Characterisation of 20S Proteasome in <i>Tritrichomonas foetus</i> and Its Role during the Cell Cycle and Transformation into Endoflagellar Form

<div><p>Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite <i>Tritrichomonas foetus</i>. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of <i>T</i>. <i>foetus</i>. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the <i>T</i>. <i>foetus</i> genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from <i>T</i>. <i>foetus</i>-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a <i>T</i>. <i>foetus</i>-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in <i>T</i>. <i>foetus</i>. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of <i>T</i>. <i>foetus</i> to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of <i>T</i>. <i>foetus</i> were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in <i>T</i>. <i>foetus</i>.</p></div

TEM of lactacystin-treated PS <i>T</i>. <i>foetus</i>.

<p>Parasites were incubated with 20 μM lactacystin for 12 h. The parasites exhibit uncommon enlarged endoplasmic reticulum (ER)-derived membranes and concentric membrane whorls (asterisks), which resemble autophagy vacuoles. Other structures, such as hydrogenosomes (H), nucleus (N) and axostyle (Ax) remain unaltered. Bars, 500 nm.</p

Amino acid sequence alignment of the <i>T</i>. <i>foetus</i> proteasomal α-type subunits.

<p>The alignment was performed using the CLUSTAL W program. The α subunit from the Archaea <i>Thermoplasma acidophilum</i> (TaciA—UniProt accession: P25156) was used as a reference to identify the conserved domains in the α-type subunits from <i>T</i>. <i>foetus</i>. Dashes indicate gaps introduced in the protein sequence alignment. Residues that are functionally conserved in all subunits are shaded in black. Residues that are functionally conserved in 75% to 87.5% and 50% to 62.5% of the subunits are shaded in dark and light grey, respectively. The N-terminal Tyr residue essential for assembly of the α ring is indicated by the arrowhead. The bracket indicates the N-terminal α-helical region (helix H0) responsible for interactions between the α-type subunits and their assembly into rings. The arrow indicates the position of the N-terminal Thr residue in the α3 subunit (shaded in blue) essential to regulate the gate-opening to the central proteolytic cavity of the proteasome. The highly conserved proteasome GxxxD motif (x represents any residue) and the α-family RPxG motif (x represents a hydrophobic residue) responsible for forming the base of the loop that constricts the central pore at the level of the α rings are indicated. The putative nuclear localisation signal sequence K(K/R)xxx(K/R) (x represents any residue) is also indicated. The asterisks show the amino acids at the turn region of the <i>T</i>. <i>acidophilum</i> α subunit and the double line identifies the amino acids that border the pore of the archaeal α rings. Circles indicate Gly-34, Lys-66, Leu-81 and Val-82 residues essential for binding of the ATPase regulatory particles to the 20S proteasome from <i>T</i>. <i>acidophilum</i>.</p

Effects of lactacystin on growth, cell cycle and ubiquitinated protein levels of PS <i>T</i>. <i>foetus</i>.

<p>(A) Growth curve of parasites. The parasites were initially cultured for 12 h at 37°C (initial inoculum: 1x10⁴ parasites/mL). After this period (arrow), 50 μM E-64d,10 and 20 μM of lactacystin were added to the culture medium and parasites were incubated for up to 30 h at 37°C. The cell growth was calculated after 6, 12, 18, 24 and 30 h of incubation. Untreated parasites were used as a control. Values are expressed as the means ± SD across three independent experiments, each performed in triplicate. (B) Analyses of the DNA content of <i>T</i>. <i>foetus</i> after treatment with 50 μM E-64d, and 10 (L10) and 20 μM (L20) of lactacystin for 12 h. Data acquisition and analysis were performed using a flow cytometer. Values are expressed as means ± SD across three independent experiments. Note that lactacystin arrests the <i>T</i>. <i>foetus</i> cell cycle in the G2/mitosis phases. ***p < 0.001 compared to control. (C) Immunoblot analyses of anti-ubiquitin and anti-proteasome antibodies. C, control; E, parasites treated with 50 μM E-64d for 12 h; L10 and L20, parasites treated with 10 μM and 20 μM lactacystin for 12 h, respectively. GAPDH was used as loading control. (D) Densitometric analysis of blot of anti-ubiquitin and anti-proteasome antibodies. The results are normalised to the intensity of GAPDH bands and are expressed as the means of relative densitometry units ± SD across three independent experiments. The levels of ubiquitinated and proteasomal proteins increased significantly when the parasites were treated with 20 μM lactacystin (L20) for 12 h. ***p < 0.001 compared to control.</p

Electron micrograph of <i>T</i>. <i>foetus</i> 20S proteasome.

<p>Two μL of the F3 fraction were applied to a glow-discharge carbon-coated grid for 1 min and negatively stained with 1% uranyl acetate for 1 min. The grids were then dried and observed using transmission electron microscopy. Circles indicate examples of the typical morphology of 20S proteasome core units in a side view. Bar, 50 nm.</p

Summary of the predicted 20S-proteasome proteins identified in <i>T</i>. <i>foetus</i> shotgun (454) and mate pair (Illumina) libraries using selected protein sequences of the <i>T</i>. <i>vaginalis</i> as reference.

<p>^a The sequences were deposited in GenBank</p><p>^b Assigned according to the KEGG Orthology database</p><p>^c aa, number of amino acids</p><p>Summary of the predicted 20S-proteasome proteins identified in <i>T</i>. <i>foetus</i> shotgun (454) and mate pair (Illumina) libraries using selected protein sequences of the <i>T</i>. <i>vaginalis</i> as reference.</p

Phylogenetic analysis of the predicted 20S proteasome α- and β-type subunits from <i>T</i>. <i>foetus</i>.

<p>The amino acid sequences of the 14 proteasome subunits identified in the <i>T</i>. <i>foetus</i> genome were aligned with their respective orthologues sampled from several eukaryotic species using CLUSTAL W algorithm. The unrooted phylogenetic tree was constructed by the neighbor-joining method based on the alignment using MEGA v. 5.2.2 software. The distance matrix was obtained by calculating p-distances for all pairs of sequences. Gaps were excluded using the pairwise-deletion option. Branch points were tested for significance by bootstrapping using 1,000 replications. All seven α- and β-type subunits are marked and shown in different colours. The <i>T</i>. <i>foetus</i> 20S proteasome subunits are indicated with colourful dots. Nodes supported by high bootstrap results (≥ 95%) are indicated by black dots. Scale bar represents 0.1 substitutions per site. Organism abbreviations: Tvag, <i>Trichomonas vaginalis</i>; Tcru, <i>Trypanosoma cruzi</i>; Scer, <i>Saccharomyces cerevisiae</i>; Ddis, <i>Dictyostelium discoideum</i>; Cele, <i>Caenorhabditis elegans</i>; Atha, <i>Arabidopsis thaliana</i>; Dmel, <i>Drosophila melanogaster</i>; Hsap, <i>Homo sapiens</i>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129165#pone.0129165.t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129165#pone.0129165.s009" target="_blank">S1 Table</a> for the accession numbers of the proteasome sequences.</p

Biochemical characterisation of the fractions obtained during the <i>T</i>. <i>foetus</i> 20S proteasome isolation procedure.

<p>(A) Coomassie brilliant blue-stained 12.5% SDS–PAGE of TE, F1, F2, F3 and F4-fractions (1 X 10⁹ cells). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129165#pone.0129165.s005" target="_blank">S5 Fig</a> for a description of each fraction. S, molecular weight standard. Similar results were obtained for PS and EFF. (B) Immunoblot and densitometric analyses using an anti-proteasome antibody. The antibody reacted specifically with two bands near 27-kDa in each fraction. The results of densitometry were normalised to the intensity of bands in the TE fraction and are expressed as the means of relative densitometric units ± SD across three independent experiments. The proteasomal protein level increased approximately 4.5-fold in the F3-fraction. **p<0.01; ***p<0.001 compared to TE-fraction. Similar results were obtained for PS and EFF. (C) Fluorogenic substrate assay of proteasome activity of each fraction obtained from PS (light grey bars) and EFF (dark grey bars). The caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (CT-L) activities were measured by spectrofluorometry using the fluorogenic substrates Z-LLE-AMC, Z-ARR-AMC and Z-LLL-AMC, respectively. Data are expressed as means of fluorescence units ± SD across three independent experiments performed in triplicate. The peptidase activities of proteasome from EFF were significantly higher than those from PS parasites. **p<0.01; ***p<0.001 compared to PS. In both parasite forms, the higher proteasome activity levels were found in the F3-fraction. (D) Effects of 20 μM lactacystin (Lac), 100 μM E-64 and 100 μM TPCK on the T-L, C-L and CT-L activities of F3-fractions obtained from PS (light grey bars) and EFF (dark grey bars). Data are expressed as means of relative percentage of control (without inhibitors) ± SD across three independent experiments performed in triplicate. In both forms, the peptidase activities were inhibited by lactacystin only. The proteasomal activities of the EFF were significantly more susceptible to lactacystin when compared to those of the PS. Similar results were obtained for other fractions. **p<0.01; ***p<0.001 compared to PS.</p

Proteasome proteolytic activity and the expression of ubiquitinated proteins during the EFF induction assay.

<p>(A) Time-course of the trypsin-like (T-L), caspase-like (C-L) and chymotrypsin-like (CT-L) proteasome during EFF induction assays. T-L (•), C-L (■) and CT-L (▲) activities were measured by spectrofluorometry using the fluorogenic substrates Z-ARR-AMC, Z-LLE-AMC and Z-LLL-AMC, respectively. The peptidase activity assays were performed using samples of the TE fractions of parasites (1 X 10⁹ cells) from different times during the EFF induction assay. Data are expressed as means of fluorescence units ± SD across three independent experiments performed in triplicate. The three peptidase activities increased over the course of EFF induction (columns). (B) Immunoblot analyses of anti-proteasome and anti-ubiquitin antibodies. GAPDH was used as a loading control. (C) Densitometric analysis of blot of anti-proteasome and anti-ubiquitin antibodies. The results are normalised to the intensity of GAPDH bands and are expressed as the means of relative densitometry units ± SD across three independent experiments. The levels of proteasomal protein levels remained unaltered, whereas the ubiquitinated proteins decreased significantly during the course of EFF induction. *p < 0.05; ***p < 0.001 compared to time 0.0.</p

Amino acid sequence alignment of the proteasomal β-type subunits from <i>T</i>. <i>foetus</i>.

<p>The alignment was performed using the CLUSTAL W program. The catalytically active β subunit from the Archaea <i>T</i>. <i>acidophilum</i> (TaciB—UniProt accession: P28061) and β5-subunit of <i>S</i>. <i>cerevisiae</i> were used as references to identify the conserved domains in the β-type subunits from <i>T</i>. <i>foetus</i>. For the purpose of clarity, the alignment was performed using the sequences that were previously identified by the NCBI CD-Search software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129165#pone.0129165.s003" target="_blank">S3 Fig</a> for details). Dashes indicate gaps introduced in the protein sequence alignment. Residues that are functionally conserved in all subunits are shaded in black. Residues that are functionally conserved in 75% to 87.5% and 50% to 62.5% of the subunits are shaded in dark and light grey, respectively. The box indicates the three possible catalytically active subunits of the <i>T</i>. <i>foetus</i> 20S proteasome. The functionally conserved residues found in the proteolytic active subunits are shaded in blue. Asterisks indicate the N-terminal nucleophile Thr-1 residue as well the catalytically essential Lys-33, Gly-128, Ser-129, and Gly-130 residues in the proteolytic active β-type subunits. The highly-conserved proteasome GxxxD motif (x represents any residue) is indicated. The positions of residues responsible for caspase-like, trypsin-like and chymotrypsin-like activities in the yeast subunits β1, β2 and β5, respectively, are shaded in red. Circles indicate the conserved Thr-1, Thr-21, Met-45, Gly-47 and Ala-49 residues of subunit β5 from yeasts that interact with the inhibitor lactacystin.</p