Human 20s proteasome

The human 20S proteasome was obtained from Enzo Life Sciences, and 1 µg of purified protein was preincubated for 30 min at 37 °C with the indicated concentrations of brusatol or MG132 in 50 mM Hepes (pH 7.8). Probe substrates for trypsin-like (Boc-LSTR-AMC, 50 µM; Sigma–Aldrich), chymotrypsin-like (Suc-LLVY-AMC, 50 µM; Enzo Life Sciences, UK), or caspase-like (Z-LLE-AMC, 400 µM; Enzo Life Sciences) activity were subsequently added and substrate cleavage was determined by quantification of 7-amino-4-methylcoumarin (AMC) fluorescence (excitation 360 nm, emission 460 nm) over 8 h and referenced to an AMC standard curve.

Susceptibility of the designed modulators to degradation by the 20S proteasome was examined by reversed-phase high-performance liquid chromatography (S10 Fig). The compounds were dissolved in water or DMSO to 10 mM concentration and then diluted with 25 mM Tris buffer containing 0.25 mM EDTA (pH 8.0) to the final concentration of 1 mM. 2.5 μg of human 20S proteasome (Enzo Life Sciences) was added to the mixture, which then, after adjustment of its final volume to 100 μl, was incubated at 37°C for 3 h. The reaction was stopped by adding 5 μl of 10% aqueous trifluoroacetic acid. Next, 40 μl of the mixture, along with the control not containing the 20S, were injected onto C8 Kromasil column (4,6 x 250 mm, 100 Å, 5 μm). The linear gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid was performed with SIL-20AHT UFLC system equipped with SPD-M20A PDA detector (SHIMADZU), and the elution was observed at the wavelength of 223 nm.