Genetically encoding ε-N-methacryllysine into proteins in live cells

Lysine acylation is a ubiquitous post-translational modification (PTM) that plays pivotal roles in various cellular processes, such as transcription, metabolism, protein localization and folding. Thousands of lysine acylation sites have been identified based on advances in antibody enrichment strategies, highly sensitive analysis by mass spectrometry (MS), and bioinformatics. However, only 27 lysine methacrylation (Kmea) sites have been identified exclusively in histone proteins. It is hard to separate, purify and differentiate the Kmea modification from its structural isomer lysine crotonylation (Kcr) using general biochemical approaches. Here, we identify Kmea sites on a non-histone protein, Cyclophillin A (CypA). To investigate the functions of Kmea in CypA, we develop a general genetic code expansion approach to incorporate a non-canonical amino acid (ncAA) ε-N-Methacryllysine (MeaK) into target proteins and identify interacting proteins of methacrylated CypA using affinity-purification MS. We find that Kmea at CypA site 125 regulates cellular redox homeostasis, and HDAC1 is the regulator of Kmea on CypA. Moreover, we discover that genetically encode Kmea can be further methylated to ε-N-methyl-ε-N-methacrylation (Kmemea) in live cells. It is challenging to purify and prepare large amount of methacrylated proteins from cells. Here, the authors develop genetic code expansion technology to site-specifically incorporate ε-N-Methacryllysine into proteins and identify the post-translational modification ε-N-methyl-ε-N-methacrylation.