A semi-synthetic pathway to SUMOylated protein mimics
Résumé
Native chemical ligation (NCL) is the gold-standard reaction for chemical protein synthesis, enabling the chemoselective assembles of unprotected peptide segments by forming an amide bond from a C-terminal thioester and an N-terminal cysteine. Nowadays, mainly due to continuousmethodological improvements in NCL, chemical protein synthesis represents an appealing alternative for obtaining protein targets that are inaccessible through recombinant methods, particularly those carrying a specific post-translational modification. The study of SUMOylation is laborious. Indeed, the isolation of the modified protein is compromised by the sensitivity of the isopeptide bond towards hydrolysis by SUMO-proteases. Moreover, in vitro enzymatic SUMOylation is difficult to control. In order to overcome these problems, we aimed to develop a methodology for generating chemical biology tools designed to decipher the role of SUMOylation. This methodology relies on conjugating a synthetic SUMO-2 protein equipped with a C-terminal alkyne to a recombinant protein containing an azide function, through a copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC). The formed triazole is an excellent mimic of the isopeptide bond and resists hydrolysis by proteases. After the development of an efficient and clean method to synthesize the native SUMO-2 protein by NCL and its analogue equipped with a C-terminal alkyne, we pursued our investigations with this latter to synthesize a SUMOylated-peptide mime by the CuAAC-reaction. Although the SUMOylated-peptide mime was recognized by SUMO-protease, it was completely resistant towards hydrolysis. Afterwards, the methodology was extended to the synthesis of a SUMOylated-protein, involving the recombinant production of a protein containing a non-canonical amino acid possesing an azide group.
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