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目的:探讨E3泛素连接酶(SIAH)在α-突触核蛋白(α-synuclein)降解和细胞死亡中的作用。方法:用MTT法检测雷帕霉素和SIAH-1抗体处理24 h后的细胞活性,Western blot检测LC3-Ⅱ、E1、SIAH-1、P53和α-synuclein蛋白表达,RT-PCR检测相关蛋白的m RNA水平,用免疫荧光探讨SIAH抗体处理后α-synuclein、SIAH-1和LC3在细胞的分布和共定位情况。结果:MPP+处理增加α-synuclein、E1表达和SIAH-1的活性,然而LC3-Ⅱ、P53和α-synuclein的蛋白质水平显著降低,而抗SIAH-1抗体处理扭转了这一趋势,E1水平明显增加。雷帕霉素处理在MPP+组中降低SIAH-1和α-synuclein水平。SIAH-1抗体显著降低α-synuclein的免疫阳性染色,而α-synuclein和LC3在细胞内失去共定位。结论:SIAH-1抗体降低了α-synuclein单泛素化和聚集,并有效地促进其通过泛素蛋白酶体系统通路降解。因此,SIAH功能对α-synuclein聚集性及降解通路的选择有明显的影响。
Objective: To investigate the role of E3 ubiquitin ligase (SIAH) in α-synuclein degradation and cell death. Methods: The cell viability was measured by MTT assay after 24 h treatment of rapamycin and SIAH-1 antibody. The protein expression of LC3-Ⅱ, E1, SIAH-1, P53 and α-synuclein were detected by Western blot. The mRNA and protein levels of α-synuclein, SIAH-1 and LC3 in SIAH-treated cells were detected by immunofluorescence. Results: MPP + treatment increased the expression of α-synuclein, E1 and the activity of SIAH-1. However, the protein levels of LC3-Ⅱ, P53 and α-synuclein were significantly decreased, while the anti-SIAH-1 antibody reversed this trend. increase. Rapamycin treatment reduced the levels of SIAH-1 and α-synuclein in the MPP + group. SIAH-1 antibody significantly reduced the immunoreactive staining of α-synuclein, whereas α-synuclein and LC3 lost colocalization within the cell. Conclusion: The SIAH-1 antibody reduced α-synuclein mono-ubiquitination and aggregation and effectively promoted its degradation through the ubiquitin proteasome pathway. Therefore, SIAH function has a significant effect on the aggregation of α-synuclein and the choice of degradation pathway.