A new paper published this week by researchers at the University of Leeds, in Science Signaling (link is external), describes the identification and characterisation of isoform-specific Affimer binders to SUMO1, SUMO2/3 and a pan-SUMO binder, that are suitable for the analysis of SUMO dependent cellular processes both in vitro and within the cell, opening the door to new insights into the function of this pathway and many other signalling events across the cell.
Sumoylation is the attachment of SUMO proteins to a target protein, for the control of target protein function and localisation within the cell. Proteins can be mono-SUMOylated or poly-SUMOylated with the three highly homologous SUMO units that show only 3 amino acid differences between SUMO2 and SUMO3 and a 47% homology between these isoforms and SUMO1. SUMO1 operates only as a monomeric protein modification, whereas SUMO 2 and 3 can also form polySUMO chains on target proteins. Within the cell SUMOylated proteins are recognised by SUMO-interaction motifs (SIMs) that facilitate non-covalent interactions with the SUMO moiety and ensure function.
Although researchers have long recognised the importance of SUMO dependent protein-protein interactions to cell signalling, methods to directly investigate these interactions, and many like them in other signalling pathways, are not well established. Interruption via chemical methods or protein mutation can be expensive and complicated to accurately interpret, with many proteins resulting in truncated or misfolded forms. While antibodies can be successfully used to specifically interrupt protein-protein interactions extracellularly, their complex structure relying on disulphide bonds means they fail to function within the reducing environment of the cell. Affimer binders allow specific targeting of protein interactions and can block function without affecting protein abundance, affording the possibility of examining discrete protein domains without affecting additional functions. As proteins act across intracellular networks with many binding partners, the ability to examine discrete domains may shed light on previously unknown functions and isoform specific functionality.
As part of the new study the authors used Affimer technology to rapidly isolate binders specific to the SUMO1 and SUMO2/3 isoforms in addition to a binder that recognised all three isoforms. Through structure guided studies, involving swapping the Affimer binder loop sequences, the researchers developed a molecular understanding of the isoform specificity of the SUMO specific Affimers for SUMO1, SUMO2/3 and the pan-SUMO binder. Intracellular expression of the Affimer binders demonstrated that the binders did not affect cell function and were specifically localised to nucleoli and PML bodies, which are the major sites of SUMOylation in cells.
Researchers performed in vitro SUMOylation assays to show that the isoform specific Affimer binders for SUMO did not interfere with SUMO conjugation or deSUMOylation either in vitro or when expressed within the cell. Rather, the Affimer binders interrupt the non-covalent binding of SUMO and SIMs and can inhibit the cellular stress response that requires SUMO-mediated protein-protein interactions. It is crucial that any tool to study SUMOylation does not interrupt SUMO conjugation to ensure the normal functioning of the SUMO pathway under investigation, and has previously been shown to be a problem with a SUMO1 specific Monobody (link is external).
In addition to generating SUMO-Affimers as useful tools for analysis of SUMO dependent cellular processes, this study also shows the applicability of this technology for generating reagents that interfere with specific protein-protein interactions for basic research and potentially for clinical development.