人肝细胞脂筏区蛋白质组学研究揭示肝活素新机制  

Proteomics Studies of Lipid Rafts in Human Hepatocytes Reveal New Mechanisms of Hepassocin

作　　者：乔倩 刘贤 陈慧 翟华立 葛志强[1] 杨晓明[1,2] 于淼 QIAO Qian;LIU Xian;CHEN Hui;ZHAI Hua-Li;GE Zhi-Qiang;YANG Xiao-Ming;YU Miao(Department of Pharmaceutical Engineering,School of Chemical Engineering,Tianjin University,Tianjin 300072,China;Special Injury Omics Laboratory,Beijing Institute of Lifeomics,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 102206,China)

机构地区：[1]天津大学化工学院制药工程系,天津300072 [2]军事科学院军事医学研究院生命组学研究所特种损伤组学研究室,北京102206

出　　处：《中国生物化学与分子生物学报》2020年第9期1071-1079,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：北京市自然科学基金(No.7182124)资助项目。

摘　　要：肝活素(hepassocin, HPS)是一种重要的肝细胞活性因子,主要表达于肝组织,少量表达于胰腺。目前研究表明,肝活素通过非受体酪氨酸激酶(Src proto-oncogene, non-receptor tyrosine kinase, Src)/表皮生长因子受体(epithelial growth factor receptor, EGFR)/细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK)途径诱导L02细胞增殖,小窝蛋白1(caveolin 1, CAV1)在肝活素刺激下调节L02细胞G1/S期细胞周期行进。但肝活素诱导细胞增殖的分子机制仍不明确。Src、EGFR和CAV1均定位在脂筏中。因此推测,肝活素可能通过脂筏发挥作用。本研究首先验证了一种快速有效的分离脂筏区蛋白质提取方法。基于此方法提取脂筏区蛋白质,以磷酸酪氨酸单克隆抗体(p-Tyr)进行免疫共沉淀分析,通过对人肝细胞脂筏区蛋白质组学研究和分析后,聚焦于信号传导与转录活化因子3(signal transducers and activators of transcription 3, STAT3)通路。Western印迹和间接免疫荧光结果表明,肝活素刺激可显著活化STAT3通路,促进STAT3入核,上调其下游靶基因MYD88天然免疫信号转导适配器(innate immune signal transduction adaptor, MYD88),蛋白酶体20亚基β8(proteasome 20S subunit beta 8, PSMB8), TRIM21 (tripartite motif containing 21)和内质网氨基肽酶1(endoplasmic reticulum aminopeptidase 1, ERAP1)(^****P<0.0001,^***P<0.001,^**P<0.01,^*P<0.05)的表达。分别抑制p-EGFR和Src后,肝活素对STAT3的活化作用消失。阻断STAT3通路明显抑制肝活素的促肝细胞增殖功能(^P<0.05)。综上所述,肝活素刺激可活化STAT3通路。本研究为肝活素的功能和作用机制提供新的线索。Hepassocin(HPS)is an important hepatocyte active factor,which is mainly expressed in the liver and a small amount in the pancreas.Current research showed that HPS induced L02 cell proliferation through the Src proto-oncogene,and the Src/epithelial growth factor receptor(EGFR)/extracellular regulated protein kinases(ERK)pathway.Under HPS stimulation,Caveolin 1(CAV1)would regulate the G1/S cell cycle progression of L02 cells.However,the molecular mechanism of HPS-induced cell proliferation is still unclear.As Src,EGFR and CAV1 are localized in lipid rafts,we speculate that HPS may function through lipid rafts.This study first validated a fast and effective method for lipid raft protein extraction.Based on this method,lipid raft proteins were extracted,and immunoprecipitation experiments were performed with p-Tyr antibody.After proteomics research and analysis of the lipid raft region of human hepatocytes,we focused on the signal transducers and activators of transcription 3(STAT3)pathway.Western blotting and indirect immunofluorescence results showed that HPS stimulation significantly activates the STAT3 pathway,translocates STAT3 into the nucleus,and up-regulates the expression of its downstream target genes MYD88,PSMB8,TRIM21 and ERAP1(^***P<0.0001,^***P<0.001,^**P<0.01,^*P<0.05).Inhibiting EGFR kinase activity and Src prevent HPS-induced activation of STAT3 phosphorylation.Blocking the STAT3 pathway inhibited the hepatocellular proliferation function of HPS(^*P<0.05).In summary,HPS stimulation can activate the STAT3 pathway.This study provides new clues to the function and mechanism of HPS.

关 键 词：肝活素 脂筏 信号传导与转录活化因子3通路 

分 类 号：Q71[生物学—分子生物学]