Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis*

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetylCoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

“…A majority of modified proteins are found to be associated with metabolism and are cytosolic. Also, negative regulation of protein function was found in three such studies (88,90,91). Analysis of the mycobacterial acetylome for the presence of other modifications such as pupylation and phosphorylation reveal novel signaling nodes in Mycobacterium.…”

Discovery and Identification of Serum Succinyl‐Proteome for Postmenopausal Women with Osteoporosis and Osteopenia

“…In terms of the multiple differentially expressed protein classification, cluster analysis offered the chance to authenticate the accuracy of potential connections and differences in specific functions, we collected the functional classification information and obtained P ‐values of the protein groups, we screened the functional classification for significant enrichment ( P ‐value <0.05) in at least one cluster. Immediately after, the screened data matrix was transformed by the function x = −log10 ( P value), and then classified by Z ‐transformation.…”

“…Subsequently, western blotting analysis was used to identify the lysine succinylated peptides derived from the proteins in vivo. Increasing evidence has revealed that succinylation locating on critical metabolic networks, such as enzyme activity, central metabolism pathway and cell stress response to aging and apoptosis. As a result of the mitochondrial sirtuin SIRT5‐mediated lysine desuccinylation, lysine succinylation sites overlapped to differential extents and regulations compared with other diverse post‐translational modifications.…”

Nε- and O-Acetylation in Mycobacterium tuberculosis Lineage 7 and Lineage 4 Strains: Proteins Involved in Bioenergetics, Virulence, and Antimicrobial Resistance Are Acetylated

“…15,17,33 Furthermore, bacterial acetylome studies have been limited to the analysis of lysine residues. O-acetylation of proteins has been shown to be involved in regulating key functions in eukaryotes.…”