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. 2019 May 28;9(6):207.
doi: 10.3390/biom9060207.

Effect of Protein Denaturation and Enzyme Inhibitors on Proteasomal-Mediated Production of Peptides in Human Embryonic Kidney Cells

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Free PMC article

Effect of Protein Denaturation and Enzyme Inhibitors on Proteasomal-Mediated Production of Peptides in Human Embryonic Kidney Cells

Sayani Dasgupta et al. Biomolecules. .
Free PMC article

Abstract

Peptides produced by the proteasome have been proposed to function as signaling molecules that regulate a number of biological processes. In the current study, we used quantitative peptidomics to test whether conditions that affect protein stability, synthesis, or turnover cause changes in the levels of peptides in Human Embryonic Kidney 293T (HEK293T) cells. Mild heat shock (42 °C for 1 h) or treatment with the deubiquitinase inhibitor b-AP15 led to higher levels of ubiquitinated proteins but did not significantly increase the levels of intracellular peptides. Treatment with cycloheximide, an inhibitor of protein translation, did not substantially alter the levels of intracellular peptides identified herein. Cells treated with a combination of epoxomicin and bortezomib showed large increases in the levels of most peptides, relative to the levels in cells treated with either compound alone. Taken together with previous studies, these results support a mechanism in which the proteasome cleaves proteins into peptides that are readily detected in our assays (i.e., 6-37 amino acids) and then further degrades many of these peptides into smaller fragments.

Keywords: Proteasome; bortezomib; epoxomicin; mass spectrometry; peptide; peptidomics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Analysis of the effect of b-AP15 and heat shock on the ubiquitination of proteins, using anti-ubiquitin antibody. Top panels: representative Western blots for ubiquitination and α-tubulin (as loading control) in HEK 293T cells after various treatments. Lower panels: the broad “smear” of ubiquitinated proteins from 50 kDa to the top of the gel was quantified using ImageJ and normalized to α-tubulin. Treatments: 1 µM b-AP15 in 0.05% DMSO for 1 h or control (0.05% DMSO alone) for 1 h (A,B); heat shock at 42 or 37 °C control for 20 min (C,D); heat shock at 42 or 37 °C control for 1 h (E,F). Error bars represent the standard error of mean (n = 6). ***, p ≤ 0.001 and **, p ≤ 0.01 using Student’s t-test; ns: not significant.
Figure 2
Figure 2
Percentage of peptides present in groups divided according to relative level. For samples subjected to b-AP15 (1 µM, 1 h), heat shock (42 °C for 20 min and 1 h), and cycloheximide (0.2 mM, 35 min) treatment, the ratios for both replicates were averaged for the analysis and the peptides were subsequently grouped based on the average relative levels. For peptides found multiple times with different charge states and/or numbers of tags, the peak intensities were summed and expressed as the ratio of average control such that a single value for peptide ratio could be obtained for each replicate, which were then averaged. For the controls, peptide ratios of each replicate of every peptide from all the experiments were combined together for the analysis. Data for each of the peptides are provided in Table S1.
Figure 3
Figure 3
Mean relative levels of peptides from proteins in response to treatment with 1 µM b-AP15 for 1 h (A), 42 °C heat shock for 20 min (B) or 1 h (C), and 0.2 mM cycloheximide for 35 min (D). For all the proteins that gave rise to at least two peptides, the relative levels of all peptides (including different charge states and/or number of tags) derived from a single protein were averaged. Ratios of 0.8 and 1.25 are marked with dashed lines, and 1.0 is marked with a dotted line. Error bars indicate the range between the largest (upwards error bar) and smallest (downwards error bar) peptide ratio from each protein. * p < 0.05; ** p < 0.01. Mean relative levels of peptides derived from these proteins, statistical significance, and number of peptides from each protein are provided in Table S2.
Figure 4
Figure 4
Summary plots of the peptidome of HEK293T cells in response to proteasome inhibitors. (A) Cells were treated for 30 min with a combination of 0.2 µM epoxomicin and 0.5 µM bortezomib or with 0.5 µM bortezomib alone (all in the presence of 0.05% DMSO). (B) Data from a previously published study in which cells were treated with 0.2 µM epoxomicin in 0.05% DMSO, or 0.05% DMSO alone for 1 h [21]. (C) Cells were treated for 30 min with a combination of 0.2 µM epoxomicin and 0.5 µM bortezomib or with 0.2 µM epoxomicin alone; all samples included 0.05% DMSO. (D) Data from a previously published study in which cells were treated with 0.5 µM bortezomib in 0.05% DMSO or with 0.05% DMSO for 30 min [27]. The y-axis represents the relative levels of peptides (log-scale) and the x-axis represents the rank order of peptides sorted according to the relative level. If the ratio was <0.20 or >5.0, the value was capped at 0.20 or 5.0 to reflect the typical signal-to-noise ratio. Red circles indicate the ratio of each replicate of identified peptides in cells treated with the combination of epoxomicin and bortezomib in panels A and C and either inhibitor alone in panels B and D, expressed relative to the average of the respective controls (i.e., for panel A, the red circles represent the relative level of each peptide in cells treated with both inhibitors relative to cells treated with bortezomib alone, while, for panel C, the red circles represent the relative level of each peptide in cells treated with both inhibitors relative to cells treated with epoxomicin alone). Black circles indicate the ratio of each control replicate expressed relative to the average control value (either bortezomib or epoxomicin alone in panels A and C; DMSO in panels B and D). See Figure S2 for details on the treatments of cells for each of these peptidomic studies.
Figure 5
Figure 5
Heat map analysis of selected peptides. Peptides commonly detected in most experiments were selected for this analysis. Each row denotes a specific peptide, and each column represents a different experiment described in Figure 4. Peak intensities of peptides found with multiple charge states and/or numbers of tags were summed and expressed as a ratio of average control such that a single value for the peptide ratio could be obtained for each replicate. The ratio was color-coded using the scheme shown in Figure 2, with green representing decreases and red representing increases. Grey represents peptides that did not change substantially. White corresponds to peptides that were either not detected or which could not be accurately quantified due to peak overlap with another co-eluting peptide. Names of protein precursors, peptide sequences, mass, cleavage sites, and peptide ratios are provided in Table S4.
Figure 6
Figure 6
Relative levels of all peptides derived from heat shock 10-kDa protein 1 in HEK293T cells. Cells were treated as described in Figure 4 and the ratio for each peptide was color-coded using the scheme shown in Figure 2, with green representing decreases, red representing increases, and grey representing no substantial change.
Figure 7
Figure 7
Correlation of the effect of proteasome inhibitors with the location of the peptide within the protein precursor. The N-terminal group includes peptides lacking the initiation Met. Cells were treated as described in Figure 4 and color coded as described in Figure 2.

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