Generation of DiUb-AMC Fluorescent Substrates and Fluorescence-Based DUB Assays.

AMC-derived Ub, and Ub-like proteins (SUMO1, NEDD8, ISG15) (Boston Biochem) were used in fluorescence-based DUB assays to measure the activity of USP9X CD. Assays were carried out in 10-μL reaction volumes in 384-well black polypropylene PCR plates (PCR-384-BK; Axygen) and the fluorescence was measured on a Synergy plate reader (Biotek). For AMC assays, 10 nM USP9X was mixed with 0.5 μM AMC substrate in 20 mM Tris (pH 8.0), 50 mM NaCl, 0.005% Triton X-100, and 2 mM DTT, and the excitation and emission wavelengths were set at 360 and 460 nm, respectively, with a 40-nm bandwidth filter. All kinetic parameters were determined using Michaelis–Menten regression analysis in Prism 6 software (GraphPad).

Continuous diUb cleavage assays were performed using 50 nM USP9X CD and 500 nM K11- (Pos4, #UF-440; Boston Biochem) or 200 nM K63- (K63-2, #DU6302; LifeSensors) IQF-diUb substrates in an optimized USP9X buffer (20 mM Bicine, pH 8.0, 0.004% Triton X-100, 0.6 mM DTT), with excitation and emission wavelengths at 530 and 575 nm, respectively. Alternatively, for full kinetic analysis of K11 IQF-diUb and Ub-AMC substrate, 10–25 nM WT and mutant USP9X CD was used. Assays were performed in 50 mM Hepes (pH 7.8), 50 mM NaCl, 0.5 mM EDTA, 0.1 mg/mL BSA, and 1 mM DTT at 25 °C. Fluorescence was measured using a Fluoromax-4 fluorescence spectrometer (Horiba). For Ub-AMC, excitation and emission wavelengths at 355 and 440 nm were used, respectively. For IQF-diUb, excitation and emission wavelengths at 544 nm and 572 nm were used, respectively.

K11-, K48-, and K63-linked diUb-NC1-AMC2 fluorescent substrates were generated by first reacting Ub_1–75-MESNA containing a respective cysteine mutation at K11, K48, or K63 with 32 mM glycine-AMC in the presence of 0.5 M MESNA, 0.17 M collidine and 5 mg/mL N-hydroxysuccinimide for 40 h. The resulting reaction mixture was treated with 10 mM DTT overnight, purified using an SP column with a salt gradient (50–500 mM NaCl) and buffer exchanged into a 20 mM MES (pH 6.5) buffer. Following purification, the cysteine-containing Ub-AMC was reacted with Ub-NC to generate diUb-NC1-AMC2 substrate (Fig. 2A) and further purified using an SP column with a salt gradient from 100 mM to 1 M NaCl. Fluorogenic assay was performed in a reaction buffer containing 50 mM Hepes (pH 7.8), 50 mM NaCl, 0.5 mM EDTA, 0.1 mg/mL BSA, and 1 mM DTT at 25 °C. Fluorescence was measured using a Fluoromax-4 fluorescence spectrometer (Horiba) with excitation and emission wavelength at 355 and 440 nm, respectively.