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Review
. 2015 Oct;282(19):3669-80.
doi: 10.1111/febs.13378. Epub 2015 Jul 31.

Mapping the SUMOylated landscape

Karolin Eifler  1 , Alfred C O Vertegaal  1
Affiliations
Review

Mapping the SUMOylated landscape

Karolin Eifler et al. FEBS J. 2015 Oct.

Abstract

SUMOylation is a post-translational modification that regulates a multitude of cellular processes, including replication, cell-cycle progression, protein transport and the DNA damage response. Similar to ubiquitin, SUMO (small ubiquitin-like modifier) is covalently attached to target proteins in a reversible process via an enzymatic cascade. SUMOylation is essential for nearly all eukaryotic organisms, and deregulation of the SUMO system is associated with human diseases such as cancer and neurodegenerative diseases. Therefore, it is of great interest to understand the regulation and dynamics of this post-translational modification. Within the last decade, mass spectrometry analyses of SUMO proteomes have overcome several obstacles, greatly expanding the number of known SUMO target proteins. In this review, we briefly outline the basic concepts of the SUMO system, and discuss the potential of proteomic approaches to decipher SUMOylation patterns in order to understand the role of SUMO in health and disease.

Keywords: SUMO; cross-talk; group modification; mass spectrometry; post-translational modification; proteomics; site-specific; small ubiquitin-like modifier; ubiquitin.

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Figures

Figure 1
Figure 1
The SUMOylation cycle. First, the SUMO precursor protein is cleaved to its mature form by SUMO proteases, exposing a C‐terminal di‐glycine motif. In an ATP‐dependent reaction, the C‐terminus of SUMO is attached via a thioester bond to the SUMO activating enzyme, consisting of the two subunits SAE1 and SAE2. It is then further transferred to an internal cysteine of the SUMO conjugating enzyme (UBC9). In the third step, SUMO is attached to a lysine residue of a target protein, a process that is facilitated by the presence of a SUMO ligase. Finally, SUMO proteases cleave SUMO from its substrate, resulting in free SUMO that re‐enters the SUMOylation cycle.
Figure 2
Figure 2
Proteomic approaches to identify SUMO targets. (A) Purification via SUMO‐specific antibodies. Cells are lysed under denaturing conditions to inactivate SUMO proteases. Afterwards, samples are diluted to obtain mild buffer conditions and SUMOylated proteins are purified using SUMO‐specific antibodies. Proteins are subsequently trypsinized and subjected to mass spectrometry. (B) Purification via SIM traps. Cells are lysed in a mild buffer supplemented with iodoacetamide, and SUMOylated proteins are purified using the SIM‐containing protein RNF4 as bait. Proteins are subsequently trypsinized and subjected to mass spectrometry. (C) Purification with epitope tags. Cells expressing a tagged SUMO fusion protein are lysed in denaturing buffer. For subsequent immunoprecipitation of the SUMO target proteins using antibodies targeting the protein tag, samples are diluted to obtain mild buffer conditions. Finally, they are trypsinized and analysed via mass spectrometry. (D) Purification with affinity tags. Cells expressing SUMO tagged with affinity tags are lysed in denaturing buffer, and SUMO targets are purified using affinity matrices that specifically bind to the tag. Subsequently, proteins are trypsinized and analysed via mass spectrometry. (E) After trypsin digestion, the C‐terminal fragments of mammalian SUMO family members are too large to efficiently map the SUMO‐conjugated lysines in target proteins. To enable site‐specific purification, protease cleavage sites are introduced in the C‐termini of mammalian SUMO family members. SUMO target proteins modified with these mutant versions of SUMOs are fused to specific protein tags and purified as previously described for epitope or affinity tags. Subsequent digestion with either trypsin or the endoproteinase LysC, depending on the SUMO mutant employed, results in shorter SUMO peptides to facilitate identification of SUMO sites via mass spectrometry. (F) Alignment of the C‐termini of mature SMT3 from yeast and mature human ubiquitin, SUMO1, SUMO2 and SUMO3, demonstrating the various lengths of the tryptic remnants remaining after cleavage. Arginine and lysine residues are highlighted in red. The mutations used to facilitate identification of SUMO2 sites are indicated by arrows.
Figure 3
Figure 3
Protein group modification via SUMO. In response to specific cellular or external stimuli, the activity and localization of the SUMO conjugation machinery is altered, leading to SUMOylation of target proteins with similar functions during the cellular response. This protein group modification triggers the formation of larger protein complexes via specific SUMO–SIM interactions. Increased activity of SUMO proteases reverses this process, leading to disassembly of these protein complexes.
Figure 4
Figure 4
Cross‐talk between post‐translational modifications as identified via mass spectrometry. (A) Several SUMO target proteins contain a so‐called phosphorylation‐dependent SUMO motif, in which the modified lysine residue is followed by a phosphorylated serine, usually five amino acids further downstream. A serine residue situated in a phosphorylation‐dependent SUMO motif of the nuclear protein NOP58 is phosphorylated via casein kinase 2 (CK2), promoting UBC9 binding and subsequent SUMOylation of the indicated lysine residue. (B) Similar to phosphorylation, acetylation via specific acetyl transferases may induce SUMOylation of a protein, as described for histone H3. (C) Many lysine residues that were found as SUMO acceptor sites have also been shown to be ubiquitylated or acetylated, suggesting extensive competition between these modifiers. (D) Dozens of enzymes regulating post‐translational protein modifications have been identified as SUMO target proteins in proteomic screens, including SUMO pathway components (S), or enzymes regulating other post‐translational modifications, such as phosphorylation (P), ubiquitylation (Ub), methylation (Me) or acetylation (Ac).
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