突变及SUMO化修饰对DJ-1线粒体定位的影响

The effects mutant DJ-1 and sumoylation of DJ-1 protein on DJ-1 mitochondrial localization

目的探究帕金森病(Parkinson’s disease,PD)致病基因DJ-1致病突变A39S以及DJ-1的SUMO化修饰对DJ-1蛋白在线粒体亚细胞定位的影响。方法构建可稳定表达野生型、A39S突变型、K130R突变型及A39S-K130R双突变型DJ-1蛋白及SUMO-1蛋白的COS-7细胞系。再通过Western印记、激光共聚焦及免疫荧光技术观察DJ-1蛋白表达情况及亚细胞定位,免疫荧光共定位观察DJ-1和SUMO-1是否存在相互作用。结果免疫荧光表明DJ-1蛋白主要分布在细胞浆中,也存在于细胞核中,野生型和K103R型DJ-1蛋白部分分布在线粒体上,A39S及A39S-K130R突变型DJ-1蛋白大部分转移至线粒体上,SUMO-1主要分布于细胞核及核周,呈斑点状分布。免疫荧光共定位发现四种DJ-1蛋白均与SUMO-1蛋白存在共定位。结论DJ-1致病突变A39S使DJ-1蛋白从胞浆转移至线粒体上;DJ-1与SUMO-1存在共定位,两者可能存在相互作用。

Objective To investigate the effects of DJ-1 A39S mutation in Parkinson’s disease(PD)and SUMO modification of DJ-1 on DJ-1 mitochondrial localization.Methods Five COS-7 cell lines that can stably express wild-type,A39S mutant,K130R mutant,A39S-K130R double mutant DJ-1 protein and SUMO-1 protein were constructed.The transfection results were detected by Western blot,double enzyme digestion and PCR sequencing.The expression and subcellular localization of DJ-1 protein were observed by Western blotting,confocal microscope and immunofluorescence.Results Immunofluorescence showed that DJ-1 protein was mainly distributed in the cytoplasm and also in the nucleus.Some wild-type and K130R DJ-1 proteins were distributed in mitochondria,most of A39S and A39S-K130R mutant DJ-1 proteins were transferred to mitochondria,and SUMO-1 was mainly distributed in the nucleus and around the nucleus.Immunofluorescence co localization showed that four DJ-1 proteins were co-localize with SUMO-1 protein.Conclusion The pathogenic mutation A39S of DJ-1 transferred DJ-1 protein from cytoplasm to mitochondria;DJ-1 and SUMO-1 have co localization,and they may interact.