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Abstract
Chemically diverse acylations on protein lysines have emerged as important posttranslational modifications (PTMs) that regulate many cellular processes such as gene transcription, cell cycle, and apoptosis. Lysine acylations are driven by lysine acetyltransferases (KATs) that can covalently deposit acyl group to the epsilon amino group of lysine residues from acyl-CoA molecules. Many studies have demonstrated that dysregulation of lysine acylation due to the dysfunction of KATs or abnormal fluctuation of acyl-CoA level may lead to the occurrence and progression of various diseases. Elucidation of the regulatory mechanisms of lysine acylations and their biological outcomes has profound significance to understand the pathophysiological mechanisms of the related diseases and develop effective therapeutic approaches towards them. Here, we report our efforts in the studies of novel functions and activities of KAT enzymes. We designed and synthesized an acetyl-CoA surrogate 3-azidopropionyl-CoA (3AZ-CoA) that can specifically react with the wild type KAT p300 and the mutant KAT GCN5-T612G. Based on the application of 3AZ-CoA, we developed an activity based protein profiling (ABPP) approach for valid profiling of KAT sub-acylome and identified hundreds of substrates for p300 and GCN5 KAT enzymes. By welding the application of 3AZ-CoA with fluorescence resonance energy transfer (FRET), we designed and validated a mix-and-read method for KAT activity measurement. This method can directly quantify the production of acylated protein in a fast, sensitive, and accurate way. We also discovered the novel activity of KAT enzymes from the MYST family as lysine propionyltransferases. The western blot and proteomic data suggested that one of the MYST members, MOF is able to propionylate both histone and non-histone proteins and has a partially distinct propionylome than other lysine propionyltransferases. The crystal structure of MOF bound with propionyl-CoA provides structural insights of how MYST KATs carry out lysine propionylation. Last but not least, we defined lysine isobutyrylation as a novel PTM on histone lysines and HAT1 as a potential isobutyryltransferase. Together, the present work provides valuable insights in understanding the regulatory mechanism of lysine acylations and the functions of KATs.