Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System.

Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids

<i>Tb</i>SENP peptidase and isopeptidase activity.

<p><b>(A)</b> SDS-PAGE followed by Coomassie Blue staining (left panel) or Western blot analysis using anti-GST antibodies of <i>Tb</i>SENP purification (right panel). In: input, cell-free extract from bacteria overexpressing <i>Tb</i>SENP, FT: Flow through, fraction not retained by the resin and El: eluate, sample retained and eluted from the resin. The full length protein is marked with an arrowhead. Faster migrating bands likely correspond to <i>Tb</i>SENP-GST degradation products. <b>(B)</b> SUMO precursor cleavage by <i>Tb</i>SENP was evaluated <i>in vitro</i> using a <i>Tb</i>SUMO precursor produced in <i>E</i>. <i>coli</i> tagged at the N-terminus with His-HA and fused at the C-terminus to the GST protein. After purification on glutathione-agarose resin 7.5 μg of His-HA-<i>Tb</i>SUMO-GST protein was mixed with 0.75 μg of purified recombinant <i>Tb</i>SENP (produced as described in Materials and Methods) in 30 μl of TBS containing 1 mM DTT in the absence (lane 2) or presence (lane 3) of the general cysteine peptidase inhibitor N-ethylmaleimide 20 mM final concentration (NEM) and incubated at 37°C for 1 hr. Samples were analyzed by Western blot using anti-HA monoclonal antibodies. The substrate without the addition of the protease was run as a control (lane 1). <b>(C)</b> Broad-specificity SUMO deconjugation ability of <i>Tb</i>SENP was analyzed on purified HA-tagged <i>Tb</i>SUMO conjugates from parasites (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134950#sec008" target="_blank">Materials and Methods</a>). Isopeptidase activity was evaluated in reaction mixtures containing 3 μg of <i>Tb</i>SUMO conjugates and 0.75 μg of purified <i>Tb</i>SENP in 30 μl of TBS containing 1 mM DTT in the presence (lane 2) or in the absence (lane 3) of 20 mM NEM incubated at 37°C for 1 hr. Samples were analyzed by Western blot using anti-HA monoclonal antibodies. The substrates without the addition of the protease was run as a control (lane 1).</p

SUMO polymeric chains are involved in nuclear foci formation and chromatin organization in Trypanosoma brucei procyclic forms.

SUMOylation is a post-translational modification conserved in eukaryotic organisms that involves the covalent attachment of the small ubiquitin-like protein SUMO to internal lysine residues in target proteins. This tag usually alters the interaction surface of the modified protein and can be translated into changes in its biological activity, stability or subcellular localization, among other possible outputs. SUMO can be attached as a single moiety or as SUMO polymers in case there are internal acceptor sites in SUMO itself. These chains have been shown to be important for proteasomal degradation as well as for the formation of subnuclear structures such as the synaptonemal complex in Saccharomyces cerevisiae or promyelocytic leukemia nuclear bodies in mammals. In this work, we have examined SUMO chain formation in the protozoan parasite Trypanosoma brucei. Using a recently developed bacterial strain engineered to produce SUMOylated proteins we confirmed the ability of TbSUMO to form polymers and determined the type of linkage using site-directed mutational analysis. By generating transgenic procyclic parasites unable to form chains we demonstrated that although not essential for normal growth, SUMO polymerization determines the localization of the modified proteins in the nucleus. In addition, FISH analysis of telomeres showed a differential positioning depending on the polySUMOylation abilities of the cells. Thus, our observations suggest that TbSUMO chains might play a role in establishing interaction platforms contributing to chromatin organization

<i>In vitro</i> deconjugation of SUMOylated <i>Sc</i>PCNA.

<p>Cell lysates of <i>E</i>. <i>coli</i> heterologously expressing <i>Sc</i>PCNA and the complete <i>T</i>. <i>brucei</i> SUMOylation system (lane 1) were incubated at 28°C in the absence (lane 2) or presence of recombinant <i>Tb</i>SENP (lane 3) as described in Material and Methods. Deconjugation ability of <i>Tb</i>SENP was specifically inhibited by the addition of 20 mM NEM (lane 4). Reaction mixtures were analyzed by Western blot using anti-Flag monoclonal antibodies.</p

Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms

SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitinlike MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. HisHA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a HisHA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others

<i>Tb</i>SUMO chain formation.

<p><b>(A)</b> Anti-HA Western blot analysis of soluble cell extracts from induced cultures of <i>E</i>. <i>coli</i> transformed with only one plasmid pCDFDuet-1-<i>Tb</i>SUMO-<i>Tb</i>E2; pACYCDuet-1-<i>Tb</i>E1a-<i>Tb</i>E1b or both pCDFDuet-1-<i>Tb</i>SUMO-<i>Tb</i>E2 and pACYCDuet-1-<i>Tb</i>E1a-<i>Tb</i>E1b. Different exposure times were used to evidence the SUMO ladder which was observed at the shorter times while a more complex pattern was obtained with longer periods of exposure. <i>Tb</i>SUMO monomer, dimers, trimers and multimers are indicated. <b>(B)</b> Western blot analysis of SUMO pattern performed on soluble cell extracts from an incomplete (lanes 1 and 2) or a complete bacterial SUMOylation system (lane 3) using a Lys deficient version of SUMO (<i>Tb</i>SUMO K9R). Note the complete absence of SUMO conjugates implying the absence or artificial SUMOylation of bacterial proteins.</p

Reconstitution of the <i>T</i>. <i>brucei</i> SUMOylation system in <i>E</i>. <i>coli</i>.

<p><b>(A)</b> Schematic representation of the plasmids used to express the multiple components of the <i>T</i>. <i>brucei</i> SUMOylation system in bacteria. pACYCDuet-1 carrying the P15A replicon and chloramphenicol resistance gene (<i>cat</i>) was designed to direct the co-expression of <i>T</i>. <i>brucei</i> activating enzyme subunits a and b (<i>Tb</i>E1a and <i>Tb</i>E1b) as fusions to an N-terminal His-tag or a C-terminal S-tag, respectively. pCDFDuet-1 carries the CloDF13 replicon and the streptomycin/spectinomycin resistance gene (<i>aadA</i>) and drives the co-expression of mature SUMO (<i>Tb</i>SUMO) and its conjugating enzyme (<i>Tb</i>E2) both tagged at the N-terminus either with an HA epitope or a His tag, respectively. Finally, pET28 carries the ColE1 origin of replication and the kanamycin resistance gene (<i>Kmr</i>) and is used for individual expression of SUMOylation target proteins. <b>(B)</b> Analysis of recombinant protein expression by Coomassie Blue staining of SDS-PAGE gels. Equal amount of protein (30 μg) was loaded in each lane. Samples correspond to the soluble fraction of <i>E</i>. <i>coli</i> BL21 (DE3) host cells transformed with the empty vector pACYCDuet-1 (lanes 1 and 2),pACYCDuet-1-<i>Tb</i>E1a-<i>Tb</i>E1b (lane 3 and 4), pCDFDuet-1 (lane 5 and 6), pCDFDuet-1-<i>Tb</i>SUMO-<i>Tb</i>E2 (lane 7 and 8), or with the complete SUMOylation system (lane 9) and induced (I) or not (UI) for protein expression during 5 hr at 37°C using 1mM IPTG. The predicted molecular masses of the recombinant proteins (including tags) are: 14 kDa for <i>Tb</i>SUMO, 28 kDa for <i>Tb</i>E2, 40 kDa for <i>Tb</i>E1a and 97 kDa for <i>Tb</i>E1b. Recombinant proteins are marked with an asterisk in the figure and labeled with an arrowhead at the right of the gel. <b>(C)</b> Immunoblot detection of the recombinant proteins was performed on the same samples using anti-HA antibodies for <i>Tb</i>SUMO and anti-His antibodies for <i>Tb</i>E2 and <i>Tb</i>E1a.</p

In-bacteria SUMOylation of <i>Sc</i>PCNA.

<p><b>(A)</b> Anti-Flag Western blot analysis of <i>Sc</i>PCNA performed on soluble cell extracts from induced cultures of <i>E</i>. <i>coli</i> transformed with pET28-<i>Sc</i>PCNA-3xFlag alone (lane 1) or in the background of an incomplete (lane 2, pACYCDuet-1-<i>Tb</i>E1a-<i>Tb</i>E1b; lane 3, pCDFDuet-1-<i>Tb</i>SUMO-<i>Tb</i>E2) or a complete (lane 4, pCDFDuet-1-<i>Tb</i>SUMO-<i>Tb</i>E2 plus pACYCDuet-1-<i>Tb</i>E1a-<i>Tb</i>E1b) SUMOylation system. <b>(B)</b> Mutational analysis of <i>Sc</i>PCNA was performed in a background of <i>E</i>. <i>coli</i> Bl21 (DE3) cells (C) or in a complete SUMOylation system (S). The band corresponding to SUMO conjugated to K127 and the higher molecular weight band corresponding to poliSUMOylated PCNA at K127 or K164 are marked with asterisks. (<b>C</b>) Bacterial lysate overexpressing SUMOylated <i>Sc</i>PCNA or the complete SUMOylation system as a control, were subjected to Ni⁺² affinity chromatography. The inputs (lane 1 and 3) and the eluates (lane 2 and 4) were analyzed by Coomassie staining or immunoblotting using monoclonal anti-Flag antibodies.</p

<i>TbS</i>UMO-SIM binding assays.

<p><b>(A)</b> Illustration of the domain arrangement in SIMx4 and mutSIMx4 probes. <b>(B)</b> Coomassie Blue stained SDS-PAGE analysis of probe purifications. <i>E</i>. <i>coli</i> BL21 DE3 cells expressing SIMx4 or mutSIMx4 probes were lysed and cleared lysates were subjected to Ni⁺² chromatography. Inputs, In; Eluates, El. <b>(C)</b> <i>E</i>. <i>coli</i> BL21 DE3 cells expressing HA-<i>Tb</i>SUMO chains (Inputs) were incubated with the SIMx4 or the mutSIMx4 probes. Samples were pulled-down using Ni⁺²-Sepharose beads. Proteins were visualized by Western blot using anti-HA antibodies. Coomassie stained gels of the inputs are shown. <b>(D)</b> Cell free extract from <i>T</i>. <i>brucei</i> 427 PCF was incubated with SIMx4 or mutSIMx4 probes, which were subsequently purified by Ni⁺² chromatography. The capture of SUMOylated proteins was determined by Western blot analysis using anti-<i>Tb</i>SUMO antibodies, while anti-α-tubulin was used as the loading control for inputs.</p

SUMO chain mutant <i>T</i>. <i>brucei</i> procyclic parasites.

<p><b>(A)</b> Schematic representation of the SUMO variants expressed in PCF parasites. GG, diglycine motif essential for SUMO processing, activation and conjugation. <b>(B)</b> Growth of HisHA-<i>Tb</i>SUMO<i>all</i>KR strain (<i>Tb</i>SUMO<i>all</i>KR) compared to HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) and wild-type parasites. Wild-type and transgenic parasites were cultured up to one month without observing significant differences in growth rate. <b>(C)</b> Conjugating capacity of HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) and HisHA-<i>Tb</i>SUMO<i>all</i>KR (<i>Tb</i>SUMO<i>all</i>KR) parasites. Whole-cell extracts were boiled in Laemmli sample buffer immediately after harvesting, separated in a 7.5%-12.5% discontinuous acrylamide gel (3,5x10⁷ cells/lane), and analysed by Western blot using anti-HA antibodies. An unspecific cross-reacting band of ∼50 kDa from anti-<i>Tc</i>SUMO antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193528#pone.0193528.ref022" target="_blank">22</a>] was used as loading control. <b>(D)</b> Immunofluorescence analysis of wild-type, HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) and HisHA-<i>Tb</i>SUMO<i>all</i>KR (<i>Tb</i>SUMO<i>all</i>KR) parasites. Nuclear and kinetoplast DNA were visualized by DAPI staining (blue). Representative images of anti-<i>Tb</i>SUMO (green), anti-HA (green) and <i>Tb</i>SUMO/anti-HA-DAPI merged images are shown.</p