Z lle amc

Tissue homogenates containing proteasome were prepared just after dissection using ice-cold homogenization buffer containing: 20 mM Tris-HCl (pH 7.6), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA and 2 mM DTT (Hovhannisyan et al., 2019 (link)). Samples were minced with scissors and homogenized using plastic pestles, incubated 30 min on ice, then centrifuged at 12,000 g for 15 min at 4°C. Protein concentration was measured using the Bradford method with bovine serum albumin as a standard. The proteasomal chymotrypsin-like, trypsin-like and caspase-like activities of the 20S catalytic core were assayed using the fluorogenic substrates N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC, Enzo Life Sciences), Bz-Val-Gly-7-amino-4-methylcoumarin (Bz-VGR-AMC, Enzo Life Sciences) and Z-Leu-Leu-Glu-7-amino-4-methylcoumarin (Z-LLE-AMC, Enzo Life Sciences), respectively. The mixture, containing 10 μg of total protein in 20 mM Tris (pH 8) and 10% gylcerol, was incubated at 37°C with 20 μM peptide substrates in a final volume of 100 μl. Enzymatic kinetics were monitored in a temperature-controlled microplate fluorimetric reader (FLUOstar Galaxy, BMG labtech). Excitation/emission wavelengths were 350/440 nm. The difference between assays with or without MG-132, a proteasome inhibitor, represented the proteasome-specific activity.

Blood cells were sonicated in lysis buffer containing 50 mM Tris-HCl (pH = 7.5), 1 mM EDTA, 1 mM ethylene-bis(oxyethylenenitrilo)tetraacetic acid EGTA, 0.5% Triton X, and centrifuged at 12,000× g for 15 min at 4 °C. The supernatants were diluted to 2 mg protein/mL and assayed for proteasome activity in the assay buffer containing 100 mM Tris/HCl pH = 7.5, 1 mM EDTA, 1 mM EGTA, using the chymotrypsin-like activity substrate, Suc-LLVY-7-amino-4-methylcoumarin (Suc-LLVY-AMC; Sigma-Aldrich, St. Louis, MO, USA), the trypsin-like activity substrate, Bz-VGR-AMC (Enzo Life Sciences, Inc., Farmingdale, NY, USA), or the caspase-like activity substrate, Z-LLE-AMC (Enzo Life Sciences, Inc., Farmingdale, NY, USA). All substrates were at a concentration of 100 μM in a final volume of 50 μL [75 (link)]. All components were added to a black 96 well plate and incubated at 37 °C for 30 min. The released fluorogenic 7-amino-4-methylcoumarin (AMC) concentration was measured spectrofluorometrically [355 nm excitation and 460 nm emission] using a fluorometric plate reader (EnSpire 2300 Multilabel Reader, PerkinElmer, Waltham, MA, USA). The amount of released AMC was evaluated from the AMC reference curve, which allowed expression of proteasome activity in pmol AMC/min/mg protein to be assessed. All tests were carried out in triplicate.

Proteasome chymotryptic and caspase activity was assayed as the rate of hydrolysis of the fluorogenic peptide suc-LLVY-AMC (Sigma Aldrich) or Z-LLE-AMC (Enzolifesciences, Farmingdale, New York), respectively. Cell extracts were prepared in 25 mM Tris HCl, pH 7.5 using sonication. Protein lysate (20 µg) was incubated with 12.5 µM suc-LLVY-AMC or Z-LLE-AMC in a total volume of 200 µl. MG132 was spiked into the reaction well at a final concentration of 0.1 µM where indicated. AMC fluorescence was measured using 355 nm excitation and 460 nm emission filters with free AMC (Sigma Aldrich) as standard every min for 30 min at 37°C.