Development of genetically expanded yeast-two-hybrid system for acetylome analysis

Abstract

Yeast-two-hybrid (Y2H) is most widely used system to detect protein-protein interaction (PPI), and it has greatly contributed to proteomic analysis. Proteins generally function in different ways and interact with novel proteins via post-translational modifications (PTMs), yet Y2H system cannot verify PTM-dependent interactions. Genetic code expansion (GCE) site-specifically incorporates unnatural amino acids (UAAs) into proteins with the help of orthogonal aminoacyl-tRNA synthetase and its cognate tRNA, and it leads to direct expression of post-translationally modified proteins. This study accomplished site-specific acetylation on proteins and proposed the novel system, genetically expanded Y2H (GEY2H) system, to detect acetylation-dependent interaction for acetylomic analysis. Y2H system can be also used for screening peptide inhibitors of target proteins, yet its library size is small due to necessary transformation process. Warhead strategy is a traditional detour to discover authentic peptide drugs from small size of library by aiming to active sites of target proteins. 2-Amino-8-oxodecanoic acid (AODA), which is non-hydrolysable analogue, is an adequate warhead to mimic acetylation in nature and to inhibit the enzymatic activity of target proteins at the same time. Using GEY2H system which can incorporate AODA into peptides at in-frame stop codon, cyclic peptide inhibitors containing AODA have been successfully discovered. GEY2H system can be applied not only to find novel acetylation-dependent PPIs from high-throughput acetylome analysis but also to discover cyclic peptide inhibitors bearing warhead UAAs to aim to target proteins.