Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington’s Disease

<div><p>Huntington’s disease is the result of a long polyglutamine tract in the gene encoding huntingtin protein, which in turn causes a large number of cellular changes and ultimately results in neurodegeneration of striatal neurons. Although many theories have been proposed, the precise mechanism by which the polyglutamine expansion causes cellular changes is not certain. Some evidence supports the hypothesis that the long polyglutamine tract inhibits the proteasome, a multiprotein complex involved in protein degradation. However, other studies report normal proteasome function in cells expressing long polyglutamine tracts. The controversy may be due to the methods used to examine proteasome activity in each of the previous studies. In the present study, we measured proteasome function by examining levels of endogenous peptides that are products of proteasome cleavage. Peptide levels were compared among mouse striatal cell lines expressing either 7 glutamines (ST<i>Hdh</i>^Q7/Q7) or 111 glutamines in the huntingtin protein, either heterozygous (ST<i>Hdh</i>^Q7/Q111) or homozygous (ST<i>Hdh</i>^Q111/Q111). Both of the cell lines expressing huntingtin with 111 glutamines showed a large reduction in nearly all of the peptides detected in the cells, relative to levels of these peptides in cells homozygous for 7 glutamines. Treatment of ST<i>Hdh</i>^Q7/Q7 cells with proteasome inhibitors epoxomicin or bortezomib also caused a large reduction in most of these peptides, suggesting that they are products of proteasome-mediated cleavage of cellular proteins. Taken together, these results support the hypothesis that proteasome function is impaired by the expression of huntingtin protein containing long polyglutamine tracts.</p></div

Summary plots of the peptidome of Q7Q7 cells in response to proteasome inhibitors.

<p>A: Cells were treated with 200 nM epoxomicin for 1 hour. B: Cells were treated with 500 nM bortezomib for 1 hour. The relative levels of all peptides identified by MS/MS analysis in each of the inhibitor-treated replicates was compared to the average level of the peptide in the untreated control replicates. The <i>y</i>-axis represents the relative peptide levels (log-scale) and the <i>x</i>-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 inhibitor-treated cells, expressed relative to the average control value. Black circles indicate the ratio of each control replicate expressed relative to the average control value.</p

Representative MS data from peptidomics experiment comparing peptide levels between Q7Q7, Q7Q111 and Q111Q111 cells.

<p>In this experiment, the two replicates of Q7Q7 cells were labeled with D0- and D3-TMAB-NHS, one replicate of the Q7Q111 cells was labeled with D9-TMAB-NHS and two replicates of Q111Q111 cells were labeled with D6- and D12-TMAB-NHS. A: Example of a peptide that is present in the Q111-expressing cell lines at levels much lower than in Q7Q7 cells. This peptide was identified by MS/MS analysis as TLLIKTVETRDGQVINETSQHHDDLE, derived from vimentin. B: Example of a peptide that is present in the Q111-expressing cell lines at levels slightly lower than in Q7Q7 cells. This peptide was identified by MS/MS analysis as Ac-MDTSRVQPIKLAR, derived from the 40S ribosomal protein S28. C: Example of a peptide that is present in all cell lines at comparable levels. This peptide was identified by MS/MS analysis as Ac-ADEIAKAQVAQPGGDTIFGKIIRK, derived from histidine triad nucleotide-binding protein 1.</p

Analysis of amino acids in the P1 position of the cleavage site.

<p>All identified peptides in each experiment were grouped into categories based on levels relative to those in untreated Q7Q7 cells. Decrease (green bars) represent ratio ≤0.80, no change (grey bars) represent ratios of 0.81 to 1.24, increase (red bars) represent ratio ≥1.25. The residue in the P1 position of the cleavage site was considered for every peptide; peptides found in multiple replicates were counted each time found. For peptides that represent the N-terminal or C-terminal fragment of the protein, a single cleavage is sufficient to generate the peptide. Peptides that represent internal fragments of proteins require two cleavages, and so the number of cleavage sites is greater than the number of peptides. A: Cleavage site analysis of peptides in Q111-expressing cells. The data for peptides in heterozygous Q7Q111 cells were pooled with the data for peptides in homozygous Q111Q111 cells, resulting in 1441 cleavage sites for peptides that decreased (relative to Q7Q7 cells) and 73 cleavage sites for peptides in the “no change” group. Only 12 cleavage sites from 7 peptides were in the “increased” group, so it was not included in the graph. B: Cleavage site analysis of peptides in Q7Q7 cells treated with 200 nM epoxomicin for 1 hour. Peptides found to decrease represent 775 cleavage sites, the “no change” group represent 82 cleavage sites, and the increase group represents 51 cleavage sites. C: Cleavage site analysis of peptides in Q7Q7 cells treated with 500 nM bortezomib for 1 hour. There were 417 cleavage sites in the group that decreased, 59 cleavage sites in the unchanged group, and 183 cleavage sites in the group that increased after the treatment.</p

Measurement of proteasome activity in Q7Q7, Q7Q111 and Q111Q111 cells.

<p>Cell extracts were incubated with fluorogenic peptide substrates for 1 hour at 37°C, following which enzyme activity was determined by fluorescence measurement of AMC. The activity in Q7Q111 and Q111Q111 cells is expressed as percent enzyme activity relative to the Q7Q7 cells. Activities of the chymotrypsin-like (β5) and trypsin-like (β1) proteolytic subunits were measured by cleavage of Suc-Leu-Leu-Val-Tyr-AMC (A) and Ac-Arg-Leu-Arg-AMC (B) respectively. The error bars show standard error of mean (n = 10 for Suc-Leu-Leu-Val-Tyr-AMC and n = 5 for Ac-Arg-Leu-Arg-AMC). Statistical analysis was performed using Student’s t-test: *, p ≤ 0.05; ns, no significant difference (p > 0.05) versus Q7Q7 cells.</p

Summary plots of the peptidome of Q7Q7, Q7Q111 and Q111Q111 cells.

<p>The relative levels of all peptides identified by MS/MS analysis in the Q7Q111 (A) and Q111Q111 (B) cells were compared to the average level of the peptide in Q7Q7 cells. The <i>y</i>-axis represents relative peptide levels (log-scale) and the <i>x</i>-axis represents the rank order of peptides sorted according to the relative level. For those peptides detected multiple times (different charge states and/or tag numbers), the relative ratio for each form was considered separately. If the ratio was <0.20, the value was capped at 0.20 to reflect the typical signal to noise ratio. The red circles represent the ratio of each replicate of the identified peptides in Q7Q111 or Q111Q111 cells, expressed relative to the level in Q7Q7 cells for that LC-MS run. The black circles represent the ratio of each Q7Q7 replicate expressed relative to the average level of Q7Q7 replicates in that LC-MS run (i.e. run 1 and 2; the other two runs did not include replicates of Q7Q7 cells).</p

Effect of bortezomib or epoxomicin on relative levels of peptides in yeast.

<p>Wild type yeast (top panel) or yeast deleted for drug transporter genes <i>PDR5</i> (second panel) or <i>SNQ2</i> (bottom two panels) were treated with proteasome inhibitors for 1 hour and then processed for peptidomics as described in Materials and Methods. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163312#pone.0163312.s003" target="_blank">S1 Fig</a> for labeling scheme used for quantitative peptidomics. Wild-type yeast were treated with 1 μM bortezomib, the <i>pdr5</i>Δ and <i>snq2</i>Δ strains were treated with 10 μM bortezomib, the <i>snq2</i>Δ strain was treated with 4 μM epoxomicin and control replicates were treated with a comparable amount of drug vehicle alone (maximum 0.1% DMSO). Each replicate was compared to the average level of that peptide in the control replicates and the individual ratios sorted by rank and plotted. The y-axis represents the relative level of each replicate, comparing either drug-treated to the average control values (red) or the control replicates to the average control values (black). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163312#pone.0163312.s009" target="_blank">S5 Table</a> for relative peptide levels of each peptide in the various treatments.</p

Analysis of the turnover rate of proteins found in the analysis of the yeast peptides.

<p>Protein half-life data were reported by Christiano et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163312#pone.0163312.ref032" target="_blank">32</a>] and were determined by growing yeast in heavy [13C6/15N2] L-lysine for many generations, then growing in light lysine for various lengths of time and measuring the ratio of heavy/light proteins using mass spectrometry-based proteomics. The half-life was capped by Christiano et al at 100 hours. Out of 75 proteins found in our peptidomics analysis, 73 corresponded to proteins detected by Christiano et al. The relative half life (log scale) for these 73 proteins and for all 3772 proteins reported by Christiano are plotted as a percentile of the half-life for each group of proteins.</p

Analysis of the Yeast Peptidome and Comparison with the Human Peptidome

<div><p>Peptides function as signaling molecules in species as diverse as humans and yeast. Mass spectrometry-based peptidomics techniques provide a relatively unbiased method to assess the peptidome of biological samples. In the present study, we used a quantitative peptidomic technique to characterize the peptidome of the yeast <i>Saccharomyces cerevisiae</i> and compare it to the peptidomes of mammalian cell lines and tissues. Altogether, 297 yeast peptides derived from 75 proteins were identified. The yeast peptides are similar to those of the human peptidome in average size and amino acid composition. Inhibition of proteasome activity with either bortezomib or epoxomicin led to decreased levels of some yeast peptides, suggesting that these peptides are generated by the proteasome. Approximately 30% of the yeast peptides correspond to the N- or C-terminus of the protein; the human peptidome is also highly represented in N- or C-terminal protein fragments. Most yeast and humans peptides are derived from a subset of abundant proteins, many with functions involving cellular metabolism or protein synthesis and folding. Of the 75 yeast proteins that give rise to peptides, 24 have orthologs that give rise to human and/or mouse peptides and for some, the same region of the proteins are found in the human, mouse, and yeast peptidomes. Taken together, these results support the hypothesis that intracellular peptides may have specific and conserved biological functions.</p></div

Primary function of proteins found in the peptidomic analysis of human and yeast cells.

<p>For each protein, the major function reported in UniProt was used.</p