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