Sumo1 amc

In vitro deSUMOylation assay was carried out in a 96-well plate in a 10 μl reaction mixture containing 5 μM SUMO1-AMC (UL-551, Boston Biochem), GST-SENP1 (400 pM), GST-UXT (various concentrations), and assay buffer (50 mM Tris–HCl pH 7.8, 100 μg/μl BSA, and 10 mM DTT). GST protein in varying concentrations was used as a negative control. The reaction was measured in a fluorometer plate reader Wallac Victor2 (PerkinElmer). The concentration of the fusion proteins was adjusted using SENP buffer (30 mM Tris–HCl, 100 mM KCl, 5 mM MgCl2, and 2 mM DTT). In addition to end-point enzymatic activity measurement, a continuous measurement of SENP1 enzymatic activity was made using LS50B Luminescence spectrometer (PerkinElmer) in a reaction mixture containing SENP1 at a concentration of 1000 pM, SUMO1-AMC at a concentration of 5 μM and varying concentrations of UXT.

IC₅₀ measurements were performed with at least two replications using eight concentrations starting at 200 μM and for the fragments at 500 μM with three-fold dilution. Human recombinant, His6-SENP1 Catalytic Domain (Boston Biochem Cat# E-700, Bio-Techne AG, Zug, Switzerland) was prepared in reaction buffer (50 mM HEPES, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, pH 7.5, 0.01% CHAPS). To this solution, a 20 mM or 50 mM inhibitor stock solution in DMSO was added and the mixture was incubated for 15 min at ambient temperature. The substrate SUMO1/2 or 3-AMC (SUMO1-AMC; Boston Biochem Cat# UL-551; SUMO2-AMC; Boston Biochem Cat# UL-758; SUMO3-AMC; Boston Biochem Cat# UL-768; Bio-Techne AG, Zug, Switzerland) was delivered to a 96 well-plate to initiate the reaction. The enzyme activities were monitored (Ex/Em = 355/460 nm) as a time-course measurement of the increase in fluorescence signal from SUMO1/2 or 3-AMC for 60 min at 25 °C. The data were analyzed by taking the slope (signal/time) of the linear portion of measurement. The slope was calculated using Excel and the curve fits were performed using GraphPad Prism7 with a four-parameter least-squares fit. The final reaction conditions contained 0.02 nM SENP1 and 300 nM SUMO1/2 or 3-AMC).

Activity assays of DUBs against AMC-labeled Ub/UbL substrates were performed using reaction buffer (150 mM NaCl, 20 mM TRIS pH 7, 10 mM DTT) 1 µM DUBs and 10 µM zRLRGG-AMC (BACHEM AG, Switzerland), 1 µM Sumo1-AMC, 1 µM Sumo2-AMC (Boston Biochem, Inc., USA), 1 µM ISG15-AMC (Boston Biochem, Inc., USA), 1 µM Nedd8-AMC (ENZO Life Sciences GmbH, Germany), LC3A-AMC (Boston Biochem, Inc., USA), or 5 µM Ub-AMC (UbiQ-Bio, The Netherlands). The reaction was performed in black 96-well plates (Corning) at 30 °C and released fluorescence was measured using the Infinite F200 Pro plate reader (Tecan) equipped for excitation wavelength of 360 nm and an emission wavelength of 465 nm. The measurements were performed in triplicate and the mean is presented.