白细胞分化抗原36的棕榈酰化位点突变对HepG2细胞自噬的影响  

Effect of CD36 palmitoylation modification site mutation on autophagy of HepG2 cells

作　　者：俞婷 夏珺 赵蕾 YU Ting;XIA Jun;ZHAO Lei(Chongqing Key Laboratory of Lipid and Glucose Metabolism,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学脂糖代谢性疾病重庆市重点实验室,重庆400016

出　　处：《中国病理生理杂志》2020年第7期1269-1273,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81970510)。

摘　　要：目的:探讨白细胞分化抗原36(cluster of differentiation 36,CD36)的棕榈酰化位点突变对HepG2细胞自噬的影响。方法:构建过表达野生型CD36(WT-CD36)和棕榈酰化位点突变型CD36(AA-SS)的HepG2细胞系。自噬双标腺病毒(GFP-mRFP-LC3腺病毒)感染细胞,用共聚焦显微镜观察自噬;细胞免疫荧光染色和共聚焦显微镜检测转录因子EB(transcription factor EB,TFEB)核转位;Western blot检测肝激酶B1(liver kinase B1,LKB1)和AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)总蛋白表达及其磷酸化水平;免疫共沉淀检测CD36与蛋白酪氨酸激酶Fyn的共聚体形成。结果:与WT-CD36组相比,AA-SS组细胞内GFP-mRFP-LC3腺病毒绿色荧光明显减弱,红色荧光明显增强,表明AA-SS-HepG2细胞的自噬-溶酶体流增强(P<0.01)。同时,与WT-CD36组相比,AA-SS组CD36与Fyn共聚体的形成显著减少(P<0.01),LKB1蛋白的磷酸化水平降低(P<0.01),而AMPK的磷酸化水平升高(P<0.01)。此外,TFEB在AA-SS组细胞的核内定位也较WT-CD36组显著增多(P<0.05)。结论:CD36的棕榈酰化位点突变能减少CD36/Fyn共聚体形成,抑制LKB1蛋白磷酸化,从而激活AMPK-TFEB通路,促进TFEB核转位,增强HepG2细胞自噬。AIM:To investigate the effect of cluster of differentiation 36(CD36)mutation at palmitoylation modification sites on autophagy of HepG2 cells.METHODS:The HepG2 cells with wild-type CD36(WT-CD36)or mutated CD36 at palmitoylation modification sites(AA-SS)were established.The HepG2 cells were transfected with microtubule-associated protein 1 light chain 3(LC3)double fluorescent(GFP-mRFP-LC3)adenovirus to determine autophagy.The nuclear translocation of transcription factor EB(TFEB)was detected by confocal microscopy.The protein expression of liver kinase B1(LKB1)and AMP-activated protein kinase(AMPK),and their phosphorylation levels were examined by Western blot.The CD36/Fyn complex was measured by co-immunoprecipitation assay.RESULTS:Compared with WT-CD36 group,the green fluorescence of LC3 double fluorescent adenovirus was reduced,while the red fluorescence was significantly increased in AA-SS group,indicating that the autophagy-lysosomal flux was enhanced in AA-SS-HepG2 cells(P<0.01).Compared with WT-CD36 group,the formation of CD36/Fyn complex in AA-SS group was significantly reduced(P<0.01).Consequently,the phosphorylation of LKB1 protein was reduced and the phosphorylated AMPK was increased in AA-SS-HepG2 cells(P<0.01).Furthermore,nuclear localization of TFEB in AA-SS group was obviously increased as compared with WT-CD36 group(P<0.05).CONCLUSION:CD36 palmitoylation plays a crucial role in the regulation of HepG2 cell autophagy.Inhibition of CD36 palmitoylation activated LKB1-AMPK-TFEB pathway via reducing the formation of CD36/Fyn complex.

关 键 词：白细胞分化抗原36 棕榈酰化 自噬 AMPK/TFEB信号通路 核转位 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R363.2[医药卫生—基础医学]