蛋白磷酸酶2A-C 亚基转录后修饰在细胞氧化应激反应中精细调控的研究

Protein phosphatase 2A(PP2A) is one of the most important protein serine/threonine phosphatase in mammalian cells. Post-transcriptional modification of PP2A catalytic subunit (PP2A-C) included phosphorylation at T304 and Y307, and methylation at L309. Previous research base on single site and single post-transcriptional modification indicated that post-transcriptional modification plays a crucial role in the regulation of PP2A function, but how it regulates PP2A function is far from clear. Our preliminary studies showed that cellular PP2A-C can be plural modified at T304, Y307 and/or L309 site. Different post-transcriptional modified PP2A-C lost its methyl group according a certain sequence by hydrogen peroxide stimulation. Therefore, we proposed a fine-tuned regulation on PP2A post-transcriptional modification induced by oxidative stress, which is related to its function. In this study, we will investigate how hydrogen peroxide finely regulates PP2A-C post-transcriptional modification in human primary fibroblast cells, and its effect on PP2A activity and PP2A binding protein. This study will provide understanding on oxidative stress induced PP2A dependent biological effect and provide new clue on prevention and treatment of oxidative stress related disease and chemical damage.

蛋白磷酸酶2A 是胞内极重要的丝/苏氨酸蛋白磷酸酶，其C亚基羧基端存在T304、Y307两个位点磷酸化和L309位点甲基化的转录后修饰方式。既往基于单一位点单一修饰方式的研究发现PP2A-C转录后修饰可调控PP2A功能，但具体影响方式仍未明了。前期研究提示细胞内PP2A-C存在磷酸化和甲基化组合的双修饰或三修饰状态，在H2O2诱导的PP2A-C甲基丢失过程中不同修饰状态的PP2A-C按一定的先后顺序去甲基，提示氧化应激诱导PP2A-C去甲基有更为精细的调控规律，并与其功能有关。本研究拟以正常人皮肤成纤维细胞为研究对象，综合考虑PP2A-C三个位点及两种修饰方式，研究H2O2对细胞内PP2A-C转录后修饰的精细调控及其对PP2A活性和PP2A结合蛋白的影响。本研究为PP2A依赖的氧化应激相关生物学调控提供依据，为氧化应激介导的相关疾病及化学损伤的防治提供新线索。

蛋白磷酸酶2A是胞内极重要的丝/苏氨酸蛋白磷酸酶，其C亚基羧基端存在磷酸化和甲基化的转录后修饰方式，对PP2A功能起重要调控作用。氧化应激是细胞内重要的生理病理过程，本项目通过细胞学实验和蛋白质组学探讨氧化应激对PP2A-C转录后修饰的精细调控作用，研究结果显示①不同修饰状态的PP2A-C在氧化应激反应过程中敏感性不一，磷酸化-去甲基化PP2A-C变化最敏感，甲基化而非磷酸化是氧化应激作用的敏感作用靶点；②LCMT1高表达可调控PP2A-C甲基化，对抗氧化应激损伤，其保护作用可能与非红细胞血红蛋白表达，MAPK3失活和Rho信号转导相关蛋白有关。③氧化应激诱导PME从胞核转运到胞浆中，从而导致去甲基化-磷酸化PP2A-C核质重分布。④利用巨噬细胞分化模型探讨细胞内源性氧化应激对PP2A-C甲基化的作用，结果显示，随着炎症因子诱导细胞内氧化应激水平增高， PP2A-C去甲基化降低，促进M1型巨噬细胞分化，并增强巨噬细胞促炎性细胞因子表达和吞噬功能。