Chen Tao, Liao Xiao-ping, Wen Guo-qiang, Nong Zhi-gang, Ouyang Feng, Deng Yi-dong, Guo Min, Wu Hui-ling, Zhou Peng
Department of Neurology, Hainan Provincial People's Hospital, Haikou, Hainan, 570311 PR China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2010 Jun;27(3):267-71. doi: 10.3760/cma.j.issn.1003-9406.2010.0.007.
To investigate the effect of sumoylation of alpha-synuclein by SUMO-1 on the mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system.
Primers of wild-type, A53T pathogenic mutant and K96R mutant of human alpha-synuclein were designed to amplify the corresponding cDNAs without stop codon. The cDNAs were cloned into pGEM T-easy vector, analyzed by using enzyme mapping and DNA sequencing, and subcloned into pEGFP-N1 vector. The recombinant plasmids of pEGFP-alpha-synuclein-WT, pEGFP-alpha-synuclein-A53T and pEGFP-alpha-synuclein-K96R were transfected into HEK293 cells by lipofectamine method. The expression of the alpha-synuclein protein was measured by immunofluorescence and confocal microscope. Then mitochondria staining as well as immunofluorescence were utilized to investigate the effect of wild-type, A53T mutant and sumoylation of alpha-synuclein on mitochondria subcellular localization of alpha-synuclein. The effect of sumoylation of alpha-synuclein on its degradation via the ubiquitin-proteasome system in the cells was assayed by Western-blot.
The enzyme mapping suggested that the eukaryotic expression plasmids for human wild-type, A53T and K96R mutants of the alpha-synuclein gene were constructed successfully. By immunofluorescence and confocal microscope, it was observed that alpha-synuclein-WT and alpha-synuclein-A53T proteins aggregated in cytoplasm, and alpha-synuclein-K96R protein aggregation was decreased in cytoplasm of cultured cells. The alpha-synuclein proteins of wild-type, A53T and K96R mutants were co-localized with mitochondria. Western-blot analysis revealed that both wild-type and A53T mutant affected the amount of the ubiquitinated proteins.
Neither overexpression of wild-type and A53T pathogenic mutant alpha-synuclein, nor sumoylation of alpha-synuclein, affected the subcellular localization in the mitochondria. However, overexpression of wild-type and A53T mutant alpha-synuclein affected the amount of the ubiquitinated proteins.
研究SUMO-1介导的α-突触核蛋白的SUMO化修饰对其线粒体亚细胞定位及通过泛素-蛋白酶体系统降解的影响。
设计人α-突触核蛋白野生型、A53T致病突变体和K96R突变体的引物，扩增无终止密码子的相应cDNA。将cDNA克隆到pGEM T- easy载体中，通过酶切图谱和DNA测序进行分析，然后亚克隆到pEGFP-N1载体中。采用脂质体法将重组质粒pEGFP-α-突触核蛋白-WT、pEGFP-α-突触核蛋白-A53T和pEGFP-α-突触核蛋白-K96R转染至HEK293细胞。通过免疫荧光和共聚焦显微镜检测α-突触核蛋白的表达。然后利用线粒体染色和免疫荧光研究野生型、A53T突变体及α-突触核蛋白的SUMO化修饰对α-突触核蛋白线粒体亚细胞定位的影响。通过蛋白质免疫印迹法检测α-突触核蛋白的SUMO化修饰对其在细胞内通过泛素-蛋白酶体系统降解的影响。
酶切图谱显示成功构建了人α-突触核蛋白基因野生型、A53T和K96R突变体的真核表达质粒。通过免疫荧光和共聚焦显微镜观察到，α-突触核蛋白-WT和α-突触核蛋白-A53T蛋白在细胞质中聚集，而α-突触核蛋白-K96R蛋白在培养细胞的细胞质中的聚集减少。野生型、A53T和K96R突变体的α-突触核蛋白均与线粒体共定位。蛋白质免疫印迹分析显示，野生型和A53T突变体均影响泛素化蛋白的含量。
野生型和A53T致病突变体α-突触核蛋白的过表达以及α-突触核蛋白的SUMO化修饰均不影响其在线粒体中的亚细胞定位。然而，野生型和A53T突变体α-突触核蛋白的过表达影响泛素化蛋白的含量。
