环氧合酶2蛋白质稳态调控线粒体偶联的内质网功能介导肝毒性的机制研究

The integrity of mitochondria-associated endoplasmic reticulum membrane (MAM) mediates the coupling interaction of organelles to keep cell homeostasis. We have found the regulatory proteostasis of COX2 influences xenobiotics-mediated cell outcomes. Recently, we found COX2 is translocated on mitochondria, participates endoplasmic reticulum (ER) stress and autophagy, also related to protein phosphatase 2A (PP2A) expression. But the regulatory mechanism for the proteostasis of COX2 and its relation with the structure and function of MAMs is yet unclear. This project will for the first time propose the hypothesis that “dephosphorylational regulation of COX2 proteostasis is a new pathway to modulate the integrity of MAMs interaction”. We plan to conduct in vitro and in vivo experiments on regulation model of hepatocytes and PHH-FRG mice. We will confirm the proteostasis of COX2 and the PP2A’s effect on the regulation of targeted dephosphorylation with the treatment of xenobiotics. The character proteostasis of COX2 will be investigated, which participates the regulation of the structure and function integrity of MAMs. It will be elucidated that a novel pathway of PP2A mediated proteostasis of COX2, and its mechanism affects the integrity of MAMs interaction induced cytotoxicity and outcomes. The aim of the project is to explore a new molecular interaction and regulation pathway and to provide new MAM molecular events and targets for early stress evaluation and intervention of toxic effects caused by environmental exposure.

线粒体关联内质网膜(MAM)完整性介导细胞器偶联以维持细胞稳态。我们证实环氧合酶2(COX2)蛋白质稳态影响外源物诱导细胞转归；新发现COX2转位到线粒体并调节内质网(ER)应激和自噬、且与蛋白磷酸酶2A(PP2A)调控有关。但COX2蛋白质稳态的质量控制及其与MAM的关系尚未明。本项目首次提出“COX2去磷酸化调控蛋白质稳态是调节MAM互作完整性的新通路”假设。采用人肝细胞株和嵌合小鼠体内外试验，确定与外源物诱导细胞毒性有关的COX2蛋白质稳态和分布及其PP2A亚基靶向去磷酸化调控；探讨COX2参与MAM互作结构和功能完整性的调节及其对外源物诱导肝损伤效应的影响。阐明COX2蛋白质稳态调节MAM互作完整性的PP2A调控新通路及其介导外源物诱导毒性转归的机制。探明以COX2蛋白质稳态调控为核心的分子互作和毒性通路，为应用MAM靶点和事件进行环境暴露的早期应激识别、评价和有效干预提供新思路。

环氧合酶2(COX-2)可受外源物诱导并引发蛋白质稳态调控。蛋白磷酸酶2A(PP2A)靶向底物蛋白去磷酸化调节以维持其蛋白质稳态。线粒体关联内质网膜(MAM)分子机器中特定组分的蛋白质质量控制参与维持其结构和功能完整性和调控细胞命运。本项目聚焦“PP2A调控COX-2去磷酸化调节MAM动态介导肝毒性”的科学问题。探明了“PP2A-COX-2-MAM-肝毒性”信号通路级联的调控机制。项目完成了线粒体(mito-)COX-2蛋白质稳态的PTM调节、PP2A-B亚基靶向调控COX-2去磷酸化、MAM上分子互作重塑其结构和功能、介导外源物诱导肝细胞RCD关联性肝毒性等研究内容。.主要结果：(1)探讨COX-2蛋白质稳态调控线粒体质量控制(MQC)介导外源物诱导肝毒性作用。研究发现线粒体COX-2调节AFB1诱导HBx表达肝细胞坏死性凋亡和线粒体动力学紊乱触发肝脂肪变性；表明靶向线粒体COX-2调控经由介导Drp1依赖性线粒体动力学重塑增强肝细胞癌抗肿瘤敏感性；提示mito-COX-2及其介导线粒体自噬反应性可作为潜在敏感性生物标志应用于安全性评价。(2)探究COX-2经由MAM转位调控外源物诱导肝细胞RCD的机制。体内外试验表明COX-2参与MAM结构和功能调控介导SPIO-NPs诱导肝细胞凋亡依赖性毒性损伤；研究发现Mfn2的S-谷胱甘肽化修饰经由扰乱MAM促进坏死性凋亡介导镉诱导毒性的分子机制。(3)探索PP2A靶向调节COX-2介导MAM功能的调控机制。提示PP2A-B55δ调控p-COX-2Ser601去磷酸化及其MAM调节介导AFB1诱导肝细胞RCD和促进肝脏炎症损伤的机制；表明PP2A-B56γ靶向p-COX-2Ser584去磷酸化调控MAM介导HBx表达肝细胞脂质蓄积的机制。.本项目阐明了“PP2A靶向COX-2去磷酸化调控蛋白质稳态和互作、经由调节MAM结构和功能重塑的级联新通路、介导外源物诱导肝毒性转归”的新分子机制。在“COX-2关联性肝毒性调控的MAM新通路”方面具有创新性。研究结果提供了筛选线粒体-ER细胞器间PP2A-COX-2-MAM调控轴作为外源物诱导RCD相关肝毒性损伤新的生物标志、和潜在干预新靶点的依据。