Genetic code expansion reveals aminoacylated lysine ubiquitination mediated by UBE2W

Protein post-translational modification (PTM) regulates nearly every aspect of cellular processes in eukaryotes. However, the identification of new protein PTMs is very challenging. Here, using genetically encoded unnatural amino acids as chemical probes, we report the identification and validation of a previously unreported form of protein PTM, aminoacylated lysine ubiquitination, in which the modification occurs on the α-amine group of aminoacylated lysine. We identify more than 2,000 ubiquitination sites on all 20 aminoacylated lysines in two human cell lines. The modifications can mediate rapid protein degradation, complementing the canonical lysine ubiquitination-mediated proteome degradation. Furthermore, we demonstrate that the ubiquitin-conjugating enzyme UBE2W acts as a writer of aminoacylated lysine ubiquitination and facilitates the ubiquitination event on proteins. More broadly, the discovery and validation of aminoacylated lysine ubiquitination paves the way for the identification and verification of new protein PTMs with the genetic code expansion strategy.