SUMOylation plays a key role in genome stability and transcription as well as controlling the cell’s dynamic response to stress. A recent paper by Hendriks et al. offers new insight into the global targeting of this post-translational modification across the cell’s proteome1. Using high resolution mass spectrometry the authors were able to map over 4,300 sites of SUMOylation in over 1,600 proteins.
Mass spectrometry based techniques have been used previously to probe SUMO function across the proteome, but the use of a new purification strategy in this paper enabled the Dutch-based team to identify SUMOylation sites more efficiently. HeLa cells were used as a model system and stably transfected with a His10-SUMO-2 mutant with a short C-terminus to allow easier identification of the lysine residues at the acceptor sites. This method offers a step forward for the SUMO research field, allowing analysis of proteome-wide SUMOylation sites in a site-specific manner and allows SUMO function to be probed in cells under standard growth conditions.
Among the 1,606 proteins undergoing SUMOylation identified in this study there were a number of well-known SUMO target-proteins including RanGAP1, PML, and BRCA1, further verifying the new SUMO purification method. These results have expanded the number of known SUMOylation sites by over 3000, whilst reconfirming many of the 1000 sites recently mapped under heat shock conditions and providing over 1000 new SUMOylation sites utilised by the cell under standard growth conditions.
This is the first study that has identified SUMO target-proteins under standard growth conditions, with 1069 SUMO binding sites identified under these conditions. The authors also examined SUMO binding sites under protease inhibition, proteasome inhibition and heat shock conditions and identified a further 3292 SUMOylation sites active in these stress situations, displaying the dynamic nature of SUMOylation in response to different situations. Interestingly, whilst over half the SUMO target-proteins identified contained only one SUMOylation site, 96 displayed ten or more SUMO binding sites, and 11 proteins contained over twenty SUMO binding sites, with more SUMO proteins binding per site following cellular stress.
SUMO and its cousin ubiquitin are known to carry out cross-talk, but this study has uncovered further points at which this may occur within the proteome, with many of the lysine residues which are acceptor sites of SUMO and ubiquitin also being able to be modified by acetylation and methylation. An example of this is the first visualisation of SUMOylation of histone H3, which is carried out in an acetylation dependent manner, suggesting cross-talk that may hint at a mechanism for transient activation of this region of the genome.
The way in which SUMO and ubiquitin can interact within the cell was also seen to increase according to the results of this study, with the identification of five different lysines in ubiquitin that can be used as SUMO-acceptors, indicating that mixed chain formation between the ubiquitin family members may be more extensive than previously thought.
The increased insight into SUMOylation and its function offered by this study opens up exciting new avenues to investigate the mechanisms and biological relevance of this signal transduction – Affimer technology offers the perfect tools to help researchers explore these questions.