Succinylation Sites Research Articles

Site-Specific Installation of Succinyl Lysine Analog into Histones Reveals the Effect of H2BK34 Succinylation on Nucleosome Dynamics

Posttranslational modifications of histones play key roles in the dynamic regulation of chromatin structure. Lysine succinylation is a new type of histone modification, but its biological significance in chromatin structure and dynamics remains unknown. Here we develop a chemical approach to site-specifically install a succinyl lysine analog into histones. This analog serves as an ideal structural and functional mimic to natural succinyl lysine. The incorporation of this succinylation mimic into histone H2B at lysine 34, a succinylation site at the nucleosomal DNA-histone interface, leads to significant decrease in nucleosome stability invitro, which is consistent with the defects in chromatin structure of a budding yeast strain containing a lysine-to-glutamate mutation at the corresponding residue of yeast histone H2B. This study provides a simple method for the rapid generation of histones with site-specific succinylation mimics, and reveals novel regulatory mechanisms of histone succinylation in the dynamic organization of chromatin.

Involvement of the Vitamin D Receptor in Energy Metabolism Revealed by Profiling of Lysine Succinylome of White Adipose Tissue

Lysine succinylation, emerging as a novel post-translational modification, is closely related to the regulation of diverse biological processes, including many aspects of metabolism. Growing evidence suggests that low vitamin D status might exert an adverse impact on energy balance, adipogenesis and inflammation in white adipose tissue (WAT). However, whether there are any interactions between vitamin D and lysine succinylation still remains unknown. Here, combining high-affinity enrichment of lysine succinylated peptides with mass spectrometry and bioinformatics analysis, we reported the systematic profiling of the lysine succinylome, identifying 209 sites occurring on 159 proteins were up-regulated, 3 sites in 3 proteins were down-regulated in vitamin D receptor (VDR)−/− mice. Bioinformatics analysis reveals potential impacts of lysine succinylation on diverse biological processes and molecular functions, especially on carbon biotransformation, fatty acid metabolism and TCA cycle. Furthermore, eight unique motifs surrounding the succinylation sites were validated. Collectively, our findings demonstrate the first comprehensive profiling of WAT succinylome in VDR−/− mice, and provide crucial clues for further elucidating the underlying mechanisms of the involvement of the VDR in energy metabolism.

Succinyl-proteome profiling of Dendrobium officinale, an important traditional Chinese orchid herb, revealed involvement of succinylation in the glycolysis pathway

BackgroundLysine succinylation is a ubiquitous and important protein post-translational modification in various eukaryotic and prokaryotic cells. However, its functions in Dendrobium officinale, an important traditional Chinese orchid herb with high polysaccharide contents, are largely unknown.ResultsIn our study, LC-MS/MS was used to identify the peptides that were enriched by immune-purification with a high-efficiency succinyl-lysine antibody. In total, 314 lysine succinylation sites in 207 proteins were identified. A gene ontology analysis showed that these proteins are associated with a wide range of cellular functions, from metabolic processes to stimuli responses. Moreover, two types of conserved succinylation motifs, ‘***Ksuc******K**’ and ‘****EKsuc***’, were identified. Our data showed that lysine succinylation occurred on five key enzymes in the glycolysis pathway. The numbers of average succinylation sites on these five enzymes in plants were lower than those in bacteria and mammals. Interestingly, two active site amino acids residues, K103 and K225, could be succinylated in fructose-bisphosphate aldolase, indicating a potential function of lysine succinylation in the regulation of glycolytic enzyme activities. Furthermore, the protein–protein interaction network for the succinylated proteins showed that several functional terms, such as glycolysis, TCA cycle, oxidative phosphorylation and ribosome, are consisted.ConclusionsOur results provide the first comprehensive view of the succinylome of D. officinale and may accelerate future biological investigations of succinylation in the synthesis of polysaccharides, which are major active ingredients.

A systematic identification of species-specific protein succinylation sites using joint element features information.

Lysine succinylation, an important type of protein posttranslational modification, plays significant roles in many cellular processes. Accurate identification of succinylation sites can facilitate our understanding about the molecular mechanism and potential roles of lysine succinylation. However, even in well-studied systems, a majority of the succinylation sites remain undetected because the traditional experimental approaches to succinylation site identification are often costly, time-consuming, and laborious. In silico approach, on the other hand, is potentially an alternative strategy to predict succinylation substrates. In this paper, a novel computational predictor SuccinSite2.0 was developed for predicting generic and species-specific protein succinylation sites. This predictor takes the composition of profile-based amino acid and orthogonal binary features, which were used to train a random forest classifier. We demonstrated that the proposed SuccinSite2.0 predictor outperformed other currently existing implementations on a complementarily independent dataset. Furthermore, the important features that make visible contributions to species-specific and cross-species-specific prediction of protein succinylation site were analyzed. The proposed predictor is anticipated to be a useful computational resource for lysine succinylation site prediction. The integrated species-specific online tool of SuccinSite2.0 is publicly accessible.

Mild metabolic perturbations alter succinylation of mitochondrial proteins.

Succinylation of proteins is widespread, modifies both the charge and size of the molecules, and can alter their function. For example, liver mitochondrial proteins have 1,190 unique succinylation sites representing multiple metabolic pathways. Succinylation is sensitive to both increases and decreases of the NAD+ -dependent desuccinylase, SIRT5. Although the succinyl group for succinylation is derived from metabolism, the effects of systematic variation of metabolism on mitochondrial succinylation are not known. Changes in succinylation of mitochondrial proteins following variations in metabolism were compared against the mitochondrial redox state as estimated by the mitochondrial NAD+ /NADH ratio using fluorescent probes. The ratio was decreased by reduced glycolysis and/or glutathione depletion (iodoacetic acid; 2-deoxyglucose), depressed tricarboxylic acid cycle activity (carboxyethyl ester of succinyl phosphonate), and impairment of electron transport (antimycin) or ATP synthase (oligomycin), while uncouplers of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine or tyrphostin) increased the NAD+ /NADH ratio. All of the conditions decreased succinylation. In contrast, reducing the oxygen from 20% to 2.4% increased succinylation. The results demonstrate that succinylation varies with metabolic states, is not correlated to the mitochondrial NAD+ /NADH ratio, and may help coordinate the response to metabolic challenge.

Quantitative Succinyl-Proteome Profiling of Camellia sinensis cv. \u2018Anji Baicha\u2019 During Periodic Albinism

Lysine succinylation is a novel dynamic and evolutionarily conserved post-translational modification (PTM) that regulates various biological processes. ‘Anji Baicha’ is an albino tea variety that exhibits temperature-based variability of leaf colour and amino acid concentrations. However, the mechanism underlying albinism in ‘Anji Baicha’ has not been investigated at the level of succinylation. Here, we identify 3530 lysine succinylation sites mapped to 2132 proteins in ‘Anji Baicha’, representing the first extensive data on the lysine succinylome in the tea plant. Eleven conserved succinylation motifs were enriched among the identified succinylated peptides. The protein-protein interaction maps were visualized using Cytoscape software. Comparison across three typical developmental stages of ‘Anji Baicha’ revealed that proteins exhibiting differential succinylation levels were primarily involved in photosynthesis, carbon fixation, biosynthesis of amino acids and porphyrin and chlorophyll metabolism, suggesting that these succinylated proteins are involved in ‘Anji Baicha’ leaf colour variability. These results not only deepen our understanding of the mechanism underlying ‘Anji Baicha’ albinism and the regulatory role of succinylation in the tea plant but also provide new insight into molecular breeding for leaf colour variety.

Quantitative global proteome and lysine succinylome analyses provide insights into metabolic regulation and lymph node metastasis in gastric cancer

With the rapid development of high-throughput quantitative proteomic and transcriptomic approaches, the molecular mechanisms of cancers have been comprehensively explored. However, cancer is a multi-dimensional disease with sophisticated regulations, and few studies focus on the crosstalk among multiomics. In order to explore the molecular mechanisms of gastric cancer (GC), particularly in the process of lymph node metastasis (LNM), we investigated dynamic profiling changes as well as crosstalk between long non-coding RNAs (lncRNAs), the proteome, and the lysine succinylome. Our study reports the first qualitative and quantitative profile of lysine succinylation in GC. We identified a novel mechanism through which the TCA cycle and pentose phosphate pathway might be regulated through lysine succinylation in their core enzymes. We then examined the potential of using lysine succinylation as a biomarker for GC and successfully developed a succinylation-dependent antibody for the K569 site in Caldesmon as putative biomarker. Finally, we investigated the relationship between the lysine succinylome and lncRNAs, identifying potential crosstalks between two lncRNAs and one succinylation site. These results expand our understanding of the mechanisms of tumorigenesis and provide new information for the diagnosis and prognosis of GC.

Proteomic analysis of lysine succinylation of the human pathogen Histoplasma capsulatum

Histoplasma capsulatum, the causative agent of histoplasmosis (also called “Darling's disease”), can affect both immunocompetent and immunocompromised hosts. Post-translational protein modification by lysine succinylation (Ksuc) is a frequent occurrence in eukaryote and prokaryote. Recently, the roles of succinylation and its regulatory enzymes in regulating metabolic pathway in bacteria, mammalian and fungus were highlighted. Here, we report the first global profiling of lysine succinylation, with 463 modification sites in 202 proteins from H. capsulatum NAM1 identified, coupling immune-affinity enrichment using an anti-succinyllysine antibody with mass spectrometry. The bioinformatics results including GO functional and enrichment analysis showed that these succinylated proteins are mainly involved in central metabolism and protein synthesis, consistent with previous reports. 13 lysine succinylation sites on histones including H2A, H2B, H3 and H4 in H. capsulatum were firstly reported. The data is a good resource for further functional characterization of lysine succinylation in H. capsulatum. Biological significanceH. capsulatum is the causative agent of lung disease histoplasmosis. The ability of H. capsulatum yeasts to infect and proliferate within macrophages as an intracellular pathogen can be contributed to several virulence factors and metabolic regulation. Lysine succinylation was recently shown to play a critical role in the metabolism regulation of Candida albicans. H. capsulatum succinylated proteins were firstly characterized in this work, and bioinformatics results showed that this modification may also be relevant with central metabolism in H. capsulatum. New succinylation sites on histones were reported. This represents an important resource to address the function of H. capsulatum lysine succinylation.

DV-iSucLys: Decision Voting to Improve Protein Lysine Succinylation Site Identification from Sequence Data

Protein Post Translation Modification identification is one of the important steps in conducting disease-associated mutation studies. Though multiple chemical alterations happen in a protein after translation, the addition of succinyl group to lysine residue plays a vital role in regulating cellular metabolism and thus disease. Use of a classification algorithm on some features, driven either from protein structural, physicochemical or even biochemical information becomes a common approach that can yield a satisfactory result up to a certain level. Although, researchers already developed many computational methods to identify whether a lysine residue modified with succinyl group after translation, most of them focused on the improvement either on a single decision using a single method or feature enrichment or even development of a benchmark dataset. Therefore, there still exists scope for further improvement to characterise lysine residues of a protein sequence by considering multiple predictors at a time. In this study, an ensemble based approach called DV-iSucLys has been designed to characterise the lysine residue by adapting three well known and conceptually different classifiers and ensembling their decisions. Also, a benchmark succinylation dataset was extracted from existing benchmark datasets and recently updated succinylation data from UniProt consortium to investigate the performance of the proposed approach as well as contribute to further research. Analysing rigorous cross-validation results show that DV-iSucLys can characterise succinyl lysine residue better than the existing predictors.

Proteome-wide identification of lysine succinylation in thermophilic and mesophilic bacteria

Lysine succinylation, one of post-translational acylations conserved from eukaryotes to bacteria, plays regulatory roles in various cellular processes. However, much remains unknown about the general and specific characteristics of lysine succinylation among bacteria, and about its functions different from those of other acylations. In this study, we characterized lysine succinylation, a newly discovered widespread type of lysine acylation in five bacterial species with different characteristics such as optimal growth temperature and cell wall structure. This study is the first to demonstrate that succinylation is general phenomenon occurring not only in mesophiles but also in thermophiles. Mapping of succinylation sites on protein structures revealed that succinylation occurs at many lysine residues important for protein function. Comparison of the succinylation sites in the five bacterial species provides insights regarding common protein regulation mechanisms utilizing lysine succinylation. Many succinylation sites were conserved among five bacteria, especially between Geobacillus kaustophilus and Bacillus subtilis, some of which are functionally important sites. Furthermore, systematic comparison of the succinyl-proteome results and our previous propionyl-proteome results showed that the abundance of these two types of acylations is considerably different among the five bacteria investigated. Many succinylation and propionylation events were detected in G. kaustophilus, whereas Escherichia coli and B. subtilis exhibited high succinylation and low propionylation; low succinylation and high propionylation were identified in Thermus thermophilus, and low succinylation and propionylation were observed in Rhodothermus marinus. Comparison of the characteristics of lysine succinylation and lysine propionylation suggested these two types of acylation play different roles in cellular processes.

Crosstalk among proteome, acetylome and succinylome in colon cancer HCT116 cell treated with sodium dichloroacetate

Protein lysine acetylation and succinylation play important regulatory roles in cells, both of which or each other has a close relationship. Dichloroacetate (DCA), a well-known pyruvate dehydrogenase kinase (PDK) inhibitor, has the potential to be used as anti-cancer drugs for several tumors including colorectal cancer. However, little is known about the potential mechanism of DCA-based cancer therapy by protein posttranslational modifications (PTM) including global proteome, acetylome and succinylome. Here the combinations with stable isotope labeling (SILAC), antibody affinity enrichment and high resolution LC-MS/MS analysis were performed in human colon cancer HCT116 cells. The quantifiable proteome was annotated using bioinformatics. In total, 4,518 proteins, 1,436 acetylation sites, and 671 succinylation sites were quantified, respectively to DCA treatment. Among the quantified acetylated sites, 158 were with increased level (quantification ratio >1.5) and 145 with decreased level (quantification ratio <0.67). Meanwhile, 179 up-regulated and 114 down-regulated succinylated sites were identified. The bioinformatics analyses initially showed acetylation and succinylation were involved in a wide range of cellular functions upon DCA-based anti-cancer effects. Notably, protein-protein interaction network analyses demonstrated widespread interactions modulated by protein acetylation and succinylation. Taken together, this study may shed a light on understanding the mechanism of DCA-based cancer treatment.

Systematic Analysis of the Lysine Succinylome in Candida albicans.

Candida albicans is the most common human fungal pathogen for both immunocompetent and immunocompromised individuals. Lysine succinylation is a frequently occurring post-translational modification that is found in many organisms; however, the role of succinylation is still under investigation. Here, we initiated a first screening of lysine succinylation in C. albicans. We identified 1550 succinylation sites from 389 proteins in C. albicans, demonstrating that succinylation is conservative in this organism. However, the lysine succinylation sites showed some difference in C. albicans, with the overlapping rates between C. albicans and other species ranging from 55% for Saccharomyces cerevisiae, 40% for human, 35% for mouse, and to only 16% for Escherichia coli. The further bioinformatics analysis indicated that the succinylated proteins were involved in a wide range of cellular functions with diverse subcellular localizations. Furthermore, we discovered that lysine succinylation could coexist with phosphorylation and/or acetylation in C. albicans. The KEGG enrichment pathway analysis of these succinylated proteins suggested that succinylation may play an indispensable role in the regulation of the tricarboxylic acid cycle. The bioinformatic data obtained from this study therefore enable the depth-resolved physiological roles of lysine succinylation in C. albicans.

Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress.

Lysine acetylation and succinylation are major types of protein acylation that are important in many cellular processes including gene transcription, cellular metabolism, DNA damage response. Malfunctions in these post-translational modifications are associated with genome instability and disease in higher organisms. In this study, we used high-resolution nano liquid chromatography-tandem mass spectrometry combined with affinity purification to quantify the dynamic changes of protein acetylation and succinylation in response to ultraviolet (UV)-induced cell stress. A total of 3345 acetylation sites in 1440 proteins and 567 succinylation sites in 246 proteins were identified, many of which have not been reported previously. Bioinformatics analysis revealed that these proteins are involved in many important biological processes, including cell signalling transduction, protein localization and cell metabolism. Crosstalk analysis between these two modifications indicated that modification switches might regulate protein function in response to UV-induced DNA damage. We further illustrated that FEN1 acetylation at different sites could lead to different cellular phenotypes, suggesting the multiple function involvement of FEN1 acetylation under DNA damage stress. These systematic analyses provided valuable resources and new insight into the potential role of lysine acetylation and succinylation under physiological and pathological conditions.

Succinyl-proteome profiling of a high taxol containing hybrid Taxus species (Taxus × media) revealed involvement of succinylation in multiple metabolic pathways.

Protein lysine succinylation, a ubiquitous protein post-translational modification among eukaryotic and prokaryotic cells, represents a vital regulator of various metabolic processes. However, little is known about its functions and cellular distribution in Taxus × media, which is a hybrid Taxus species containing a high content of taxol. In this study, LC-MS/MS was used to identify peptides enriched by immune-purification with high-efficiency succinyl-lysine antibody. A total of 193 succinylated proteins and 325 succinylation sites were identified. The bioinformatics analysis indicated that these succinylated proteins were involved in a wide range of cellular functions from metabolism to protein binding and showed diverse subcellular localizations. Furthermore, our findings suggested that lysine succinylation in Taxus × media involved a diverse array of metabolic processes and protein–protein interactions. Many enzymes involved in multiple metabolic pathways, such as glycolysis, pyruvate metabolism, the tricarboxylic acid cycle and carbon fixation, were identified as substrates for lysine succinylation, suggesting the presence of a common mechanism underlying the participation of succinylation in metabolic regulation. These results provide the first comprehensive view of the succinylome of Taxus × media and may catalyze future biological investigation of succinylation.

Quantitative succinylome analysis in the liver of non-alcoholic fatty liver disease rat model.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a clinical frequent disease. However, its pathogenesis still needs further study, especially the mechanism at the molecular level. The recent identified novel protein post-translational modification, lysine succinylation was reported involved in diverse metabolism and cellular processes. In this study, we performed the quantitative succinylome analysis in the liver of NAFLD model to elucidate the regulatory role of lysine succinylation in NAFLD progression.MethodsFirstly, experimental model of NAFLD was induced by carbon tetrachloride injection and supplementary high-lipid and low-protein diet. Then series histochemical and biochemical variables were determined. For the quantitative succinylome analysis, tandem mass tags (TMT)-labeling, highly sensitive immune-affinity purification, liquid chromatography-tandem mass spectrometry techniques were applied. Bioinformatics analysis including gene ontology annotation based classification; Wolfpsort based subcellular prediction; function enrichment; protein-protein interaction network construction and conserved succinylation site motifs extraction were performed to decipher the differentially changed succinylated proteins and sites and p-value < 0.05 was selected as threshold.ResultsTotally, 815 succinylation sites on 407 proteins were identified, of which 243 succinylation acetylation sites on 178 proteins showed changed succinylation level with the threshold fold change > 1.5. Theses differentially changed succinylated proteins were involved in diverse metabolism pathways and cellular processes including carbon metabolism, amino acid metabolism, fat acid metabolism, binding and catalyzing, anti-oxidation and xenobiotics metabolism. Besides, these differentially changed succinylated proteins were prominently localized to cytoplasm and mitochondria. Moreover, 8 conserved succinylation site motifs were extracted around the succinylation sites.ConclusionsProtein succinylation was an extensive post-translation modification in rat. The changed succinylation level in diverse proteins may disturb multiple metabolism pathways and promote non-alcoholic fatty liver disease development. This study provided a basis for further characterization of the pathophysiological role of lysine succinylation in NAFLD progression, which laid a foundation for the innovation of novel NAFLD drugs and therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0092-y) contains supplementary material, which is available to authorized users.

PSuc-Lys: Predict lysine succinylation sites in proteins with PseAAC and ensemble random forest approach

Being one type of post-translational modifications (PTMs), protein lysine succinylation is important in regulating varieties of biological processes. It is also involved with some diseases, however. Consequently, from the angles of both basic research and drug development, we are facing a challenging problem: for an uncharacterized protein sequence having many Lys residues therein, which ones can be succinylated, and which ones cannot? To address this problem, we have developed a predictor called pSuc-Lys through (1) incorporating the sequence-coupled information into the general pseudo amino acid composition, (2) balancing out skewed training dataset by random sampling, and (3) constructing an ensemble predictor by fusing a series of individual random forest classifiers. Rigorous cross-validations indicated that it remarkably outperformed the existing methods. A user-friendly web-server for pSuc-Lys has been established at http://www.jci-bioinfo.cn/pSuc-Lys, by which users can easily obtain their desired results without the need to go through the complicated mathematical equations involved. It has not escaped our notice that the formulation and approach presented here can also be used to analyze many other problems in computational proteomics.

Quantitative Analysis of the Sirt5-Regulated Lysine Succinylation Proteome in Mammalian Cells.

Lysine (Lys) succinylation is a recently discovered protein posttranslational modification pathway that is evolutionarily conserved from bacteria to mammals. It is regulated by Sirt5, a member of the class III histone deacetylases (HDACs) or the Sirtuins. Recent studies demonstrated that Lys succinylation and Sirt5 are involved in diverse cellular metabolic processes including urea cycle, ammonia transfer, and glucose metabolism. In this chapter, we describe the general protocol to identify Sirt5-regulated Lys succinylation substrates and a computational method to calculate the absolute modification stoichiometries of Lys succinylation sites. The strategy employs Stable Isotope Labeling of Amino acid in Cell culture (SILAC) and the immunoaffinity enrichment of Lys succinylated peptides to identify the Lys succinylation sites that are significantly upregulated in Sirt5 knockout mouse embryonic fibroblast cells.

SuccinSite: a computational tool for the prediction of protein succinylation sites by exploiting the amino acid patterns and properties.

Lysine succinylation is an emerging protein post-translational modification, which plays an important role in regulating the cellular processes in both eukaryotic and prokaryotic cells. However, the succinylation modification site is particularly difficult to detect because the experimental technologies used are often time-consuming and costly. Thus, an accurate computational method for predicting succinylation sites may help researchers towards designing their experiments and to understand the molecular mechanism of succinylation. In this study, a novel computational tool termed SuccinSite has been developed to predict protein succinylation sites by incorporating three sequence encodings, i.e., k-spaced amino acid pairs, binary and amino acid index properties. Then, the random forest classifier was trained with these encodings to build the predictor. The SuccinSite predictor achieves an AUC score of 0.802 in the 5-fold cross-validation set and performs significantly better than existing predictors on a comprehensive independent test set. Furthermore, informative features and predominant rules (i.e. feature combinations) were extracted from the trained random forest model for an improved interpretation of the predictor. Finally, we also compiled a database covering 4411 experimentally verified succinylation proteins with 12 456 lysine succinylation sites. Taken together, these results suggest that SuccinSite would be a helpful computational resource for succinylation sites prediction. The web-server, datasets, source code and database are freely available at http://systbio.cau.edu.cn/SuccinSite/.

ISuc-PseOpt: Identifying lysine succinylation sites in proteins by incorporating sequence-coupling effects into pseudo components and optimizing imbalanced training dataset

Succinylation is a posttranslational modification (PTM) where a succinyl group is added to a Lys (K) residue of a protein molecule. Lysine succinylation plays an important role in orchestrating various biological processes, but it is also associated with some diseases. Therefore, we are challenged by the following problem from both basic research and drug development: given an uncharacterized protein sequence containing many Lys residues, which one of them can be succinylated, and which one cannot? With the avalanche of protein sequences generated in the postgenomic age, the answer to the problem has become even more urgent. Fortunately, the statistical significance experimental data for succinylated sites in proteins have become available very recently, an indispensable prerequisite for developing a computational method to address this problem. By incorporating the sequence-coupling effects into the general pseudo amino acid composition and using KNNC (K-nearest neighbors cleaning) treatment and IHTS (inserting hypothetical training samples) treatment to optimize the training dataset, a predictor called iSuc-PseOpt has been developed. Rigorous cross-validations indicated that it remarkably outperformed the existing method. A user-friendly web-server for iSuc-PseOpt has been established at http://www.jci-bioinfo.cn/iSuc-PseOpt, where users can easily get their desired results without needing to go through the complicated mathematical equations involved.

Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction

The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more easily than acetylation in such conditions where the substrates for both acetylation and succinylation are limited. This is the first study investigating the acetylome and succinylome of C. glutamicum, and it provides new insight into the roles of acyl modifications in C. glutamicum biology.