Studying lysine aminoacylation of human metabolic enzymes - Lysine aminoacylation is an emerging protein post-translational modification, which was first observed in human liver cancer tissues and the human HEK293T cell line. Approximately 3,500 proteins have been identified to carry lysine residues which are aminoacylated by all 20 standard amino acids. These substrate proteins are mostly involved in gene regulation, cell signaling, and metabolism with a proposed role in sensing and transmitting intracellular amino acid signals. However, only limited studies have been reported to further validate the function of lysine aminoacylation. Current gaps in knowledge of lysine aminoacylation include its substrate specificity, the necessity of aminoacyl-tRNA synthetases (AARS) in this process, the tools to study it, and its role in cellular metabolism. Our preliminary studies have shown that lysine aminoacylation has a strong preference for lysine or glutamate residues flanking aminoacylation-sensitive lysine residues. We have also established orthogonal translation systems (OTS) for four types of lysine aminoacylation to generate homogeneously aminoacylated proteins at target lysine sites by the genetic code expansion strategy. In addition, we have identified a series of essential metabolic enzymes associated with human diseases, which have been observed as substrate proteins of lysine aminoacylation. Our hypothesis is that lysine aminoacylation is an AARS-mediated enzymatic process with specific targets and plays roles in human metabolism by modifying essential metabolic enzymes. To test this hypothesis, three linked but mutually independent specific aims are proposed. Aim 1 is to determine the AARS specificity for lysine aminoacylation. Aim 2 is to develop OTSs for lysine aminoacylation by genetic code expansion. Aim 3 is to determine the role of lysine aminoacylation in cellular metabolism. This proposal is innovative because: 1) It is expected to demonstrate the noncanonical function of AARSs in aminoacylating lysine residues in proteins; 2) We propose lysine aminoacylation of metabolic enzymes plays roles in regulating cellular metabolism, which is a novel mechanism of lysine modifications associated with human metabolism; 3) The state-of-the-art genetic code expansion technique will be used in this project, and we will apply advanced approaches for OTS development such as fluorescence-activated cell sorting (FACS). The proposed research is significant because it will provide direct evidence of the role of lysine aminoacylation in regulating metabolism in human cells. It will also develop tools for studying lysine aminoacylation, which benefits other researchers in this field. For medical relevance, this project could determine the potential association of lysine aminoacylation with human diseases.
