Identification of Lysine Succinylation Substrates and the Succinylation Regulatory Enzyme CobB in Escherichia coli

Abstract

Lysine succinylation is a newly identified protein posttranslational modification pathway present in both prokaryotic and eukaryotic cells. However, succinylation substrates and regulatory enzyme(s) remain largely unknown, hindering the biological study of this modification. Here we report the identification of 2,580 bacterial lysine succinylation sites in 670 proteins and 2,803 lysine acetylation (Kac) sites in 782 proteins, representing the first lysine succinylation dataset and the largest Kac dataset in wild-type E. coli. We quantified dynamic changes of the lysine succinylation and Kac substrates in response to high glucose. Our data showed that high-glucose conditions led to more lysine-succinylated proteins and enhanced the abundance of succinyllysine peptides more significantly than Kac peptides, suggesting that glucose has a more profound effect on succinylation than on acetylation. We further identified CobB, a known Sir2-like bacterial lysine deacetylase, as the first prokaryotic desuccinylation enzyme. The identification of bacterial CobB as a bifunctional enzyme with lysine desuccinylation and deacetylation activities suggests that the eukaryotic Kac-regulatory enzymes may have enzymatic activities on various lysine acylations with very different structures. In addition, it is highly likely that lysine succinylation could have unique and more profound regulatory roles in cellular metabolism relative to lysine acetylation under some physiological conditions. Molecular & Cellular

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

“…The clarified lysate was precipitated by using 10% trichloroacetic acid and 1% sodium deoxycholate, and then washed twice with ice cold acetone. The precipitated proteins (2 mg) were redissolved in 50 mM ammonium bicarbonate, then in-solution digested by trypsin according to previously described (14,42). The protein concentration was determined with the Bradford assay (BioRad, Hercules, CA).…”

“…Enrichment and Identification of Lysine Succinylation Sites in Mtb-To identify the lysine succinylation sites in Mtb, we used a similar procedure as previously described (7,14,16) (Fig. 2A).…”

“…Peptides-Succinylated peptides were enriched using agarose-conjugated anti-succinyl lysine antibody (PTM Biolabs Inc., Chicago, IL) as previously described (14). Briefly, tryptic peptides were redissolved in NETN buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), and then incubated with anti-succinyllysine antibody conjugated protein A agarose beads at 4°C for 6h with gentle rotation.…”

“…It was recently identified and comprehensively validated in both bacterial and mammalian cells (8,14,16). It was also identified in core histones, suggesting that lysine succinylation may regulate the functions of histones and affect chromatin structure and gene expression (7).…”

“…Accumulating evidence suggests that lysine succinylation is a widespread and important PTM in both eukaryotes and prokaryotes and regulates diverse cellular processes (16). The system-wide studies involving lysine-succinylated peptide immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS/MS) have been employed to analyze the bacteria (E. coli) (14,16), yeast (S. cerevisiae), human (HeLa) cells, and mouse embryonic fibroblasts and liver cells (16,19). These succinylome studies have generated large data sets of lysine-succinylated proteins in both eukaryotes and prokaryotes and demonstrated the diverse cellular functions of this PTM.…”