、与外源物诱导细胞毒性评价的研究进展分析；PP2A调控Drp1Ser616介导MQC相关蛋白质稳态、p53线粒体转位经由Drp1依赖性MQC，参与外源物诱导肝细胞坏死性凋亡和肝脂质稳态失衡、肝毒性损伤。.本项目阐明了“PP2A靶向p53/RB去磷酸化调节线粒体定位及其蛋白互作、经由MQC关联性转录非依赖性通路调控外源物诱导肝毒性转归”的分子机制。为探查线粒体PP2A-p53/RB-MQC轴作为外源物引发RCD相关肝毒性损伤的生物标志和有效干预新靶点提供依据。