The Effect Of Ubiquitination And Sumoylation Modification On α-Synuclein Degradation

α-Synuclein(α-Syn), a small molecules acidic protein, contains 140 amino acids, in which nearly disperses in the brain neurons. Deletion or mutation of α-Syn gene causes a variety of neurological diseases, especially induced autosomal dominant Parkinson’s disease. However, the exact mechanism of mutant α-Syn-induced Parkinson’s disease is largely not clear.It focuses on the relations among protein post-translational modification and neurodegenerative diseases, especially on SUMOylation, ubiquitination and phosphorylation in recent years, althought it is unclear what the role of α-Syn SUMOylation and ubiquitination on the degradation of α-Syn.Therefore, in this study, we investigate the role of SUMO and ubiquitin on the degradation pathway of α-Syn, revealing the effect of SUMOylation and Ubiquitination on conversion mechanism of α-Syn degradation pathways. The results will enrich the degradation mechanism of Parkinson’s disease- associated proteins.Firstly, we reconstructed the eukaryotic expression recombinant plasmids by two-step PCR, the Ubiquitin wild-type(WT) and mutants with Flag-tagged, and detected these plasmids by double enzyme digestion, gene sequencing and western blot. Then we explored the distribution of Flag-Ubiquitin WT and mutants by immunofluorescence. We found that Flag-Ubiquitin WT and mutants nearly dispersed in the cytoplasm but the minority in the nucleus, and the mutants had greater cytotoxicity by LDH and WST-1 assay.Secondly, to establish the stable expressing α-Syn cell line, we transfected α-Syn plasmid in B103 cells using 200 μg/mL of Hygromycin B as resistance screening, and identified the expression level of stable cells line by western blot.Finally, in α-Syn stable cells, we investigated the relations among α-Syn with SUMO, Ubiquitin and proteasome by immunofluorescence, respectively. The results showed that there was no obvious colocalization between Ubiquitin and SUMO in B103 cells, whereas a significant co-localization appeared in the stable expressing synuclein cells. We find that a fraction of modified α-Syn can be degraded via the ubiquitin-proteasome system.In summary, the difference of Flag-Ubiquitin WT and mutants-induced cytotoxicity attributs to their distribution and the formation of aggregates in the cells. α-Syn simultaneously associates with Ubiquitin and SUMO, and then forms the complexes of α-Syn, ubiquitin and SUMO. Obviously, there is somewhat mechanism between SUMO and Ubiquitin modification on α-Syn. In addition, we find that a fraction of modified α-Syn can be degraded via the ubiquitin-proteasome system.