Proteasome (trypsin-like and caspase-like) activity assay

Proteasome activity assay to evaluate trypsin-like and caspase-like activity of the proteasome in immortalized human cardiomyocytes was used. Trypsin-like and caspase-like activities were determined utilizing an AMC-tagged peptide substrate, which releases free highly fluorescent AMC (7-amido-4-methyl coumarin) in the presence of proteasome proteolytic activity. The assay was performed in the presence and absence of bortezomib (Santa Cruz Biotechnology, Dallas, TX, USA) proteasome inhibitor. Briefly, cells were plated (seeding density 2.5 × 10⁴ cells/cm²) in 10% FBS-supplemented medium w/o phenol red into a flask T75 cm². The next day, the culture medium was replaced with 1% FBS-supplemented medium w/o phenol red, this culture medium was daily replaced until the seventh day of culture. On the seventh day of culture, the cells were exposed, for 24 h, to 100 and 200 μM of NSAIDs in 1% FBS-supplemented medium w/o phenol red. After exposure to treatments, the cell monolayer was detached by trypsin and centrifuged 6 min at 250 g. The cell pellets were washed with cold PBS and transferred into 1.5 ml tubes, then centrifuged 6 min at 250 g. Proteasome lysis buffer (50 mM Tris–Hcl pH 7.5, 250 mM sucrose, 5 mM MgCl₂, 0.5 mM EDTA free acid, 1 mM DTT, 2 mM ATP, 0.025% digitonin, 10% glycerol, all chemicals were purchased from all chemicals purchased from Sigma, St. Louis, Mo, USA) in Milli-Q-water was used to suspend cell pellets, to obtain crude protein extract. About 120 μl, for cell pellets, of proteasome lysis buffer were used. After homogenization by pipetting up and down ten times, the extracts were incubated 10 min at 4 °C. After centrifugation 30 min at 20,000 g (by refrigerated mini centrifuge), the supernatants (protein crude extract) were collected and maintained at 4 °C, ready for the assay. The total protein content was determined by extrapolation from a BSA standard curve (0.025–2 mg/ml). Protein crude extracts were dilute in proteasome assay buffer (50 mM Tris–HCl pH 7.5, 40 mM KCl, 5 mM MgCl₂, 0.5 mM ATP, 1 mM DTT, 0.05 mg/ml BSA in Milli-Q-water) to obtain 5–10 μg/100 μl of final protein concentration into the well. Extract samples and AMC standards (1–10 μM) were placed in 96 black well plate in a total volume of 100 μl. In all sample wells the fluorescent substrate AMC (final concentration 200 μM, trypsin-like [Boc-LRR-AMC] and caspase-like [Z-LLE-AMC] both from R&D System, Minneapolis, MN, USA) was placed with or without bortezomib proteasome inhibitor (final concentration 100 μM). After mixing all the components in the wells, the plate was incubated at 37 °C for 30 min protected from light (T1 measure). Trypsin-like and caspase-like activity at T1 was determined by measuring the fluorescence released from the AMC substrates, using a PerkinElmer VICTOR³, at an Ex-355 and Em-460 nm. After the first measurement, the plate was incubated at 37 °C for other 30 min protected from light (T2 measure). Trypsin-like and caspase-like activity at T2 was determined by measuring the fluorescence released from the AMC substrates, using a PerkinElmer VICTOR³, at an Ex-355 and Em-460 nm, each experiment was performed in triplicate. To quantify specific proteasome activity at each T (T1 or T2), the fluorescence values from the wells without inhibitor were subtracted to the fluorescence values from the wells with inhibitor, to obtain total relative fluorescence unit (tRFU). Measurement of the well without proteasome inhibitor showed total proteolytic activity and the wells containing proteasome inhibitor showed non-proteasome activity. Then delta RFU = tRFU2–tRFU1 was calculated. Delta RFU values were applied to the AMC standard curve to obtain B, which is the amount of AMC in the sample well, expressed as pmol/well. Proteasome activity was obtained by:

where B is the amount of AMC (pmol) in the sample, calculated by the AMC standard curve. V is the total volume reaction (μl) in the well, T1 and T2 are the time (min) of the first and second reading, respectively.