Drp1缺失通过ABCB10激活线粒体未折叠蛋白反应

Drp1 deficiency activates ABCB10⁃mediated mitochondrial unfolded protein response

目的:在小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)中探讨动力相关蛋白1(dynamin related protein 1,Drp1)基因缺失激活线粒体未折叠蛋白反应(mitochondrial unfolded protein reaction,mtUPR)的分子机制。方法:采用不同浓度(0、2.5、5.0、10.0mmol/L)3-硝基丙酸(3-nitropropionic acid,3-NP)处理Drp1敲除或敲低的MEF细胞、Drp1抑制剂Mdivi-1或选择性阻断Drp1与下游蛋白相互作用的小分子多肽P110处理的MEF细胞以及相应对照,随后进行Western blot检测CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)、ATP结合盒B亚家族成员10(ATP binding cassette subfamily B member 10,ABCB10)、Lon肽酶1(Lon peptidase 1,LONP1)以及热休克蛋白60(heat shock protein 60,Hsp60)的表达。RT-qPCR检测Drp1敲低或Mdivi-1处理后MEF细胞的ABCB10的mRNA水平。同时敲低Drp1和ABCB10,Western blot检测CHOP蛋白的表达,试剂盒检测培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)含量,流式细胞术检测线粒体活性氧和线粒体膜电位水平。结果:3-NP处理后,Drp1敲除或敲低的MEF细胞以及Mdivi-1或P110处理的MEF细胞中CHOP表达呈现倍数上调。Drp1敲除或敲低的MEF细胞以及Mdivi-1或P110处理的MEF细胞中ABCB10蛋白表达上调,mtUPR效应蛋白LONP1和Hsp60表达上调。Drp1敲低的MEF细胞和Mdivi-1处理的MEF细胞中ABCB10 mRNA水平上调。同时敲低Drp1和ABCB10后,与Drp1敲低组相比,CHOP表达下调,LDH含量降低,线粒体活性氧水平降低,线粒体膜电位水平增加。结论:在MEF细胞中,Drp1表达下调可引起ABCB10表达量增加,导致mtUPR关键蛋白CHOP、LONP1和Hsp60蛋白表达上调,进而激活mtUPR。

Objective:The current study aims to investigate the molecular mechanism of mitochondrial unfolded protein reaction(mtUPR)induced by down⁃regulation of dynamin related protein 1(Drp1)expression in mouse embryonic fibroblast(MEF)cells.Methods:The expression levels of CCAAT/enhancer⁃binding protein homologous protein(CHOP),ATP binding cassette subfamily B member 10(ABCB10),Lon peptidase 1(LONP1)and heat shock protein 60(Hsp60)were detected by Western blot in Drp1 KO/KD cells,MEF cells treated with Drp1 inhibitors Mdivi1 or P110 and control cells after administration of 3⁃nitropropionic acid(3⁃NP)at different concentrations(0,2.5,5.0,10.0 mol/L).RT⁃qPCR was used to detect ABCB10 mRNA levels in Drp1 KD or Mdivi⁃1 treated MEF cells.After Drp1/ABCB10 double knocking down,the expression levels of CHOP protein was detected by Western blot,lactate dehydrogenase(LDH)release assay kit was used to detect the content of LDH in culture medium,and the levels of mitochondrial reactive oxygen and membrane potential were detected by flow cytometry.Results:The expression levels of CHOP were upregulated in Drp1 KO/KD cells or MEF cells treated with Drp1 inhibitors Mdivi⁃1 or P110.The expression levels of ABCB10 and mtUPR related proteins LONP1 and Hsp60 were upregulated in Drp1 KO/KD cells or MEF cells treated with Drp1 inhibitors Mdivi⁃1 or P110.Compared with control groups,the mRNA levels of ABCB10 were upregulated in Drp1 KD or Mdivi⁃1 treated MEF cells.After both Drp1 and ABCB10 knocking down,the expression levels CHOP was down⁃regulated,LDH content was decreased,mitochondrial reactive oxygen level was decreased,and mitochondrial membrane potential level was increased.Conclusion:Deficiency of Drp1 activates the mtUPR through ABCB10,which causes the upregulation of the mtUPR proteins CHOP,LONP1 and Hsp60.