Post-translational modifications

Functional activity of proteins is tightly controlled via reversible post-translational modifications including phosphorylation, acetylation and ubiquitylation. These modifications enable the orchestration of cellular responses to a wide variety of stimuli. Due to these modifications, proteomes are overwhelmingly complex. Progress in the field has been greatly accelerated by the development of novel approaches to study these post-translational modifications at a proteome-wide scale using the sensitivity and robustness of mass spectrometry (MS). This has enabled the identification of thousands of dynamically regulated phosphorylation, acetylation and ubiquitylation sites by MS. The functional significance of these modifications is now being addressed worldwide at an unprecedented scale. In contrast, global understanding of ubiquitin-like signalling networks is challenging.


We are studying post-translational modification by the ubiquitin-like protein SUMO. SUMOylation is critical for eukaryotic life and regulates a wide variety of cellular processes including transcription, pre-mRNA splicing, translation, transport, replication and DNA repair (Vertegaal 2011; Eifler and Vertegaal 2015). The conjugation pathway of SUMO is similar to the conjugation pathway of ubiquitin and consists of E1, E2 and E3 enzymes. SUMOylation is a reversible process; SUMO-specific proteases can remove SUMOs from target proteins.

We are studying SUMOylation at a proteome wide level and have uncovered hundreds of SUMO target proteins. SILAC technology is employed to study SUMOylation dynamics. We have developed novel methodology to study protein SUMOylation in a site-specific manner at a proteome wide level (Hendriks et al. 2014; Hendriks et al. 2015a; Hendriks & Vertegaal 2016a and b). Over 4,300 SUMO-2 acceptor lysines in over 1,600 endogenous target proteins were identified and this information was used to refine the SUMOylation consensus motif. Two novel SUMOylation consensus motifs were identified including the inverted SUMOylation consensus motif [ED]xK[VILFP] and the Hydrophobic Cluster SUMOylation Motif (HCSM) (Matic et al. 2010). Based on this project, the SUMOylation Motif Matcher is now available on the Phosida website for the prediction of SUMOylation sites in target proteins ( – tools – SUMOylation Motif Matcher).

Post-translational modification crosstalk

Direct mass spectrometric evidence was found for crosstalk between SUMOylation and phosphorylation with a preferred spacer between the SUMOylated lysine and the phosphorylated serine of four residues. Additionally, we found crosstalk between SUMOylation and acetylation to regulate histone H3 (Hendriks et al. 2014).

We also found crosstalk between SUMO-2/3 and the ubiquitin-proteasome system (Schimmel et al. 2008). A key substrate regulated by this pathway is c-Myc (Gonzalez-Prieto et al. 2015b). This pathway includes SUMO-targeted ubiquitin E3 ligases including RNF4 that bind SUMOylated proteins. This pathway is critical for genome stability (Vyas et al. 2013). We found that USP11 binds to RNF4 and counteracts its activity (Hendriks et al. 2015c).

A role for SUMO to maintain genome stability

We used our methodology to study the role of SUMO-2 in the DNA damage response (Hendriks et al. 2015b). Interestingly, the histone demethylase JARID1C/KDM5C is SUMOylated and recruited to the chromatin in response to DNA damage to demethylate histone H3K4 to reduce global transcription levels. Furthermore, SUMOylation and PARylation cooperate to recruit and stabilize the nuclease scaffold SLX4 at DNA damage sites (Gonzalez-Prieto et al. 2015a)

Selected SUMO target proteins are studied at the functional and mechanistic level to obtain novel insight in cellular processes that are regulated by SUMOylation. Recently, we have identified a novel SUMO target protein, the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation (Schimmel et al. 2014). We found that a SUMOylation-deficient FoxM1 mutant was less active compared to wild-type FoxM1, implying that SUMOylation of the protein enhanced its transcriptional activity. Mechanistically, SUMOylation blocked the dimerization of FoxM1, thereby relieving FoxM1 autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. These findings contribute to understanding the role critical of SUMOylation to maintain genome stability during mitosis.

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