去SUMO化酶SENP5的表达、纯化、活性测定及复合物重组

Expression,Purification,Biochemical Characterization and Complex Assembly of the SUMO Specific Protease SENP5

SENP5参与转录调控、蛋白质稳态、DNA修复和细胞分裂等重要生命过程,与癌症、发育缺陷和神经变性等疾病密切相关.SENP5包含N端的无规则卷曲和C端的催化结构域.研究对SENP5的催化结构域(SENP5CD)进行了表达和纯化.C末端水解活性实验表明SENP5CD对SUMO2前体(pSUMO2)的切割活性大于SUMO1前体(pSUMO1).分子筛实验证明,SENP5能够与SUMO1和SUMO2的前体、RanGap1-SUMO1和RanGap1-SUMO2形成稳定的复合物.实验构建了SUMO1-PA和SUMO2-PA活性探针,这2种活性探针都能与SENP5CD反应形成共价结合的复合物.通过比较AlphaFold预测的SENP5CD-pSUMO1和SENP5CD-pSUMO2复合物结构,发现pSUMO2与SENP5CD的结合比pSUMO1更紧密.研究表达、纯化了SENP5,鉴定了酶活性,获得了SENP5CD与底物的复合物,为进一步研究SENP5奠定了基础.

SENP5 is involved in crucial biological processes such as transcriptional regulation,protein homeostasis,DNA repair,and cell division,and it is closely associated with diseases such as cancer,developmental defects,and neurodegeneration.This enzyme consists of an N-terminal intrinsically disordered region and a C-terminal catalytic domain.In this study,the expression and purification of the catalytic domain of SENP5(SENP5CD)were performed.C-terminal hydrolysis activity experiments revealed that SENP5CD exhibited higher cleavage activity towards the precursor of SUMO2(pSUMO2)compared to the precursor of SUMO1(pSUMO1).By using molecular sieving techniques,it was demonstrated that SENP5 can form stable complexes with the precursors of SUMO1 and SUMO2,as well as RanGap1-SUMO1 and RanGap1-SUMO2.SUMO1-PA and SUMO2-PA activity probes were generated,both of which were able to react with SENP5CD and form covalent complexes.By comparing the predicted structures of the SENP5CD-pSUMO1 and SENP5CD-pSUMO2 complexes using AlphaFold,it was observed that the binding between pSUMO2 and SENP5CD was tighter than that between pSUMO1 and SENP5CD.In this study,SENP5 was expressed and purified,the enzyme activity was characterized,and the complexes of SENP5CD with substrates were obtained,which provided a foundation for further research on SENP5.