SENP1在SPOP去SUMO化修饰中的作用研究

Study on the role of SENP1 in the deSUMOylation modification of SPOP

目的:研究小泛素相关修饰物(small ubiquitin-related modifier,SUMO)修饰对SPOP(speckle type BTB/POZ protein)蛋白水平和细胞定位的影响,并尝试在肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)中探讨SUMO特异性蛋白酶1(sentrin-specific proteases 1,SENP1)和SPOP之间的相关性。方法:利用野生型(wild type,WT)和Senp1敲除的小鼠胚胎成纤维细胞研究Senp1对Spop蛋白水平和细胞定位的影响,并通过比较外源WT-Spop和Spop的SUMO修饰位点突变体的蛋白表达量,进一步确认SUMO修饰对Spop蛋白水平的影响。后续通过邻位连接技术(proximity ligation assay,PLA)研究WT-Spop及其SUMO修饰位点突变体与SUMO1的结合能力。最后通过ccRCC患者的RNA表达数据和ccRCC细胞系探讨SENP1和SPOP在ccRCC中的相关性。结果:Senp1敲除下调小鼠胚胎成纤维细胞中Spop的蛋白量和稳定性,但不影响其细胞核定位。突变Spop的SUMO修饰位点后,其与Sumo1的结合能力下降,蛋白量也有所降低。SENP1和SPOP的表达在ccRCC中呈正相关。结论:Senp1通过去SUMO化修饰稳定Spop蛋白,SENP1和SPOP的表达在ccRCC中呈正相关。

Objective:To elucidate the impact of small ubiquitin-related modifier(SUMO)modification on the protein levels and cellular localization of speckle type BTB/POZ protein(SPOP)and explore the correlation between sentrin-specific proteases 1(SENP1)and SPOP in clear cell renal cell carcinoma(ccRCC).Methods:Using wild type(WT)and Senp1 knockout murine embryonic fibroblasts(MEFs),we investigated the effects of Senp1 on Spop protein level and cellular localization.By comparing the protein expression levels of WT-Spop and its SUMO modification site mutants,the effects of SUMO modification on Spop protein levels were further confirmed.Proximity ligation assay(PLA)was employed to study the impact of Spop SUMOylation site mutation on the binding ability with small ubiquitin-related modifier 1(SUMO1).Finally,the correlation between SENP1 and SPOP in ccRCC was examined utilizing datasets and ccRCC cell lines.Results:Senp1 knockout down-regulated the protein level and stability of Spop in MEFs without affecting its nuclear localization.Mutating the SUMOylation modification site of Spop attenuated its binding affinity with Sumo1,consequently leading to diminished protein levels.Notably,the expression of SENP1 and SPOP exhibited a positive correlation in ccRCC.Conclusion:Senp1 stabilizes Spop protein through deSUMOylation modification.The expression of SENP1 and SPOP is positively correlated in ccRCC.