定量磷酸化蛋白质组解析17β-雌二醇致死效应的细胞调控过程  

Deciphering cellular processes responding to lethality of 17β-estradiol by quantitative phosphoproteomics

作　　者：李亚楠 刘晓艳[3] 王䶮 刘震[3] 叶明亮[3] 汪海林 LI Yanan;LIU Xiaoyan;WANG Yan;LIU Zhen;YE Mingliang;WANG Hailin(School of Environment,Hangzhou Institute for Advanced Study,University of Chinese Academy of Sciences,Hangzhou 310024,China;State Key Laboratory of Environmental Chemistry and Eco-toxicology,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences,Beijing 100085,China;CAS Key Laboratory of Separation Science for Analytical Chemistry,Dalian Institute of Chemical Physics,Chinese Academy of Science,Dalian 116023,China)

机构地区：[1]国科大杭州高等研究院环境学院,浙江杭州310024 [2]中国科学院生态环境研究中心,环境化学与生态毒理学国家重点实验室,北京100085 [3]中国科学院大连化学物理研究所,中国科学院分离分析化学重点实验室,辽宁大连116023

出　　处：《色谱》2024年第4期333-344,共12页Chinese Journal of Chromatography

基　　金：国家重点研发计划(2021YFA1302602);国家自然科学基金(22204033).

摘　　要：17β-雌二醇(E2)是人体内一种重要的内分泌激素,在生理浓度下(0.2~1.0 nmol/L)对生殖系统、乳腺等靶器官的生长发育起着重要的调节作用。但很多研究表明,高剂量(μmol/L~mmol/L)的E2能够诱导肿瘤组织消退和细胞凋亡,其具体调控机制尚不明确。本工作聚焦于高剂量(μmol/L)的E2致死效应,首先分析了μmol/L水平的E2对HeLa细胞表型的影响,发现在1~10μmol/L下E2以浓度依赖的形式抑制HeLa细胞增殖,并诱导HeLa细胞发生死亡,其中,用5μmol/L E2处理2天后可使约74%的HeLa细胞增殖受到抑制,并引起约50%的HeLa细胞死亡。在此基础上,为了探究高剂量E2诱导细胞死亡的内在调控过程,将基于固相萃取(SPE)的固定化钛离子亲和色谱技术(Ti^(4+)-IMAC)与基于数据非依赖采集模式(DIA)的蛋白质组定量技术结合,用于筛选HeLa细胞内参与高剂量(μmol/L)E2致死效应调控过程的磷酸化位点。最终,在5μmol/L E2和二甲基亚砜(DMSO)处理的HeLa细胞中共鉴定到超过10000个磷酸化位点;t检验分析发现,在E2处理后,有924个磷酸化位点(对应599个蛋白质)的丰度发生了显著变化(显著性水平(p)<0.01,|log 2(倍数变化)|≥1),推测其可能参与调控E2致死效应过程。此外,有453个磷酸化位点(对应325个蛋白质)仅单独发生在E2或DMSO处理后的HeLa细胞样品中,表明这些磷酸化位点在E2处理后发生了磷酸化或去磷酸化,也可能参与E2致死效应的调控过程。分别对以上两种方式筛选的E2调控的磷酸化蛋白质进行富集分析,发现这些磷酸化蛋白质主要参与细胞分裂、核糖体/核质转运、信使核糖核酸(mRNA)加工/剪接及转录等过程,表明高剂量的E2可能通过调控核糖体及mRNA加工等过程影响蛋白质转录,进而诱导细胞发生死亡。此外,我们发现表皮生长因子受体(EGFR)和丝裂原活化蛋白激酶(MAPK)家族蛋白(包括MAPK1、MAPK4和MAPK14)上多个磷酸化位点的修饰水平在�17β-Estradiol(E2),an important endocrine hormone in the mammalian body,participates in the regulation of the physiological functions of the reproductive system,mammary glands,bone,and cardiovascular system,among others.Paradoxically,despite the physiological actions of endogenous E2(0.2-1.0 nmol/L),numerous clinical and experimental studies have demonstrated that high-dose E2 treatment can cause tumor regression and exert pro-apoptotic actions in multiple cell types;however,the underlying mechanism remains undescribed.In particular,little information of the cellular processes responding to the lethality of E2 is available.In the present study,we attempted to characterize the cellular processes responding to high-dose(μmol/L)E2 treatment using quantitative phosphoproteomics to obtain a better understanding of the regulatory mechanism of E2-induced cell death.First,the cell phenotype induced by high-dose E2 was determined by performing Cell Counting Kit-8 assay(CCK8),cell cytotoxicity analysis by trypan blue staining,and microscopic imaging on HeLa cells treated with 1-10μmol/L E2 or dimethyl sulfoxide(DMSO)for 1-3 d.E2 inhibited cell proliferation and induced cell death in a dose-and time-dependent manner.Compared with the DMSO-treated HeLa cells,the cells treated with 5μmol/L E2 for 2 d demonstrated>74%growth inhibition and approximately 50%cell death.Thus,these cells were used for quantitative phosphoproteomic analysis.Next,a solid-phase extraction(SPE)-based immobilized titanium ion affinity chromatography(Ti 4+-IMAC)phosphopeptide-enrichment method coupled with data-independent acquisition(DIA)-based quantitative proteomics was employed for the in-depth screening of high-dose E2-regulated phosphorylation sites to investigate the intracellular processes responding to high-dose E2 treatment.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)identified over 10000 phosphorylation sites regulated by E2 and DMSO in HeLa cells.In comparison with the DMSO-treated cells,the cells treated with 5μmol/L E2 showe

关 键 词：液相色谱-串联质谱 固定化钛离子亲和色谱 数据非依赖采集 磷酸化蛋白质组 17Β-雌二醇 雌激素 致死效应 

分 类 号：O658[理学—分析化学]