Targeted Identification of SUMOylation Sites in Human Proteins Using Affinity Enrichment and Paralog-specific Reporter Ions

Lamoliatte, Frederic; Bonneil, Éric; Durette, Chantal; Caron-Lizotte, Olivier; Wildemann, Dirk; Zerweck, Johannes; Wenshuk, Holger; Thibault, Pierre

doi:10.1074/mcp.m112.025569

Cited by 44 publications

(35 citation statements)

References 36 publications

(22 reference statements)

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“…3). This script is different from that reported recently 30 and takes into account cleavages of residues within the SUMO3m remnant (for example, y 11 -NQ, y 11 -NQT and y 11 -NQTG in Fig. 2c).…”

Section: Tcl Mw Markercontrasting

confidence: 83%

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Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

Lamoliatte¹,

et al. 2014

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Small ubiquitin-related modifiers (SUMO) are evolutionarily conserved ubiquitin-like proteins that regulate several cellular processes including cell cycle progression, intracellular trafficking, protein degradation and apoptosis. Despite the importance of protein SUMOylation in different biological pathways, the global identification of acceptor sites in complex cell extracts remains a challenge. Here we generate a monoclonal antibody that enriches for peptides containing SUMO remnant chains following tryptic digestion. We identify 954 SUMO3-modified lysine residues on 538 proteins and profile by quantitative proteomics the dynamic changes of protein SUMOylation following proteasome inhibition. More than 86% of these SUMOylation sites have not been reported previously, including 5 sites on the tumour suppressor parafibromin (CDC73). The modification of CDC73 at K136 affects its nuclear retention within PML nuclear bodies on proteasome inhibition. In contrast, a CDC73 K136R mutant translocates to the cytoplasm under the same conditions, further demonstrating the effectiveness of our method to characterize the dynamics of lysine SUMOylation.

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Section: Tcl Mw Markercontrasting

confidence: 83%

“…Capillary HPLC columns for nano-LC-MS were packed in-house using Jupiter C18 (3 mm) particles from Phenomenex (Torrance, CA) and fused silica tubing from Polymicro Technologies (Phoenix, AZ). All tryptic peptides with SUMO remnant chains were synthesized by automated SPOT synthesis as described previously 30 .…”

Section: Methodsmentioning

confidence: 99%

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

Lamoliatte¹,

et al. 2014

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“…Of our 332 identified sites, 86 (26%) were previously reported in proteomic screens for SUMO sites (11,14,16,17,23,24), and 82 (25%) are reported in the PhosphoSitePlus database (a resource for human PTMs, including SUMOylation) (25), thereby validating our approach (Dataset S1). Thus, to our knowledge, 227 SUMO sites (203 for SUMO1 and 82 for SUMO2) were identified here for the first time (Dataset S1).…”

Section: Resultssupporting

confidence: 60%

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

Impens

Radoshevich

Cossart

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

159

164

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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...

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“…Recent proteomic studies have also revealed Ub modification by a small Ub-like modifier (SUMO) at multiple Lys residues (Galisson et al, 2011;Lamoliatte et al, 2013;Hendriks et al, 2014). Additionally, ubiquitinated SUMO has also been reported (Lamoliatte et al, 2013;Hendriks et al, 2014).…”

Section: Different Forms Of Ubiquitin Modifications Exist In Naturementioning

confidence: 99%

Resolving the Complexity of Ubiquitin Networks

Kliza

Husnjak

2020

Front. Mol. Biosci.

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Ubiquitination regulates nearly all cellular processes by coordinated activity of ubiquitin writers (E1, E2, and E3 enzymes), erasers (deubiquitinating enzymes) and readers (proteins that recognize ubiquitinated proteins by their ubiquitin-binding domains). By differentially modifying cellular proteome and by recognizing these ubiquitin modifications, ubiquitination machinery tightly regulates execution of specific cellular events in space and time. Dynamic and complex ubiquitin architecture, ranging from monoubiquitination, multiple monoubiquitination, eight different modes of homotypic and numerous types of heterogeneous polyubiquitin linkages, enables highly dynamic and complex regulation of cellular processes. We discuss available tools and approaches to study ubiquitin networks, including methods for the identification and quantification of ubiquitin-modified substrates, as well as approaches to quantify the length, abundance, linkage type and architecture of different ubiquitin chains. Furthermore, we also summarize the available approaches for the discovery of novel ubiquitin readers and ubiquitin-binding domains, as well as approaches to monitor and visualize activity of ubiquitin conjugation and deconjugation machineries. We also discuss benefits, drawbacks and limitations of available techniques, as well as what is still needed for detailed spatiotemporal dissection of cellular ubiquitination networks.

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Targeted Identification of SUMOylation Sites in Human Proteins Using Affinity Enrichment and Paralog-specific Reporter Ions

Cited by 44 publications

References 36 publications

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

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

Resolving the Complexity of Ubiquitin Networks

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