Research ResourceBiochemistry

Generation of specific inhibitors of SUMO-1– and SUMO-2/3–mediated protein-protein interactions using Affimer (Adhiron) technology

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Sci. Signal.  14 Nov 2017:
Vol. 10, Issue 505, eaaj2005
DOI: 10.1126/scisignal.aaj2005

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Artificial proteins target SUMO

SUMOylation is the covalent attachment of SUMO-1, SUMO-2, SUMO-3, or combinations thereof to target proteins to control protein function and localization. Hughes et al. screened a library of artificial proteins called Affimers to identify those that bound to SUMO-1 or SUMO-2 (which is nearly identical to SUMO-3) and incorporated a negative selection step to remove SUMO-2–binding Affimers that also bound to SUMO-1. The authors identified Affimers that recognized SUMO-1, SUMO-2 and SUMO-3 (SUMO-2/3), or all three isoforms. Biochemical and cellular assays showed that these SUMO-specific Affimers (S-Affs) did not interfere with SUMO conjugation or deconjugation but inhibited a cellular stress response that required SUMO-mediated protein-protein interactions. In addition to generating S-Affs that will be useful tools for studying 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 (see the Protocol by Tang et al.).

Abstract

Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1–mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.

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