Discussion
Collectively, our study provided an important resource on substrate characterization and functional regulation of acidic lysine acylations in Mycobacterium smegmatis, giving valuable insights into their interrelation with the biology of infectious process.
Methods
We used the high-affinity antibody enrichment combined with high-resolution LC-MS/MS analysis to systematically investigate the global proteomic characteristics of the three acidic lysine acylations in Mycobacterium smegmatis. Subsequently, we employed in vitro enzymatic assays to validate the functional impact of acylated substrates, adenylate kinase and proteasome-associated ATPase. Furthermore, we investigated the effects of overexpressing these two substrates on the in vitro growth of Mycobacterium smegmatis, its invasion of THP-1 cells, and the influence on inflammatory cytokines.
Results
We systematically investigated the global substrate characterization of 1,703 lysine malonylated sites, 5,320 lysine succinylated sites and 269 lysine glutarylated sites in the non-pathogenic model strain Mycobacterium smegmatis. Bioinformatics analysis demonstrated a correlation between these acidic lysine acylations and the functional roles of ribosomes, in addition to their roles in various metabolic pathways. Furthermore, we investigated the impact of lysine acylations on the functional activity of adenylate kinase and proteasome-associated ATPase, as well as their roles in mycobacterial infection process.