摘要
目的 探讨α-突触核蛋白（α-synuclein）的小泛素样修饰蛋白1（small ubiquitin-like modifier1,SUMO-1）化修饰对α-synuclein蛋白亚细胞线粒体定位及α-synuelein蛋白经泛素系统降解的影响.方法 构建野生型、A53T突变型和缺失SUMO-1互作氨基酸的K96R突变型α-synuclein真核表达质粒.将野生型、A53T突变型和K96R突变型α-synuclein真核表达质粒分别转染HEK293细胞;在转染后48 h通过应用线粒体染色剂和激光共聚焦技术,观察野生型、A53T突变型及缺失SUMO-1修饰的α-synuclein蛋白的亚细胞线粒体定位和蛋白聚集情况.在转染后48 h应用anti-ubiquitin抗体进行Western印迹分析,明确野生型、A53T突变型及缺失SUMO-1修饰的α-synuclein蛋白泛素化程度有无差别.结果 将构建所得EGFP-α-synuclein-WT、EGFP-α-synuclein-A53T、EGFP-α-synuclein-K96R真核表达质粒经双酶切鉴定及DNA测序证实;激光共聚焦结果显示野生型、A53T突变型、K96R突变型α-synuclein蛋白均广泛分布于细胞质和细胞核中,以细胞质为主,野生型、A53T型细胞可见绿色荧光物质在胞质积聚,形成强荧光斑块,K96R型胞质内绿色荧光物质聚集少;野生型、A53T突变型、K96R突变型α-synuclein均与线粒体存在共定位,A53T突变型、K96R突变型α-synuclein在SUMO-1修饰或缺失的情况下对α-synuclein蛋白的线粒体亚细胞定位无明显影响.Western印迹结果显示转染缺失SUMO-1互作氨基酸的K96R突变型α-synuclein真核表达质粒组的细胞泛素蛋白的含量与转染空质粒组相比无明显变化,转染野生型、A53T突变型α-synuclein真核表达质粒组HEK293细胞中泛素蛋白的含量减少.结论 α-synuclein基因过度表达及致病突变A53T对α-synuclein蛋白的线粒体亚细胞定位无明显影响;SUMO-1修饰对α-synuclein蛋白的线粒体亚细胞定位也无明显影响;SUMO-1对a-αsynuclein基因过度表达及A53T突变型α-synuclein细胞内泛素化蛋白数量产生影响.
Objective To investigate the effect of sumoylation of a-synuclein by SUMO-1 on the mitochondria subcellular localization of a-synuclein and its degradation via ubiquitin-proteasome system. Methods Primers of wild-type, A53T pathogenic mutant and K96R mutant of human a-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-α-synuclein-WT, pEGFP-α-synuclein-A53T and pEGFP-α-synuclein-K96R were transfected into HEK293 cells by lipofectamine method. The expression of the α -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 α-synuclein on mitochondria subcellular localization of α-synuclein. The effect of sumoylation of α-synuclein on its degradation via the ubiquitin-proteasome system in the cells was assayed by Western-blot. Results The enzyme mapping suggested that the eukaryotic expression plasmids for human wild-type, A53T and K96R mutants of the α-synuclein gene were constructed successfully. By immunofluorescence and confocal microscope, it was observed that α-synuclein-WT and α-synuclein-A53T proteins aggregated in cytoplasm, and α-synuclein-K96＇R protein aggregation was decreased in cytoplasm of cultured cells. The α-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. Conclusion Neither overexpression of wild-type and A53T pathogenic mutant α-synuclein, nor sumoylation of α-synuclein, affected the subcellular localization in the mitochondria. However, overexpression of wild-type and A53T mutant α-synuclein affected the amount of the ubiquitinated proteins.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2010年第3期267-271,共5页
Chinese Journal of Medical Genetics
基金
海南省自然科学基金（807080,806119）
