Abstract
Post-translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Because little is known about crosstalk among different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk among different PTMs. In addition, various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin on inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, whereas the total protein level decreased immediately afterward. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.
Highlights
IntroductionThe limited capacity of our genome is compensated for by the processes of alternative splicing and post-translational modification (PTM)1
Improved Strategy Enables Purification of Co-modified Proteins—We have developed an improved method that enables enrichment of proteins simultaneously modified by two different small protein protein modifications (PTMs)
Concluding Remarks—We have developed improved methodology that will help to increase our knowledge about crosstalk among various PTMs
Summary
The limited capacity of our genome is compensated for by the processes of alternative splicing and post-translational modification (PTM)1 The latter adds an essential additional layer of complexity to our proteome, which is necessary to provide the cell with sufficient functionally different protein states that are needed for efficient regulation of cellular processes and pathways. We have developed an improved strategy to purify and identify proteins co-modified by two different small protein PTMs, SUMO, and ubiquitin This improved method is generic and can be applied to different combinations of these PTMs and will thereby enable us to study the phenomenon of crosstalk on a more comprehensive PTM-wide level
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