S期激酶相关蛋白2（SKP2）泛素化修饰OGG1对中波紫外线诱导的晶状体上皮细胞氧化损伤作用

目的　探索E3泛素连接酶S期激酶相关蛋白2（SKP2）介导的碱基切除修复蛋白8-氧代鸟嘌呤-DNA糖基化酶（OGG1）在年龄相关性白内障（ARC）发生过程中的泛素化降解机制。方法　采用免疫沉淀实验检测OGG1的泛素化修饰及其与SKP2的结合能力。采用中波紫外线（UVB）照射人晶状体上皮细胞株SRA01/04构建细胞氧化损伤模型。蛋白免疫印迹实验检测细胞氧化损伤模型和过表达模型细胞中SKP2的蛋白表达。通过转染SKP2质粒，增加SKP2的表达并观察细胞中OGG1蛋白的表达变化。结果　OGG1蛋白存在泛素化修饰。UbiBrowser软件预测能与OGG1结合的潜在E3泛素连接酶，并通过免疫沉淀实验证明SKP2与OGG1之间存在相互作用。在氧化损伤环境下，SRA01/04细胞中SKP2蛋白表达出现先下降后逐渐升高的趋势，其中UVB照射10 min时SKP2表达下降最为显著。对照组、转染空载质粒组和转染SKP2过表达质粒组SRA01/04细胞中SKP2蛋白的相对表达量分别为1.040±0.007、0.920±0.008和1.330±0.020，与转染空载质粒组相比，转染SKP2过表达质粒组细胞中SKP2蛋白的表达显著上调（P＜0.05）。空白对照组、UVB组、UVB+转染空载质粒组和UVB+转染SKP2过表达质粒组细胞中OGG1蛋白的相对表达量分别为1.05±0.01、5.05±0.16、5.20±0.07和3.83±0.10；与UVB+转染空载质粒组相比，UVB+转染SKP2过表达质粒组SRA01/04细胞中OGG1蛋白相对表达水平明显降低，且差异有统计学意义（P＜0.05）。结论　E3泛素连接酶SKP2能够促进OGG1发生泛素化降解，导致晶状体上皮细胞内部损伤性DNA的累积，从而导致ARC的发生发展。

S-phase kinase-associated protein 2-mediated OGG1 ubiquitination in ultraviolet B-induced oxidative damage of lens epithelial cells

Objective To investigate the mechanism of ubiquitination of base excision repair protein 8-oxoguanine-DNA glycosylase(OGG1)mediated by E3 ubiquitin ligase S-phase kinase-associated protein 2(SKP2)during the development of age-related cataract(ARC).Methods Immunoprecipitation assay was used to detect ubiquitination of OGG1 and its binding ability with SKP2.The lens epithelial cell line SRA01/04 was irradiated with ultraviolet B(UVB)to construct the cell oxidative damage model.Western blot assay was performed to detect the protein expression of SKP2 in the oxidative damage model and the overexpression model.SKP2 protein expression was increased by transfection with SKP2 plasmid,and variations in OGG1 protein levels were compared.Results OGG1 protein was ubiquitinated.UbiBrowser software predicted the potential E3 ubiquitin ligases that could bind to OGG1 and demonstrated the interaction between SKP2 and OGG1 by immunoprecipitation assay.SKP2 protein expression decreased first and then increased in the UVB-induced oxidative damage model,and the most significant decrease took place at 10 min.The protein expression levels of SKP2 in the control group,empty plasmid group,and SKP2 overexpression plasmid group were 1.040±0.007,0.920±0.008,and 1.330±0.020,respectively.Compared with the empty plasmid group,the SKP2 protein expression was significantly increased in the SKP2 overexpression plasmid group(P<0.05).The protein expression levels of OGG1 in the blank control group,UVB group,UVB+empty plasmid group,and UVB+SKP2 overexpression plasmid group were 1.05±0.01,5.05±0.16,5.20±0.07,and 3.83±0.10,respectively.Compared with the UVB+empty plasmid group,the OGG1 protein expression in the UVB+SKP2 overexpression plasmid group was significantly decreased,and the difference was statistically significant(P<0.05).Conclusion E3 ubiquitin ligase SKP2 can promote ubiquitination and degradation of OGG1 and induce the accumulation of damaged DNA inside lens epithelial cells,thus resulting in the occurrence and development of ARC.