UFDS系统检测蛋白质SUMO化修饰研究

为了验证UFDS系统（Ubc9 fusion-directed sumoylation, UFDS）是否能够检测蛋白质SUMO(small ubiquitin-related modifier)化修饰，构建了Ubc9和PKCθ的融合表达载体，与SUMO1表达载体共转染293T细胞，通过免疫共沉淀和Western印迹检测Ubc9-PKCθ与SUMO1的相互作用。 结果表明，Ubc9-PKCθ与SUMO1相互作用，且SENP1共表达时导致Ubc9-PKCθ去SUMO化修饰；相较于野生型Ubc9-PKCθ，SUMO化修饰位点突变型Ubc9-PKCθ与SUMO1的相互作用显著减弱；而相较于野生型PKCθ，SUMO化修饰位点突变型PKCθ与SUMO1的相互作用则完全检测不到。 研究结果说明，应用UFDS系统能检测蛋白质的SUMO化修饰，但在鉴定潜在的SUMO化修饰位点时有一定的缺陷。

Study on Detection of Protein Sumoylation by UFDS System

To verify Ubc9 fusion-directed sumoylation (UFDS) system as a method for protein detection of sumoylation, the Ubc9-PKCθ fusion expression vector was constructed and cotransfected into 293T cells with SUMO1 expression vector. Then the sumoylation of Ubc9-PKCθ was tested by coimmunoprecipitation and Western blot. Results showed that, Ubc9-PKCθ could be modified by SUMO in UFDS system. SENP1 could induce desumoylation of Ubc9-PKCθ. Compared to wild type Ubc9-PKCθ, the sumoylation of multi-points KR mutants of Ubc9-PKCθ was significantly weakened. And compared with wild type PKCθ, the sumoylation of multi-points KR mutants of PKCθ was totally undetectable. Results indicated that UFDS system is useful for detecting protein sumoylation, but has a certain defect in identifying potential sumoylation sites.