Abstract
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.
Highlights
SUMOylation is a post-translational modification that involves the covalent conjugation of the Small Ubiquitin-like Modifier (SUMO) to a diverse number of target proteins
To promote the recombinant expression of T. brucei SUMOylated proteins in bacteria, we decided to transfer the complete set of enzymes essential for this post translational modification in this organism to E. coli
Samples correspond to the soluble fraction of E. coli BL21 (DE3) host cells transformed with the empty vector pACYCDuet-1,pACYCDuet-1TbE1a-TbE1b, pCDFDuet-1, pCDFDuet-1-T. brucei SUMO (TbSUMO)-TbE2, or with the complete SUMOylation system and induced (I) or not (UI) for protein expression during 5 hr at 37°C using 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG)
Summary
SUMOylation is a post-translational modification that involves the covalent conjugation of the Small Ubiquitin-like Modifier (SUMO) to a diverse number of target proteins. SUMOs are ~12 kDa proteins that belong to the Ubiquitin-like proteins (UbLs) family. SUMO shares low amino acid sequence identity with ubiquitin (Ub) (~20%), these proteins present an almost identical structural fold with exception of an unstructured N-terminal extension that is present in SUMO and absent in other UbLs and Ub itself [3]. SUMO proteins are conserved and ubiquitously expressed in eukaryotes, but absent in prokaryotes and archaea. Lower eukaryotes such as yeast or invertebrates have a single SUMO gene, while in vertebrates and plants different SUMO paralogs are expressed. Some SUMO isoforms, such as SMT3 in yeast and SUMO 2/3 in vertebrates, can polymerize and form polySUMO chains in vivo and in vitro usually via Lys residues that conform a SUMOylation consensus motif and are often found at the N-terminal region [4,5]
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