Fig. 4: Analysis of the SENP5 point mutants.
From: Structural basis for the human SENP5’s SUMO isoform discrimination
a Close-up view of the ribbon representation of interfaces of SENP5CD-SUMO2 (green) and SENP5CD-SUMO1 (blue) around Asn607 and Arg624. Interface residues are labelled and shown in stick representation. b Plot of the proteolytic cleavage of SUMO1-AMC and SUMO2-AMC to test the activity of SENP5CD wild type and SENP5CD N607A, R624A, K627A, and R624/K627A mutants. SUMO2-AMC was incubated with SENP5CD, and released AMC was identified by fluorescence. The plot is an average of a triplicate experiment. c SDS-PAGE of the endpoint assays of SENP5CD wild type and SENP5CD N607A, R624A K627A, and R624/K627A mutants using RanGAP1-SUMO1 and RanGAP1-SUMO2 substrates. (Below) Plot of the fraction of the RanGAP1-SUMO2 and RanGAP1-SUMO1 substrate after 30 min reaction. Data values represent the mean ± SEM, n = 3 technical replicates. Significance was measured by a two-tailed unpaired t test relative to wild-type. All data were analyzed with a 95% confidence interval. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Exact P values from left to right: 0.0005, <0.0001, <0.0001, and <0.0001. d Western Blot analysis of HEK293F cells co-transfected with Flag-PML and either the empty pcDNA3 (−) or the plasmids expressing full-length SENP5, SENP5-R624A, SENP5-C713A or SENP5-R624A/K627A. (left) Immunoblot showing the levels of endogenous SUMO1 and SUMO2 in HEK293F cells. Anti-SUMO1 and antiSUMO2 antibodies were used to detect both endogenous SUMO isoforms. Anti-tubulin antibody was used as a loading control. (right) Plot of the Western Blot triplicate showing the fraction of polySUMO conjugates for each experiment regarding to SENP5 active site mutant (C713A). Data values represent the mean ± SEM, n = 3 technical replicates. Significance was measured by a two-tailed unpaired t test relative to C713A mutant. All data were analyzed with a 95% confidence interval. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Exact P values from left to right: <0.0001 and 0.0032. Source data are provided as a Source Data file.
