Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli

Abstract

SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and highthroughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.posttranslational modification | Listeria | cortactin | actin | anillin P osttranslational modifications (PTMs) are key mechanisms used by both prokaryotes and eukaryotes to regulate protein activity specifically, locally, and temporally. Ubiquitin and ubiquitin-like proteins (UBLs) constitute a specific class of small protein modifiers that can be covalently attached to a target protein via the formation of an isopeptide bond in a reversible manner. Small ubiquitin-related modifier (SUMO), one of these UBLs, is an essential PTM in eukaryotic cells that is involved in various cellular functions including gene expression regulation, DNA repair, intracellular transport, and response to viral and bacterial infections (1-5). The human genome encodes three different functional SUMO isoforms (SUMO1, SUMO2, and SUMO3) that are conjugated to distinct but overlapping sets of target proteins (1, 2, 6). Conjugation of SUMO to its targets in humans requires an E1-activating enzyme (the SAE1/SAE2 heterodimer), an E2-conjugating enzyme (Ubc9), and several E3 SUMO enzymes. Once conjugated to its target, SUMO can be deconjugated by several different SUMO isopeptidases that tightly regulate the SUMOylation levels of proteins (7).Since the discovery of SUMO two decades ago, much effort has been dedicated to the identification of SUMO-conjugated proteins in different organisms including yeast, plants, and mammals (8). However, isolation of SUMOylated proteins has proven to be challenging. Indeed, for most SUMO substrates, only a small proportion of the total amount of protein is SUMOmodified. In addition, the high activity of SUMO isopeptidases in cell lysates results in the rapid loss of SUMO conjugation in the absence of appropriate inhibitors. Thus, the most common approach used to isolate SUMOylated proteins is based on the expression of His-tagged versions of SUMO allowing the purification of SUMO-conjugated proteins by nickel chromatography under denaturing conditions (8, 9). Denaturing conditions inactivate SUMO isopeptidases and al...

Cooling-induced SUMOylation of EXOSC10 down-regulates ribosome biogenesis

“…In accordance with the His 6 precipitation data presented here, previous reports suggest that only SUMO1 is conjugated to EXOSC10; multiple mass spectrometry analyzes of affinity tagged SUMO1 conjugates, but not SUMO2 or SUMO3, identified EXOSC10 as a SUMO target (Zhao et al 2004;Matic et al 2010;Westman et al 2010;Impens et al 2014;Lamoliatte et al 2014). In accordance with this, a screen of the endogenous SUMOylated proteome identified EXOSC10 conjugation to SUMO1 but not SUMO2 (Becker et al 2013).…”

“…Reports of dynamic changes in EXOSC10 expression are not extensive, however it has been shown that yeast EXOSC10 is destabilized by deletion of its binding partner Rrp47 (C1D in humans) and in human cell lines by the chemotherapeutic 5-fluorouracil Feigenbutz et al 2013). Interestingly, EXOSC10 has previously been identified in screens of SUMOylation (Zhao et al 2004;Golebiowski et al 2009;Westman et al 2010;Becker et al 2013;Impens et al 2014;Tammsalu et al 2014). This presents the possibility that the increased SUMOylation in the cold participates in regulation of EXOSC10 (Fig.…”

“…Of these, K168 and K583 have strong SUMOylation consensus motifs, and all three have been described as positions of SUMOylation in mass spectrometry screens (Impens et al 2014;Tammsalu et al 2014;Hendriks et al 2015). The location of these sites within EXOSC10 is of interest.…”

“…Indeed, other exosome subunits appear in screens for SUMOylated proteins, such as EXOSC5 (Becker et al 2013;Hendriks et al 2015) and EXOSC9 (Golebiowski et al 2009;Impens et al 2014;Lamoliatte et al 2014;Hendriks et al 2015). Furthermore, SUMO site prediction tools identify putative sites in EXOSC2, EXOSC3, EXOSC7, EXOSC8, and EXOSC9 (Zhao et al 2014) and SUMOplot.…”

Proteomic Analysis of Ubiquitin-Like Posttranslational Modifications Induced by the Adenovirus E4-ORF3 Protein

“…The yeast MRX complex serves as a positive regulator of sumoylation, and sumoylation significantly promotes the DDR in this system (40). Ribet and colleagues also recently reported the use of HeLa cell lines that stably express His 6 -SUMO1-T95R and His 6 -SUMO2-T91R and use of the PTMScan ubiquitin remnant motif antibody to identify cellular proteins constitutively conjugated to SUMO1 or SUMO2 (41). That analysis identified 295 SUMO1-conjugated peptides in 227 substrate proteins and 167 SUMO2-conjugated sites in 135 substrate proteins.…”