Reversible Lysine Acetylation Modification Of Enzymes Involved In Central Metabolism Of Salmonella Enterica

Protein post-translation modification is an important content of epigenetics. The pivotal importance of lysine-acetylation and its regulatory enzymes to the several fundamental cellular processes in mammalian cells, including histone modification, calorie restriction, cell apoptosis, life span extension, transcription silencing and so on, continues to be revealed at a remarkable pace. Recently, the study of lysine-acetylation in eukaryote changes with each passing day, and while the extent and function of this modification in prokaryotic remain largely unexplored because of the absence of research means and weak basis, thereby presenting a hurdle to further functional study of this modification in prokaryotic systems.Here we reported the first global scanning of lysine-acetylation in the prokaryote S. enterica by efficient affinity enrichment of lysine acetylated peptide with homemade lysine-acetylation antibody, combining with the high resolving power of immunoprecipitation and mass spectrum, and subsequently do the further study of these acetylation substrate enzymes of S. enterica central metabolism and energy metabolism from the physiological, biochemical, and genetic levels. Integrating with our collaborator's eukaryote results, we propose that reversible lysine acetylation of metabolic enzymes is the mechanism of global regulation of the central metabolism circuits and represents an evolutionarily conserved universal mechanism in metabolic regulation in both eukaryote and prokaryotes.Our study includes four parts.In the first part, we firstly applied the high sensitivity of immunoprecipitation to enrich the lysine acetylated peptides and did the proteomics screening of lysine-acetylation substrate proteins in S. enterica, whereafter used the high resolution of HPLC/mass spectrometer to identify acetylated peptides. The screening identified a series of acetylated substrate proteins correlated with energy metabolism, transcription modulation, amino acid and nucleic acid biosynthesis and so on. We discover that 190 modification sites in 166 proteins from S. enterica are acetylated, of which 77 proteins are correlated with metabolism. All these findings totally go beyond our expectation and open up a bran-new world for prokaryote lysine-acetyaltion modulation.In order to check the physiology of the lysine-acetylation on fermentation or non-fermentation carbon sources, in the second part, we constructed the null strains of S. enterica protein acetyltransferase (Pat) and the deacetylase (CobB) by using PCR mediated Red recombination technology and then cultured them on the glucose or citrate minimal medium. We find out S. enterica and its derivates exhibit different phenotypes on different carbon source minimal media and confirm the closer relation between revisable acetylation modification and energy metabolism. Moreover, we propose that so far the Pat and CobB may be the only known enzymes responsible for the revisable acetylation modification of the key metabolism enzymes.In the third part, we cloned and expressed the Pat, CobB and other proteins concerned with central energy metabolism, such as glyceraldehydes-3-phosphate dehydrogenase (GapA), isocitrate dehydrogenase kinase/phosphatase (AceK) and isocitrate lyase (AceA) in E. coli and S. enterica, then reconstructed the acetylation/deacetylation modification system in vitro by Pat or/and CobB to check whether these enzymes' activity regulated by lysine-acetylation. Furthermore, we constructed the GapA, AceK and AceA's site-directed mutant protein and null mutant strains and assayed their activities in vitro to study the specific modification sites. The results indicate that the GapA's dehydrogenation activity is directly proportioned with its acetylation level, and while the AceK's kinase activity and AceA's lyase activity are negative related with acetylation level. Moreover, the108,115,321 and 331 lysines of GapA, the 72,83 and 553 lysines of AceK, and the 13 and 308 lysines of AceA are closely correlated with acetylation modification.In the fourth part, we used the quantity Real-time PCR, choosing the 16S rRNA as internal transcriptional controls, to check the transcription of Pat and CobB in different metabolism pathways. The results reveal that S. enterica in glucose medium has higher pat and cobB transcription levels than that in citrate medium under either mid log phase or stationary phas; Furthermore, for cells grew in either citrate or glucose, the transcription levels of pat and cobB in log phase were uniformly higher than that in stationary phase. Therefore, we put forward that the change of the ratio of pat mRNA/cobB mRNA, designed acetylation potential, between cells in the mid log phase versus in the stationary phase. Through qRT-PCR study, we validate the relation between acetylation modification are coordinated with the change of carbon sources.In summary, the findings described in this thesis provide direct biochemical, physiological, and genetic data supporting the hypothesis that reversible lysine acetylation modification plays is a key and extensive role of global regulation of central metabolic circuits. Taking the advantage of a single pair of acetylase and deacetylase targeting metabolic enzymes in S. enterica, we demonstrate that the molecular mechanism achieving this regulation is through coupled transcription variation of de/acetylase (cobB/pat) genes and reversible lysine acetylation of metabolic enzymes in response to the change of carbon sources. Consistent with our collaborator's study in mammalian cells that showed a general role of acetylation in metabolism regulation, we propose that reversible lysine acetylation of metabolic enzymes represents an evolutionarily conserved universal mechanism in metabolic regulation in both eukaryote and prokaryotes.All these results open up a new field of acetyaltion modification in prokaryote.