Different proteomic strategies to identify genuine Small Ubiquitin-like MOdifier targets and their modification sites inTrypanosoma bruceiprocyclic forms

Abstract

SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitin-like 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. His-HA-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 His-HA-TbSUMO(T106K) 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.

SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes

“…The low signal of SNF2PH antibody in TbSUMO IP experiments is likely a consequence of the dynamic nature of SUMOylation, yielding a small population of SUMOylated SNF2PH form at any given time; similar behavior has been demonstrated for TbRPAI (RNA Polymerase I largest subunit) and additional SUMO proteins in T. brucei . To determine which domains of SNF2PH are SUMOylated, we used an E. coli strain expressing the complete T. brucei SUMOylation system .…”

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

“…cruzi orthologue [ 19 ]. However, Tb SUMO chain formation seems not to be an essential feature in vivo since it was possible to replace the endogenous gene with a Lys deficient version [ 39 ], similar to the situation described for S . cerevisiae [ 40 ].…”

“…Parasites employed were procyclic form (PCF) T . brucei brucei Lister 427 [ 41 ] andHis-HA- Tb SUMO, a Lister 427 cell line with both SUMO alleles replaced by a His-HA- Tb SUMO variant [ 39 ].…”

“…To obtain purified Tb SUMO conjugates to evaluate isopeptidase activity of Tb SENP we used PCF parasites expressing only a His-HA-tag version of Tb SUMO (His-HA- Tb SUMO cell line) [ 39 ].…”

SUMOylation, aging and autophagy in neurodegeneration

“…Peters and colleagues have summarised the various processes and proteins that SUMO regulates in the central nervous system as well as in neurodegeneration (Peters et al, 2017). The SUMO protein covalently modifies acceptor lysine residues of this diverse range of target proteins altering their functional properties through the process called SUMOylation (Iribarren et al, 2015;Krumova and Weishaupt, 2013). SUMO-mediated modulation can occur via the inhibition of the binding site of a protein preventing its interaction with its substrate protein as well as posing as a 'hub' for interaction by recruiting other binding partners.…”