Figure 1.
The 26S proteasome, a multicatalytic complex cleaves proteins into peptides typically 3–25 residues long, which are further degraded into amino acids by a variety of downstream endopeptidases and/or aminopeptidases.
Figure 2.
Inhibition of proteasome activity in HEK293T cells with various proteasome inhibitors.
HEK293T cell extract was treated with increasing concentrations of the indicated compound for 30-Leu-Leu-Val-Tyr-AMC and incubation for 1 hour at room temperature. Enzyme activity was determined by fluorescence measurement of AMC. The resulting activity is expressed as percent enzyme activity relative to the control reaction without inhibitor. The error bars show standard error of mean (n = 3); points without error bars had error ranges smaller than the symbol size. Statistical analysis was performed using Student’s t-test: ***, p≤0.001; ns, no significant difference (p>0.05) versus the control without inhibitor.
Figure 3.
Summary plots of the peptidome of HEK293T cells in response to proteasome inhibitors.
The relative level of all the peptides identified by MS/MS analysis in each of the drug-treated replicates was compared to the average level of the peptide in the untreated control replicates. The y-axis represents the relative peptide levels (log-scale) sorted from low to high and the x-axis represents the rank order of each peptide sorted according to the relative level. When a peptide is detected in multiple charged states, the peptide ratio for each peptide is indicated separately. If the ratio was <0.20 or >5.0 between the drug-treated and control replicates, the value was capped at 0.20 or 5.0. Each panel shows two plots; one shows the peptide level of each replicate of the identified peptides in drug-treated cells, expressed relative to the average control value (red circles), the other shows the peptide level of each control replicate expressed relative to the average control value (black circles). Panels A–H represent data from experiments on HEK 293T cells treated with (A) 0.2 µM epoxomicin for 1 hour, (B) 0.5 µM bortezomib for 1 hour, (C) 2.5 µM MG132 for 35 min, (D) 1 µM MG262 for 35 min, (E) 1 µM clasto-Lactacystin β-lactone for 35 min, (F) 1 µM MLN2238 for 35 min, (G) 1 µM carfilzomib for 35 min, (H) 1 µM AM114 for 35 min. The data used to generate the plots shown in panels A and B were previously published [18], [19].
Figure 4.
Summary plots of the peptidome of SH-SY5Y cells in response to proteasome inhibitors.
Panels A–F represent data from experiments on SH-SY5Y cells treated with (A) 2.5 µM MG132 for 35 min, (B) 0.5 µM bortezomib for 1 hour, (C) 1 µM clasto-Lactacystin β-lactone for 35 min, (D) 1 µM MG262 for 35 min, (E) 1 µM carfilzomib for 35 min, (F) 1 µM MLN2238 for 35 min. Specifications and criteria for making the summary plots are described in Figure 3. The data used to generate the plot shown in panel B was previously published [18].
Figure 5.
Heat map analysis of selected peptides.
Peptides observed in multiple experiments were chosen for this analysis. Different peptides are plotted as separate rows; each column indicates a different experiment and each sub-column represents peptide levels in the biological replicates within that experiment. Ratios of peptides found in multiple charged states were averaged together. White panels could not be accurately quantified, either due to undetectable signals or peak overlap with another co-eluting peptide. Data from the AM114 experiment was not included in the heat map since levels of most peptides were not affected by AM114. Peptide levels were color coded. Bright green indicates peptides that were present in the treated cells at levels ≤50% of those in the control cells (ratio ≤0.50). Dark green indicates peptides that showed a smaller decrease (ratio 0.51–0.80). Grey indicates peptides that were close to the levels in the control cells (ratio 0.81–1.24). Dark red indicates peptides that were elevated 25–99% (ratio 1.25–1.99). Bright red indicates peptides that were elevated ≥100% (ratio ≥2.00). The three groups in the heat map indicated by lines and numbers on the left margin indicate peptides which showed a large or partial decrease (set 1), little or no change (set 2), or a large increase (set 3) in response to the proteasome inhibitors. See Table S2 for an expanded version of this figure, including peptide sequences and ratio values.
Table 1.
Peptides decreased by treatment of cells with proteasome inhibitors.
Table 2.
Peptides not altered substantially upon treatment of cells with proteasome inhibitors.
Table 3.
Peptides elevated by treatment of cells with proteasome inhibitors.
Figure 6.
Effect of butabindide and bortezomib on Ala-Ala-Phe-AMC cleavage in HEK293T cells.
HEK 293T cell extracts were treated with increasing concentrations of butabindide (Panel A) or bortezomib (Panel B) for 30 minutes in the absence of substrate, and then substrate was added and the reaction incubated for 1 hour. Enzyme activity was determined by fluorescence measurement of AMC and expressed as percentage enzyme activity relative to the control lacking inhibitor. Error bars show standard error of mean (n = 3), points without error bars have error ranges smaller than the symbol size. Statistical analysis was performed using Student’s t-test: *, p≤0.05; ***, p≤0.001; ns, no significant difference (p>0.05) versus the control.
Figure 7.
Effect of aminopeptidase inhibitors on Ala-AMC and Leu-AMC cleavage.
HEK 293T cell extract (panels A–D) or recombinant PSAP (panels E–F) were treated with increasing concentrations of bestatin or puromycin for 30 minutes followed by the addition of substrate and incubation for 1 hour. Enzyme activity was determined by fluorescence measurement of AMC and expressed as percentage enzyme activity relative to the control without inhibitor. (A) Bestatin inhibition of HEK293T cell extract assayed with Ala-AMC. (B) Bestatin inhibition of HEK293T cell extract assayed with Leu-AMC. (C) Puromycin inhibition of HEK293T cell extract assayed with Ala-AMC. (D) Puromycin inhibition of HEK293T cell extract assayed with Leu-AMC. (E) Puromycin inhibition of purified PSAP assayed with Ala-AMC. (F) Puromycin inhibition of purified PSAP assayed with Leu-AMC. Error bars show standard error of mean (n = 4), points without error bars had error ranges smaller than the symbol size. Statistical analysis was performed using Student’s t-test: *, p≤0.05; **, p≤0.01; ***, p≤0.001; ns, no significant difference (p>0.05) versus the control.
Figure 8.
Effect of proteasome inhibitors on Ala-AMC and Leu-AMC cleavage.
HEK 293T cell extract or recombinant PSAP was treated with 10 µM of each of the proteasome inhibitors for 1 hour in addition to a 30 min pre-incubation without the substrate. Enzyme activity was determined by fluorescence measurement of AMC and expressed as percentage enzyme activity relative to the control without inhibitor. Panels A and B represent the effect of proteasome inhibitors on cleavage of Ala-AMC and Leu-AMC respectively, in HEK293T cell extract. Panel C shows effect of the inhibitors on cleavage of Ala-AMC by purified PSAP. Error bars show standard error of mean (n = 4). Statistical analysis was performed using Student’s t-test: *, p≤0.05; **, p≤0.01; ***, p≤0.001; ns, no significant difference (p>0.05) versus the control.
Table 4.
Enzyme activity of HEK293T cell extracts assayed with Ala-AMC in the absence or presence of various inhibitors.
Figure 9.
Summary plots of the peptidome of HEK293T cells in response to aminopeptidase inhibitors.
Panels A–C represent data from experiments on HEK293T cells treated with (A) 100 µM bestatin methyl ester for 75 min; (B) 100 µM butabindide for 35 min; (C) 100 µM butabindide for 75 min. Specifications and criteria for the summary plots are described in Figure 3 legend.
Figure 10.
Effect of bortezomib on the chymotryptic-like activity of different forms of the proteasome from yeast.
Purified 20S subunit, Blm10-20S, open gate mutant of the 20S subunit and 26S were combined with the indicated concentrations of bortezomib in the presence of substrate. After incubation for 15 minutes, enzyme activity was measured by monitoring the fluorescence kinetics for the next 30 minutes. Panel A represents inhibition of 26S and 20S by bortezomib in the presence of buffer containing ATP. Panel B represents inhibition of Blm10-20S, the open gate mutant and 20S by bortezomib in the presence of buffer without ATP. Enzyme activity is expressed as percent control without inhibitor. Error bars show standard error of the mean; error bars smaller than symbol size are not visible.