Raji and THP-1 cells were cultured in RPMI-1640 medium with 10% FBS and 1% penicillin-streptomycin. When the cell concentration reached 8x105 cells/mL, the cell concentration did not exceed 1 x 106 cells/mL, and the medium was renewed every 2 days. Bortezomib (s1013) and ONX-0914 (s7172) were ordered from Selleckchem, and Ac-ANW-AMCs were ordered from R&D (s-320). Digitonin (D141) and NP-40 (492016) were purchased from Sigma, and the proteasome-GLO™ kit (G8661) was purchased from Promega. A BioTek Synergy H1 Hybrid Multi-Mode Reader was used to perform all assays. We performed the analysis and statistics with GraphPad Prism 8.0 version.
The basic process of detecting β5i subunit activity in cultured cells
Raji and Jurkat cell lines are usually used to purify immunoproteasomes and screen β5i inhibitors in vitro. Bortezomib is the first FDA-approved proteasome inhibitor for the treatment of certain multiple myeloma, and ONX-0914 is the first selective β5i inhibitor as an ideal positive control. The cell-based reagents contain a specific immunoproteasome substrate in the reaction buffer and an optimized cell permeabilization buffer that retains the high activity of immunoproteasomes. The peptide substrate Ac-ANW-AMC is cleaved by immunoproteasomes and will generate fluorescent signaling detected by the microplate reader at an emission of 460 nM. We first determined the cell number and cell lysis buffer that showed the best activity to reach the highest reaction rate, so we tried to utilize three different lysis buffers to compare their slopes of reaction. Figure 1a indicates that one hundred thousand cells are good enough to make the reaction, and the immunoproteasome exhibits the best activity in the presence of buffer 1, including digitonin. Increasing digitonin to 1 mM dramatically improved the efficiency under these conditions, suggesting that completely lysed cells contribute to the release of more immunoproteasomes recognizing its substrate (Fig. 1b). We further confirm that the protease cocktail is dispensable for the reaction in a shorter period; one million cells versus 100 µL cell lysis buffer are working well, there is no difference between the 60 µL and 80 µL reaction systems, and other benefits can save the cost of experiments with the 60 µL reaction system (Fig. 1c). Indeed, more substrates will lift the steep slope than the lower concentration, a potential danger factor for substrates that might be nonspecifically recognized by other proteases. The final concentration of 40 µM substrate presented productive outcomes in our hands (Fig. 1d). Following the current protocol, we measured the inhibition of ONX-0914 in Raji cells, and the IC50 of 2.78 µM was higher than the published data (5 nM). In addition to the β5i assay, we could not obtain the ideal IC50 of β5c if the cell lysis buffer of the proteasome-GLO kits was changed to 1 mM digitonin buffer. The IC50 of 352 nM is 5-fold higher than the published data (Fig. 1e). According to the toxicity of inhibitors, these inhibitors were added and incubated for 6 hours with cultured cells; otherwise, the cell proliferation was reduced. Figure 1g.
Optimization of detergents in cell lysis buffer
We tried to combine two different detergents in cell lysis buffer to give rise to the potency of lysed cells. Digitonin (1 mM) easily resulted in some larger debris in the cell pellets after centrifugation, and 0.01% NP-40 was added to the buffer and reversed this observation. We also used 1 mM DTT to maintain the stable activity of the immunoproteasome. These changes reduced the IC50 of β5c to 72 nM (Fig. 2b), but the IC50 of β5i was still higher than the ideal value (Fig. 2a). Using this lysis buffer, the lower substrate does not affect the final IC50 except for the relative kinetic rate; however, decreasing the amount of the cell lysate leads to the delayed maximum rate (Fig. 3c). We also found a trick of this assay. The reaction buffer with 80 µM Ac-ANW-AMC mixed with the same amount of cell lysate was held for 30 minutes at room temperature before use. We wondered whether DMSO becomes insoluble at lower temperatures (Fig. 2d). If the key step was correct, the IC50 of β5i was 34 nM.
High doses of DMSO in resolving substrates limit the maximum rate
As mentioned above, we assume that DMSO tolerance is a variable factor in determining the kinetic rate. If so, we measured the IC50 of ONX-0914 with different DMSO concentrations. Figure 3a shows that lower concentrations of DMSO are useful to improve the kinetic process. The IC50 of 7.6 nM is close to the published data; conversely, the final 1% DMSO of the reaction will inhibit the activity of enzymes and increase the IC50 to 136 nM. After modification of the current protocol, we obtained perfect IC50 values of bortezomib and ONX-0914 that reached approximately 2.5 nM and 3.5 nM, respectively (Fig. 3b). The kinetic rate was in the manner of lineage, and we still obtained a similar result to the value of the endpoint to calculate IC50 (Fig. 3c). To determine whether this assay is stable to run in different cell lines, we also compared the IC50 in both Raji cells and THP-1 cells. Figure 3d shows the comparable IC50 of both inhibitors exhibited in different cells. Now, we believe that the current version is acceptable to study functional immunoproteasomes in the future. We also detected the compatibility of the β5i assay with proteasome-GLO kits. Raji cell lysate was used to measure β5i activity, which also works in the β5c assay. The IC50 of ONX-0914 was 78 nM with the substrate of β5c, so we could finish two different assays with the same lysis buffer (Fig. 3d).
The final protocol of the β5i assay kit
1. Cell lysis buffer and β5i substrate solution
Cell lysis buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCL2, 5 mM ATP, 1 mM DTT, 0.01% NP-40, 1 mM digitonin and protease cocktails. Ac-ANW-AMC working solution: 50mM Tris, pH 7.5, 150mM NaCl, 5mM MgCL2, 5mM ATP, 80uM Ac-ANW-AMC and 1mM DTT, freshly making before using, 30µL substrate solution mixed with 30µL cell lysate.
2. Preparation of cell lysates in the β5i assay
2.1 A total of 1×105 Raji cells were seeded into 48-well plates, and different doses of inhibitors were added to duplicate wells and incubated for 6 hours with cells at 37°C.
2.2 The cells were harvested and centrifuged at 10000 rpm for 2 min, the medium was removed, the cell pellet was washed once with 1 mL PBS, and the remaining PBS was removed.
2.3 One hundred microliters of cell lysis buffer was used to resuspend the cell pellet, which was pipetted up and down 20 times, left on ice for 30 minutes, and then centrifuged at 13000 rpm for 10 minutes.
2.4 Loading 30uL cell lysate into 96 wells white plate and mixed well with another 30uL substrate working solution, incubation 5 to 10min at 37 degrees, start setting up the microplate reader with the following program during incubation.
2.5 Step 1: Set up the temperature at 37 degrees; Step 2: delay 1 min; Step 3: select the dynamic assay, total time 30 min, 30 cycles; Step 4: insert fluorescent detection with Ex 380 and Em 460.
Tips1: Leave the Ac-ANW-AMC working solution at room temperature before loading.
Tips 2: The AC-ANW-AMC stock solution was made into 8 mM DMSO so that the final DMSO concentration was 0.5% during the reaction period.
2.6 The cell lysate is compatible with proteasome-GLO kits (G8661)
Loading 30uL cell lysate and then adding 30uL Suc-LLVY-Glo™ Substrate working solution, incubation 5 to 10min at room temperature, Measure the luminescence of each sample with a microplate luminometer as directed by the manufacturer(G8661).
2.7 Blank control: substrate working solution + cell lysis buffer (without cells) and vehicle control used DMSO.