Applications of Genetic Code Expansion in Studying Protein Post-translational Modification

Various post-translational modifications can naturally occur on proteins, regulating the activity, subcellular localization, interaction, or stability of the proteins. However, it can be challenging to decipher the biological implication or physiological roles of site-specific modifications due to their dynamic and substoichiometric nature. Genetic code expansion method, relying on an orthogonal aminoacyl-tRNA synthetase/tRNA pair, enables site-specific incorporation of non-canonical amino acids. Here we focus on the application of genetic code expansion to study site-specific protein post-translational modification in vitro and in vivo. After a brief introduction, we discuss possibilities of incorporating non-canonical amino acids containing post-translational modifications or their mimics into target proteins. This approach is applicable for Ser/Thr/Tyr phosphorylation, Tyr sulfation/nitration/hydroxylation, Lys acetylation/acylation, Lys/His mono-methylation, as well as Arg citrullination. The next section describes the use of a precursor non-canonical amino acid followed by chemical and/or enzymatic reactions to afford the desired modification, such as Cys/Lys acylation, ubiquitin and ubiquitin-like modifications, as well as Lys/Gln methylation. We also discuss means for functional regulation of enzymes involving in post-translational modifications through genetically incorporated non-canonical amino acids. Lastly, the limitations and perspectives of genetic code expansion in studying protein post-translational modification are described. (C) 2021 Elsevier Ltd. All rights reserved.

Automated summary

Various post-translational modifications play important roles in regulating the function of proteins. However, studying the biological implications and physiological roles of these modifications can be challenging due to their complex nature. Genetic code expansion method allows for the site-specific study of protein post-translational modifications by introducing non-canonical amino acids, including phosphorylation, sulfation, nitration, hydroxylation, acetylation, acylation, methylation, and citrullination. Additionally, this method can be used to regulate the function of enzymes involved in post-translational modifications.