![]() ![]() SUMO can also interact non-covalently with SUMO interacting motifs (SIMs) found in some proteins. While cell-wide proteomics approaches can help to understand global SUMO signaling 10, better tools are needed that allow the study of the cause and consequences of particular SUMOylation events for individual substrates. Improvements in mass spectrometry technology have led to the identification to date of more than 40,700 SUMO sites within 6,700 SUMO substrates 9. SUMO is known to control vital biological processes including development 7 and cholesterol homeostasis 8. SUMO plays crucial roles in nuclear processes underlying health and disease such as the DNA damage response, cell cycle regulation, transcription and proteostasis 6. Together, these constitute the concept of the SUMO code and the ongoing challenge is to understand how these modifications drive distinct substrate outcomes and cellular fates. Like Ub, SUMO has internal lysines that can be further modified, extended as SUMO chains, modified by Ub chains to target degradation, or even modified by smaller moieties, like acetyl groups 3, 4, 5. If required, SUMO as well as the substrate can be recycled by the action of sentrin-specific proteases (SENPs) that cleave the isopeptide bond. ![]() Briefly, the C-terminal di-glycine motif of mature SUMOs mediates modification of target lysines in substrates through the sequential action of E1 SUMO-activating enzyme SAE1/SAE2, E2 conjugating enzyme UBC9 and SUMO E3 ligases 2. ![]() Protein SUMOylation is a rigorously regulated cycle involving an enzymatic machinery that acts in a stepwise manner. Human SUMO2 and SUMO3 share 97% sequence identity, whereas they share 47% of sequence identity with SUMO1 1. The mammalian SUMO family consists of at least three major SUMO paralogues ( SUMO1, -2, -3). The UbL family includes Small Ubiquitin-like Modifiers (SUMOs). Ubiquitin-like (UbL) proteins belong to a superfamily of small proteins that attach covalently to target substrates in a transient and reversible manner. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code. ![]() Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. ![]()
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