Sumo 2 amc

For non-denatured Shigella lysates, 4 × 10⁹ bacteria were centrifuged and resuspended in 400 µL of lysis buffer (50 mM Tris, 150 mM NaCl, protease inhibitor cocktail (Roche), 0,5% Triton) and then sonicated. DTT was added to the lysate at a final concentration of 5 mM. Bacterial lysate was diluted to 1/10 within reaction buffer (50 mM Tris, 150 mM NaCl, 0,75 mg/mL BSA, 2 mM cysteine and 1 mg/mL Chaps). Recombinant GST fusion of the SENP2 catalytic domain was added in the reaction buffer at a final concentration of 40 nM. SUMO1-AMC or SUMO-2-AMC (Boston Biochem) were added in the diluted sample at a final concentration of 100 nM, in a total volume of 200 µL. Liberation of AMC at room temperature during 60 min was monitored in a fluorimetric microplate reader (Infinite 200 pro, Tecan) with excitation at 380 nm and emission at 460 nm.

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.