苹果酸酶2乙酰化调控脑胶质瘤细胞增殖的机制  

Mechanism of malic enzyme 2 acetylation regulating the proliferation of glioma cells

作　　者：张成 牛朝诗[1] 梅加明 司文霞 魏祥品 Zhang Cheng;Niu Chaoshi;Mei Jiaming(Dept of Neurosurgery,The Affiliated Provincial Hospital of Anhui Medical University,Hefei 230001;Dept of Neurosurgery,Fuyang Fifth People′s Hospital,Fuyang 236001)

机构地区：[1]安徽医科大学附属省立医院神经外科,合肥230001 [2]阜阳市第五人民医院神经外科,阜阳236001 [3]湖北理工学院医学院湖北省肾脏疾病发生与干预重点实验室,黄石435000

出　　处：《安徽医科大学学报》2021年第6期920-924,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年项目(编号:81902860);安徽省自然科学基金(编号:1708085MH189)。

摘　　要：目的研究苹果酸酶2(ME2)的乙酰化修饰在恶性脑胶质瘤细胞系U87MG增殖中的功能及其分子机制。方法基于序列保守性并结合质谱数据库,预测ME2的乙酰化修饰位点,再构建点突变体,转染细胞,通过免疫沉淀(IP)和Western blot确定修饰位点;通过293T细胞纯化ME2野生型和突变体,检测乙酰化修饰对ME2酶活的影响;通过共转染、富集纯化并结合酶活实验,检测去乙酰化酶Sirtuin3(SIRT3)对ME2酶活、细胞内活性氧(ROS)及NADH水平的影响;在U87MG细胞中用shRNA敲低内源ME2后,回转野生型和模拟乙酰化突变型,检测ME2的乙酰化对细胞增殖的影响,并通过裸鼠成瘤在动物水平验证;通过Western blot在恶性脑胶质瘤临床样本中检测癌与癌旁组织中ME2的乙酰化水平差异。结果K156是ME2主要的乙酰化修饰位点,该位点的乙酰化会抑制ME2活性并抑制U87MG细胞增殖。SIRT3去乙酰化ME2,提高ME2活性,促进NADH的生成,降低细胞内ROS水平。结论SIRT3调控的ME2乙酰化修饰通过提高自身酶的活性,上调细胞NADH水平,降低ROS水平,从而促进U87MG细胞增殖。Objective To study the function and molecular mechanism of the acetylation of malic enzyme 2(ME2) in the proliferation of malignant glioma U87 MG cells. Methods Based on the conserved sequence and combined with the mass spectrometry database, the acetylation modification sites of ME2 were predicted, point mutants were constructed, transfected cells were transfected, and the modification sites were determined by IP and Western blot. The wild-type and mutant of ME2 were transfected into 293 T cells and purified to detect the effect of acetylation on ME2 enzyme activity. The effects of deacetylate Sirtuins 3(SIRT3) on the activity of ME2, ROS and NADH were detected through co-transfection, purification, combined with enzyme activity experiment. After knockdown of endogenous ME2 with shRNA in U87 MG cells, the effects of ME2 acetylation on cell proliferation were detected by stable ectopic expressing wild type ME2 and mutant, which was verified at the animal level by tumor formation in nude mice. Western blot was used to detect the difference of the acetylation level of ME2 in the clinical samples of malignant glioma. Results Results showed that K156 was the main acetylation site of ME2, which inhibited the activity of ME2 and the proliferation of U87 MG cells. SIRT3 deacetylated ME2, improved its activity, thus promoting the formation of NADH and reducing the intracellular ROS level. Conclusion SIRT3-deacetylated ME2 can promote the proliferation of U87 MG cells by increasing the activity of its own enzymes, up-regulating the level of NADH and reducing the level of ROS.

关 键 词：代谢酶 乙酰化 苹果酸酶 脑胶质瘤 

分 类 号：R739.4[医药卫生—肿瘤]