A Genetic Engineering Solution to the ��Arginine Conversion Problem�� in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.Stable isotope labeling by amino acids in cell culture (SILAC)¹ (1) is one of the key methods for large scale quantitative proteomics (2, 3). In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing either normal (“light”) or heavy isotope-labeled amino acids, typically lysine and arginine. Peptides derived from the light and heavy cells are thus distinguishable by mass spectrometry and can be mixed for accurate quantitation. SILAC is also possible at the whole-organism level (4).An inherent problem in SILAC is the metabolic conversion of labeled arginine to other amino acids, as this complicates quantitative analysis of peptides containing these amino acids. Arginine conversion to proline is well described in mammalian cells, although the extent of conversion varies among cell types (5). When conversion is observed, typically 10–25% of the total proline pool is found to contain label (6–11). Arginine conversion has also been reported in SILAC experiments with budding yeast Saccharomyces cerevisiae (3, 12, 13).Because more than 50% of tryptic peptides in large data sets contain proline (7), it is not practical simply to disregard proline-containing peptides during quantitation. Several methods have been proposed to either reduce arginine conversion or correct for its effects on quantitation. In some cell types, arginine conversion can be prevented by lowering the concentration of exogenous arginine (6, 14–16) or by adding exogenous proline (9). However, these methods can involve significant changes to growth media and may need to be tested for each experimental condition used. Given the importance of arginine in many metabolic pathways, careful empirical titration of exogenous arginine concentration is required to minimize negative effects on cell growth (14). In addition, low arginine medium can lead to incomplete arginine labeling, although the reasons for this are not entirely clear (7). An alternative strategy is to omit labeled arginine altogether (3, 13, 17), but this reduces the number of quantifiable peptides. Correction methods include using two different forms of labeled arginine (7) or computationally compensating for proline-containing peptides (11, 12, 18). Ultimately, none of these methods address the problem at its root, the utilization of arginine in cellular metabolism.To develop a differential proteomics work flow for the fission yeast Schizosaccharomyces pombe, we sought to adapt SILAC for use in this organism, a widely used model eukaryote with excellent classical and reverse genetics. Here we describe extremely high conversion of labeled arginine to other amino acids in fission yeast as well as a novel general solution to the problem that should be applicable to other organisms. As proof of principle, we quantitated changes in protein levels before and after cell division on a proteome-wide scale. We identified both up- and down-regulated proteins, including a novel protein involved in septation.     

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

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 acetyl-CoA 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.Post-translational modifications (PTMs)¹ are complex and fundamental mechanisms modulating diverse protein properties and functions, and have been associated with almost all known cellular pathways and disease processes (1, 2). Among the hundreds of different PTMs, acylations at lysine residues, such as acetylation (3–6), malonylation (7, 8), crotonylation (9, 10), propionylation (11–13), butyrylation (11, 13), and succinylation (7, 14–16) are crucial for functional regulations of many prokaryotic and eukaryotic proteins. Because these lysine PTMs depend on the acyl-CoA metabolic intermediates, such as acetyl-CoA (Ac-CoA), succinyl-CoA, and malonyl-CoA, lysine acylation could provide a mechanism to respond to changes in the energy status of the cell and regulate energy metabolism and the key metabolic pathways in diverse organisms (17, 18).Among these lysine PTMs, lysine succinylation is a highly dynamic and regulated PTM defined as transfer of a succinyl group (-CO-CH₂-CH₂-CO-) to a lysine residue of a protein molecule (8). 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. Notably, lysine succinylation is widespread among diverse mitochondrial metabolic enzymes that are involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (19, 20). Thus, lysine succinylation is reported as a functional PTM with the potential to impact mitochondrial metabolism and coordinate different metabolic pathways in human cells and bacteria (14, 19–22).Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a major cause of mortality worldwide and claims more human lives annually than any other bacterial pathogen (23). About one third of the world''s population is infected with Mtb, which leads to nearly 1.3 million deaths and 8.6 million new cases of TB in 2012 worldwide (24). Mtb remains a major threat to global health, especially in the developing countries. Emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Mtb, and also the emergence of co-infection between TB and HIV have further worsened the situation (25–27). Among bacterial pathogens, Mtb has a distinctive life cycle spanning different environments and developmental stages (28). Especially, Mtb can exist in dormant or active states in the host, leading to asymptomatic latent TB infection or active TB disease (29). To achieve these different physiologic states, Mtb developed a mechanism to sense diverse signals from the host and to coordinately regulate multiple cellular processes and pathways (30, 31). Mtb has evolved its metabolic network to both maintain and propagate its survival as a species within humans (32–35). It is well accepted that metabolic network is a central mediator and defining feature of the pathogenicity of Mtb (23, 36–38). Knowledge of the regulation of metabolic pathways used by Mtb during infection is therefore important for understanding its pathogenicity, and can also guide the development of novel drug therapies (39). On the other hand, increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells (14, 19–22). It is tempting to speculate that lysine succinylation may play an important regulatory role in metabolic processes in Mtb. However, to the best of our knowledge, no succinylated protein in Mtb has been identified, presenting a major obstacle to understand the regulatory roles of lysine succinylation in this life-threatening pathogen.In order to fill this gap in our knowledge, we have initiated a systematic study of the identities and functional roles of the succinylated protein in Mtb. Because Mtb H37Rv is the first sequenced Mtb strain (40) and has been extensively used for studies in dissecting the roles of individual genes in pathogenesis (41), it was selected as a test case. We analyzed the succinylome of Mtb 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 render particular enrichment to metabolic process. A large proportion of the succinylation sites are present on proteins in the central metabolism pathway. We further dissected the regulatory role of succinylation on acetyl-CoA synthetase (Acs) via site-specific mutagenesis analysis and molecular dynamics (MD) simulations showed that reversible lysine succinylation could inhibit the activity of Acs. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a deacetylase and as a desuccinylase of Acs in in vitro assays. Together, our findings provide significant insights into the range of functions regulated by lysine succinylation in Mtb.     

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

Lysine succinylation is a newly identified protein post-translational 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.Lysine acetylation (Kac)¹ is a dynamic and evolutionarily conserved post-translational modification (PTM) that is known to be involved in the regulation of diverse cellular processes (1–9). The status of this modification is controlled by two groups of enzymes with opposing enzymatic activities, lysine acetyltransferases that add an acetyl group to the lysine (Lys or K) residue, and histone lysine deacetylases (HDACs) that remove the acetyl group (10–16). HDACs are grouped into several categories (17): class I (HDAC1, -2, -3, and -8), class IIA (HDAC4, -5, -7, and -9), class IIB (HDAC6 and -10), class III (Sirt1–7), and class IV (HDAC11). The weak deacetylation activities of some HDACs (e.g. Sirt4–7 and HDAC4, -5, and -7–11), as well as the demonstration of Sirt5 as a desuccinylation and demalonylation enzyme, suggest that some HDAC enzymes have activities that are independent of acetylation (18, 19).For a long period of time, lysine acetylation was considered as a protein modification that was restricted to nuclei (20). The identification of cytosolic Kac substrates and the localization of some HDACs outside nuclei suggest a non-nuclear function of lysine acetylation (13, 21, 22). The first proteomic screening identified hundreds of substrate proteins in cytosolic and mitochondrial fractions and demonstrated high abundance of Kac in mitochondrial proteins and metabolic enzymes (23). This result implies that Kac has diverse non-nuclear roles and can regulate functions of metabolism and mitochondria (23). Since then, we and others have extensively characterized the cellular acetylome (5, 9, 24–26).The lysine succinylation (Ksucc) and lysine malonylation pathways are two PTM pathways that were recently identified and comprehensively validated in both bacterial and mammalian cells, with multiple substrate proteins identified, using HPLC-MS/MS, co-elution of synthetic peptides, isotopic labeling, Western blotting analysis using pan-anti-Ksucc antibodies, and proteomics analysis (18, 27). We also showed that Ksucc is present in core histones (29). In yeast histones, some Ksucc sites are located in regions where histones make close contact with DNA, suggesting that Ksucc sites may be involved in gene regulation by changing the chromatin structure (29). We then found that Sirt5, a member of the class III family of HDACs, can function as a desuccinylation enzyme in vitro and in vivo (18, 19). In a recent study, we revealed that Sirt5 is a key regulatory enzyme of Ksucc and that Ksucc proteins are abundant among a group of mitochondrial enzymes that are predominantly involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (28). Importantly, Ksucc is very dynamic not only in mammalian cells, but also in bacteria (27, 29). These lines of evidence strongly suggest that lysine succinylation is likely an important PTM in the regulation of cellular functions.Although key elements of the Ksucc pathway are being identified in mammalian cells, their counterparts in bacteria remain largely unknown. We and others have used a proteomics approach to identify Kac substrates in bacteria (26, 30, 31, 52). The Sir2-like enzyme CobB is the best-studied protein deacetylase in bacteria (8). CobB was initially identified as an enzyme required for the activation of acetyl-CoA synthetase (8). Recently, CobB was shown to play roles in bacterial energy metabolism (31) and stress response (32). Those studies indicated that Kac is an evolutionarily conserved PTM with a role in energy metabolism in prokaryotes. Nevertheless, dynamic changes of lysine acetylation in bacteria have not been studied. In addition, substrates of lysine succinylation and their regulatory enzymes are not known.In this paper, we report a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture (SILAC) to identify and quantify changes in bacterial lysine succinylation, as well as lysine acetylation, in response to glucose, a major energy source. Our screening detected 2,580 lysine-succinylated sites in 670 proteins and 2,803 Kac sites in 782 proteins in Escherichia coli. Our quantitative proteomics data show that glucose had a more profound effect on Ksucc than on Kac. In addition, we found that CobB, a known prokaryotic deacetylase, had dual enzymatic activities to catalyze the removal of two structurally different lysine acyl groups, acetyl and succinyl, from the modified lysine residues.     

Deep,Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.Lysine acetylation (Kac)¹ is a well conserved, reversible post-translational modification (PTM) involved in multiple cellular processes (1). Acetylation is regulated by two classes of enzymes: lysine acetyltransferases (KATs) and histone deacetylases (HDACs) (2–4). This modification was originally identified as a nuclear event on histone proteins and has been long appreciated for its role in epigenetic and DNA-dependent processes. With the help of a growing number of large-scale acetylation studies, it has become evident that lysine acetylation is ubiquitous, also occurring on cytoplasmic and mitochondrial proteins and has a role in signaling, metabolism, and immunity (1, 4–6). Therefore, the examination of lysine acetylation on nonhistone proteins has gained a prominent role in PTM analysis.To date, the identification of large numbers of acetylation sites has been challenging because of the substoichiometric nature of this modification (7, 8). Additionally, global acetylation is generally less abundant than phosphorylation and ubiquitylation (1). The introduction of antibodies specific for lysine acetylation has significantly improved the ability to enrich and identify thousands of sites (9–14). A landmark study by Choudhary et al. used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome (10). A more recent study by Lundby et al. investigated the function and distribution of acetylation sites in 16 different rat tissues, and identified, in aggregate, 15,474 acetylation sites from 4541 proteins (12).Although anti-acetyl lysine antibodies have been a breakthrough for globally mapping acetylation sites (9–12), it remains a challenge to identify large numbers of lysine acetylation sites from a single sample, as is now routinely possible for phosphorylation and ubiquitylation (13, 15–18). To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for (1) alternative anti-acetyl lysine antibodies with higher specificity, (2) optimized antibody usage parameters, and (3) robust proteomic workflows that permit low to moderate protein input. In this study, we describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples.     

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys⁷⁹ in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys⁷⁹ methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.Protein methylation is emerging as one of the most prominent posttranslational modifications in the eukaryotic cell (1). Often showing high evolutionary conservation, it is increasingly recognized for its role in modulating protein–protein interactions (2). Indeed, it has been documented in protein interaction codes (3), such as those of the histones and p53 (4, 5), where it shows interplay with modifications such as acetylation and phosphorylation. Despite this, there remains a paucity of understanding of the enzymes that catalyze protein methylation. Many of the known methyltransferases target histones. However, many other methyltransferases have been discovered recently that act on nonhistone proteins (6).While protein methylation predominantly occurs on lysine and arginine residues, it is also known to occur on glutamine, asparagine, glutamate, histidine, cysteine, and the N- and C termini of proteins. Although the presence of N-terminal methylation on numerous proteins has been known for decades (7), the first enzymes responsible for this methylation have only recently been discovered (8, 9). The Saccharomyces cerevisiae protein Tae1 and its human ortholog N-terminal methyltransferase 1 (NTMT1) catalyze N-terminal methylation of proteins with an N-terminal [A/P/S]-P-K motif (after methionine removal). Yet there is evidence that these enzymes may recognize a more general N-terminal motif (10). Human NTMT2 is a monomethyltransferase that methylates the same substrates as NTMT1 and may prime substrate proteins with monomethylation to assist subsequent trimethylation by NTMT1 (11).The biological function of N-terminal methylation on some proteins has been recently revealed. For example, N-terminal methylation of regulator of chromatin condensation protein 1 (RCC1) is known to affect its binding to chromatin and thereby the correct chromosomal segregation during mitosis (12, 13), and N-terminal methylation of DNA damage-binding protein 2 (DDB2) is important for its role in UV-damaged DNA repair (14). Interestingly, there is evidence of interplay between N-terminal methylation and other posttranslational modifications (15), suggesting that, like lysine and arginine methylation, it may be incorporated into protein interaction codes (3). N-terminal methylation therefore appears to be a modification of functional importance in the cell.Eukaryotic elongation factor 1A (eEF1A), and its bacterial ortholog EF-Tu, is an essential translation elongation factor that is found in all living organisms. Its canonical function is in facilitating delivery of aminoacyl-tRNAs to the ribosome; however, it is also known to have a role in many other cellular functions, such as actin bundling, nuclear export, and proteasomal degradation (16). A number of methyltransferases have been discovered in both S. cerevisiae and human that target translation elongation factors. In yeast, four of these elongation factor methyltransferases (EFMs) act on eEF1A, namely Efm1, Efm4, Efm5, and Efm6, generating monomethylated Lys³⁰, dimethylated Lys³¹⁶, trimethylated Lys⁷⁹, and monomethylated Lys³⁹⁰, respectively (17–19). Human METTL10 is the ortholog of Efm4 in that it trimethylates eEF1A at Lys³¹⁸, which is equivalent to Lys³¹⁶ in yeast (20). Interestingly, eukaryotic elongation factor 2 (eEF2) is also methylated by a number of lysine methyltransferases. In yeast, Efm2 and Efm3 act on eEF2, generating dimethylated Lys⁶¹³ and trimethylated Lys⁵⁰⁹, respectively (21–24). Human eEF2-KMT is the ortholog of Efm3 in that it trimethylates eEF2 at Lys⁵²⁵, which is equivalent to Lys⁵⁰⁹ in yeast eEF2 (23).Here, we report the N-terminal methylation of eEF1A in S. cerevisiae and the identification of the methyltransferase that catalyzes this event. Using parallel reaction monitoring and MS/MS/MS (MS3), we unambiguously localize the modification to the N-terminal glycine and show it is conserved in the human cell. We also show that YLR285W, which we rename elongation factor methyltransferase 7 (Efm7), is responsible for this modification in yeast, as well as dimethylation at the adjacent lysine. We also characterize the methyltransferases responsible for methylation of lysine 79 in eEF1A. Human N6AMT2 is shown to be the ortholog of yeast Efm5 through its capacity to methylate yeast and human eEF1A at Lys⁷⁹ in vitro. We therefore rename N6AMT2 as eEF1A-KMT1.     

Acetylome Analysis Reveals Diverse Functions of Lysine Acetylation in Mycobacterium tuberculosis

The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD⁺-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.Mycobacterium tuberculosis was responsible for 1.3 million deaths and 8.6 million new cases of tuberculosis (TB)¹ worldwide in 2012 (1). This global public health crisis remains a serious problem, with the emergence of drug-resistant M. tuberculosis, especially multidrug-resistant and extensively drug-resistant M. tuberculosis, and also the emergence of coinfections of TB and human immunodeficiency virus (2, 3). To counter the increasing threat of TB, it is critical to understand fundamental aspects of TB-related biology. Such studies will not only provide new drug targets for the design of novel therapeutic agents, but also facilitate the development of novel diagnostic tools and new vaccines.Acetylation is one of the important protein modifications and occurs both co- and post-translationally on the α-amino group at the N terminus of the protein, so-called “N-terminal acetylation,” or on the ε-amino group on the side chain of lysine (4). Lysine acetylation is one of the most common post-translational modifications to proteins in both eukaryotes and prokaryotes. As a dynamic and reversible process, protein acetylation plays important roles in many cellular physiological processes, including cell-cycle regulation and apoptosis, cell morphology (5), metabolic pathways (6–8), protein interactions (9), and enzymatic activity (8, 10). In recent years, great advances have been made in proteomic studies, and a large number of lysine-acetylated proteins have been identified in many eukaryotes, including human (5, 11, 12), rat (13), mouse (11), Drosophila (14), Arabidopsis (15, 16), Saccharomyces cerevisiae (17), and protozoans (18, 19). The global analysis of lysine acetylation has also been reported in bacteria, including Escherichia coli (20–22), Erwinia amylovora (23), Bacillus subtilis (24), and Salmonella enterica (6). These acetylome studies have generated large datasets of bacterial proteins acetylated on lysine residues and have demonstrated the diverse cellular functions of lysine acetylation in bacteria.Increasing evidence shows that protein acetylation occurs and plays an important regulatory role in mycobacteria (8, 25–31). For example, Lange et al. reported the N-terminal acetylation of early secreted antigenic target 6 (ESAT-6) protein (31). Rv1151c is reported to be an NAD⁺-dependent protein deacetylase in M. tuberculosis that deacetylates and thus regulates the activity of acetyl-CoA synthase (25, 32). Two cyclic adenosine monophosphate (cAMP)-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) show similarity to the GNAT family of acetyltransferases and could acetylate a universal stress protein (USP, MSMEG_4207) (30). Subsequent structural studies revealed the fine mechanisms of how cAMP regulates the protein lysine acetyltransferase in mycobacteria (27, 28). Very recently, reversible lysine acetylation was shown to regulate the activity of several fatty acyl-CoA synthetases in M. tuberculosis (8, 26), and also to regulate acetate and propionate metabolism in M. smegmatis (8, 26). However, to the best of our knowledge, only a few acetylated proteins in M. tuberculosis have been identified, presenting a major obstacle to further understanding the regulatory roles of reversible lysine acetylation in this life-threatening pathogen.To fill this gap in our knowledge, we undertook a systematic study of the functional roles of lysine acetylation in M. tuberculosis. We performed an acetylomic analysis of M. tuberculosis H37Ra using high-accuracy MS combined with the identification of 226 unique lysine acetylation sites on 137 proteins. This set of M. tuberculosis proteins acetylated on lysine residues supports the emerging view that lysine acetylation is a general and fundamental regulatory process, and is not restricted to eukaryotes. It also opens the way for its detailed functional and evolutionary analysis of lysine acetylation in M. tuberculosis. The identified acetylated proteins that are involved in several important biological processes were functionally categorized into an interaction map. This is the first time that an interaction network of acetylated proteins in M. tuberculosis has been constructed, and should allow us to better understand the significance of acetylation in key cellular mechanisms in M. tuberculosis. To further explore the effects of lysine acetylation on the physiology of M. tuberculosis H37Ra, MRA_1161, the gene encoding the only known protein deacetylase in this bacterium, was deleted. The roles of MRA_1161 in the colony morphology, carbon source utilization, heat stress tolerance, and biofilm formation of M. tuberculosis were analyzed. The effect of lysine acetylation on the immunogenicity of a known immunogen, HspX, was also tested.     

Lysine Malonylation Is Elevated in Type 2 Diabetic Mouse Models and Enriched in Metabolic Associated Proteins

Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future.Post-translational modifications (PTMs)¹ have been recognized as a common feature of proteins (1–3). More than 300 types of PTMs have been identified according to the Swiss-Prot database (4, 5). Most of them use small molecular compounds as group donors. For example, adenosine-triphosphate (ATP) is used in phosphorylation, S-adenosylmethionine (SAM) in methylation, and acetyl-CoA in acetylation. Lysine acylations including malonylation (6), succinylation (7), butyrylation (8), propionylation (9), and crotonylation (10) represent a group of PTMs that use intermediates of energy metabolism like malonyl-CoA, succinyl-CoA, butyryl-CoA, propionyl-CoA, and crotonyl-CoA as group donors. Among the lysine acylations, lysine malonylation was first identified in Escherichia coli (E. coli) and HeLa cells using a specific anti-Kmal (anti-malonyllysine) antibody (6). It was found in three proteins in E. coli and 17 proteins in HeLa cells. Using a novel chemical fluorescent probe, another group identified more than 300 malonylated protein candidates in HeLa cells (11). Despite the rapid progress in detection technologies and tools, functional studies of lysine malonylation and its role in human diseases have been lagging behind.Type 2 diabetes is characterized by hyperglycemia and production of glycated proteins. For example, glycated hemoglobin A1c (HbA1c) has been clinically used as diagnostic criteria for diabetes. In addition to glycation, the role of other types of PTMs in type 2 diabetes remains to be revealed. In fact, elevated malonyl-CoA levels have been found in type 2 diabetic patients (12), and prediabetic rats (13). And hepatic overexpression of malonyl-CoA decarboxylase (MCD) decreased malonyl-CoA and reversed insulin resistance (14). Given the use of malonyl-CoA as malonyl donor in lysine malonylation, lysine malonylation is therefore anticipated to be of functional significance in the pathogenesis of type 2 diabetes.In the present study, we observed elevated lysine malonylation in liver tissues of db/db mice after unbiased screening seven types of lysine acylations. We then detected elevated levels of lysine malonylation in liver tissues of more db/db and ob/ob mice. Using an immunoaffinity based proteomic method, we identified a total of 573 malonylated lysine sites from 268 proteins in liver tissues of wt and db/db mice. Elevation of lysine malonylation in five proteins was confirmed by immunoprecipitation coupled with Western blot analysis. Functional analysis of the malonylated proteins showed an apparent enrichment in metabolic pathways, especially those involved in the glucose and fatty acid metabolism. Our study indicates the putative association between protein lysine malonylation and type 2 diabetes.