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Proteome-wide Lysine Glutarylation Profiling of the Mycobacterium tuberculosis H37Rv Longxiang Xie, Guirong Wang, Zhaoxiao Yu, Mingliang Zhou, Qiming Li, Hairong Huang, and Jianping Xie J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00917 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 23, 2016

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Proteome-wide Lysine Glutarylation Profiling of the Mycobacterium tuberculosis H37Rv Longxiang Xie

a⊥

, Guirong Wang

b⊥

, Zhaoxiao Yu a, Mingliang Zhou a, Qiming Li a,

Hairong Huang b *,Jianping Xie a *

a. Institute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Beibei, Chongqing, China. b. National Clinical Laboratory on Tuberculosis, Beijing Key laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China 101149;

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ABSTRACT Lysine glutarylation, a new protein posttranslational modification (PTM), was recently identified and characterized in both prokaryotic and eukaryotic cells. To explore the distribution of lysine glutarylation in Mycobacterium tuberculsosis, by using a comprehensive method combining the immune affinity peptide enrichment by the glutaryl-lysine antibody with LC-MS, we finally identified 41 glutarylation sites in 24 glutarylated proteins from M. tuberculosis. These glutarylated proteins are involved in various cellular functions such as translation and metabolism, and exhibit diverse sub-cellular localizations. Three common glutarylated proteins including 50S ribosomal protein L7/L12, elongation factor Tu and dihydrolipoamide succinyltransferase are shared between Escherichia coli and M. tuberculsosis. Moreover, comparison with other PTMs characterized in M. tuberculosis, 15 glutarylated proteins are found to be both acetylated and succinylated. Notably, several stress response associated proteins including HspX are glutarylated. Our data provide the first analysis of M. tuberculosis lysine glutarylated proteins. Further studies on the role of the glutarylated proteins will unveil the molecular mechanisms of glutarylation underlying M. tuberculosis physiology and pathogenesis. Keywords Mycobacterium tuberculosis; protein post-translational modification; lysine glutarylation; metabolism; stress response Running Head: Global Profiling of Protein Lysine Glutarylation in M. tuberculosis

INTRODUCTION Protein lysine acetylation or succinylation is a highly dynamic and conserved post-translational modification (PTM), which is the transfer of acetyl-CoA (Ac-CoA) or succinyl-CoA to a lysine (K) residue of a protein molecule. Increasing acetylome

1-7

and succinylome

8-11

studies in bacteria have

indicated that these modifications occur on large number of proteins and have diverse functions in bacteria.

12, 13

Beyond protein lysine acetylation and succinylation, the scope of protein PTMs is

rapidly expanding. By using mass spectrometry as a discovery tool, in combination with chemical biology and biochemistry as validation tools, Tan et al have identified and validated a new conserved 2

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five-carbon PTM, lysine glutarylation, which is present in eukaryote and prokaryote.14 They identified 23 glutarylation sites in 13 glutarylated proteins from E. coli, 10 glutarylation sites in 10 glutarylated proteins from HeLa cells. Furthermore, they identified 683 lysine glutarylation sites on 191 proteins in sirtuin 5 (SIRT5) KO mouse liver, and found that lysine glutarylation is highly enriched on proteins related with metabolic process. They also validated carbamoyl phosphate synthase 1 (CPS1) involved in urea cycle as a glutarylated protein, and demonstrated that lysine glutarylation can inhibit its enzymatic activity.

14

So far, E. coli is the sole bacterium with protein lysine glutarylation

characterized. M. tuberculosis (Mtb), one of the most successful human pathogens, can persist within hosts for a long time. Approximately one-third of human population world-wide is latently infected with Mtb, and recent reports showed that about 1.7 million people died from this terrible disease in 2013 (WHO, 2014). Global lysine acetylation7, 15 and succinylation10, 16 in M. tuberculosis have been characterized, however, the extent of lysine glutarylation remain unknown. Herein, we performed a global glutarylome analysis on M. tuberculosis, and identified 41 glutarylation sites on 24 glutarylated proteins. These identified glutarylated proteins are involved in various biological processes, and are highly enriched in metabolic process and protein folding. Only three glutarylated proteins are shared by E. coli and M. tuberculosis. To the best of our knowledge, this work provides the first M. tuberculosis dataset on lysine glutarylation.

MATERIALS AND METHODS Protein Extraction, Trypsin Digestion and Affinity Enrichment The mid-log phase M. tuberculosis H37Rv cells were harvested and then sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer including 8 M urea, 1% Triton-100, 65 mM dithiothreitol (DTT) and 50 mM nicotinamide (NAM), 0.1% Protease Inhibitor Cocktail (Merck Millipore), 3µM trichostatin A (TSA). The remaining debris was discarded by centrifugation at 20,000g at 4 °C for 10 min and the supernatant was precipitated at -20 °C with cold 15% trichloroacetic acid (TCA) for 2 h. The sample was centrifugated at 20,000g at 4 °C for 10 min. The 3

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supernatant was removed and the pellet was washed with cold acetone for three times. Finally, the contents of the pellet was re-dissolved in buffer (8 M urea, 100 mM NH4CO3, pH 8.0) and the protein concentration was determined with 2-D Quant kit (GE Healthcare) according to the manufacturer’s instructions. The detailed following procedure was almost similar with our previous article except for several small places such as antibody and parameter. 10 For digestion, the protein solution was reduced with 10 mM DTT at 37 °C for 1 h and alkylated with 20 mM iodoacetamide (IAA) at room temperature for 45 min in dark. For trypsin digestion, the protein sample was diluted by adding 100 mM NH4CO3 to urea concentration less than 2M. Finally, two digestions were performed, adding trypsin (Promega, WI, USA) at 1:50 trypsin-to-protein mass ratio as the first digestion overnight and at 1:100 trypsin-to-protein mass ratio at 37 °C for 4 h as the second digestion. To enrich lysine glutarylation (Kg) peptides, tryptic peptides dissolved in NETN buffer which includes 1 mM ethylene diamine tetraacetic acid (EDTA), 100 mM sodium chloride (NaCl), 0.5% NP-40, 50 mM Tris-HCl, pH 8.0) were incubated with pre-washed antibody beads (Pan antiglutaryl-lysine antibody beaded agarose, Cat#PTM-904, from PTM Biolabs) at 4°C overnight with gentle shaking. Then the beads were washed with NETN buffer four times and with ddH2O twice, and the bound glutarylated peptides were eluted from the beads by using 0.1% trifluoroacetic acid (TFA). The eluted fractions were combined and vacuum-dried, and the obtained peptides were cleaned with C18 ZipTips (Millipore) according to the manufacturer’s instructions, followed by LC-MS/MS analysis LC-MS/MS Analysis Peptides were dissolved in 0.1% formic acid, directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, 75µm×2cm, 3µm, 100Å, Thermo Scientific), and then these separated peptide was performed using a C18 reversed-phase analytical column (Thermo, betasil c18, 10×250). The gradient was comprised of an increase from 7% to 20% solvent B (0.1% formic acid in 98% acetonitrile) for 20 min, 20% to 35% for 7 min and climbing to 80% in 5 min then holding at 80% for the last 3 min, all at a constant flow rate of 300 nl/min on an EASY-nLC1000 UPLC system, and the resulting peptides were analyzed by Q ExactiveTM hybrid quadrupole-Orbitrap mass spectrometer 4

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(ThermoFisher Scientific). The obtained peptides were first subjected to nanospray ion source, and then subjected to tandem mass spectrometry (MS/MS ) in Q ExactiveTM plus (Thermo) coupled to the online Ultra Performance Liquid Chromatography (UPLC). Intact peptides at a resolution of 70,000 were detected in the Orbitrap Peptides were selected for MS/MS using NCE (normalization collision energy) setting as 30 and ion fragments at a resolution of 17,500 were detected in the Orbitrap. A data-dependent process alternating between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions. MS data were acquired using following parameters: MS threshold ion count of 2×104; 10.0s dynamic exclusion; electrospray voltage of 2.0 kV; MS/MS threshold ion of 5×104. Database Search and Bioinformatics Analysis The resulting MS/MS data was conducted using MaxQuant with integrated Andromeda search engine (v.1.4.1.2) (Table S1) and then were searched against Uniprot M. tuberculosis H37Rv (6168 sequences) database. Trypsin/P was served as cleavage enzyme allowing up to 5 modifications per peptide, 5 charges and 4 missing cleavages. The parameters for mass error were 10 ppm for precursor ions and 0.02 Da for fragment ions. Fixed modification was carbamidomethylation on Cys and variable modifications were glutarylation on lysine, glutarylation on protein N-terminal, and oxidation on Met. False discovery rate (FDR) thresholds for modification site, peptide and protein were designated at 1% and minimum peptide length was set at 7. The other parameters of MaxQuant were not changed (using default values) and the site localization probability was set as > 0.75. Gene Ontology annotation and functional enrichment analysis of glutarylated proteins were performed. The detailed procedure of bioinformatics analysis was described as previous article. 10

RESULTS AND DISCUSSION Functional Classification of M. tuberculosis glutarylated proteins In order to investigate the relative abundance of lysine glutarylation on M. tuberculosis, we used immunoaffinity enrichment and a MS-based high-throughput proteomic approach combined with nano-LC to identify glutarylated proteins and their glutarylation sites. In total, we identified 41 5

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glutarylated sites in 24 glutarylated proteins in M. tuberculosis. Detailed information for all of identified glutarylation peptides and the matched proteins is shown in Table S1, and the representative MS/MS spectra of six lysine glutarylation peptides of Rv1347c, Rv3648c, Rv1259, dnak, rpsC and Rv0576, respectively, are presented in Figure S1. Notably, twenty of these glutarylated proteins just have one glutarylation site, while other proteins contain more than one glutarylation sites, for example, there are four glutarylation sites in Rv3418c. The most extensively glutarylated protein is the Rv0440 protein, which is glutarylated at 12 independent lysine residues. The distribution of the molecular size of the glutarylated proteins is wide. The molecular weight of 33% glutarylated proteins was less than 28kDa; 42% were between 28kDa to 50kDa; 25% were larger than 50kDa (Figure 1A and Table S2). To better characterize the glutarylated proteins of M. tuberculosis, all identified glutarylated proteins were

functionally

classified

according

to

the

TubercuList

website

(http://tuberculist.epfl.ch/index.html). The largest class of glutarylated proteins was associated with virulence, detoxification, adaptation, accounting for 33% of all annotated glutarylated proteins (Figure 1B and Table S2). The second class of glutarylated proteins was involved in information pathways, accounting for 29% of the all annotated glutarylated proteins (Figure 1B and Table S2). Another large glutarylated proteins class was involved in intermediary metabolism and respiration, accounting for 21% of all identified proteins (Figure 1B and Table S2). Overall, these findings implicate potential vital roles of lysine glutarylation in most fundamental cellular processes of M. tuberculosis. We further conducted GO enrichment analyses (biological process and cellular component categories), KEGG pathway, and protein domain (Figure 2 and Table S3). Our GO enrichment analysis showed that the glutarylated proteins were markedly enriched in protein folding and cellular metabolic process. This data set implied that glutarylation may play an important role in metabolic process. Furthermore, the identified glutarylated proteins were also mapped to KEGG metabolic pathways and protein domains (Figure 2 and Table S3). We found that glutarylation occurs on many proteins involved in RNA degradation, ribosome and carbon metabolism and most glutarylated proteins were enriched with GroEL-like apical domain (Figure 2 and Table S3). To examine the relative abundances of the amino acids flanking the glutarylation sites, we used Motif-X soft to 6

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perform a motif analysis of the identified Kg sites. Unfortunately, there is no specific motif found in the data. Defining the Glutarylated Catalytic Binding and Active sites To determine whether these glutarylation sites located in functional domain or active sites, we used the ScanProsite (http://prosite.expasy.org) website. Finally, we identified the following motifs and profiles among these glutarylated peptides (Table 1), such as Rv1872c (FMN-dependent alpha-hydroxy acid dehydrogenase domain profile), Rv1932 (Thioredoxin domain profile), Rv3418c (Translational (tr)-type guanine nucleotide-binding (G) domain profile), Rv3418c (Chaperonins cpn10 signature). Given that these glutarylation sites located in the important functional domain structure, lysine glutarylation may affect the function of these proteins. Some Glutarylated Proteins are Also Acetylated and Succinylated in M. tuberculosis Recently, proteome-wide lysine acetylation (658 acetylated proteins, 1128 acetylation sites) succinylation (626 succinylated proteins, 1545 succinylation sites)

16

15

and

have been identified in M.

tuberculosis H37Rv. To determine whether the three PTMs (glutarylation, acetylation and succinylation) occur at the same lysine site, we compared the glutarylation sites identified in this study with previously determined acetylation and succinylation sites in M. tuberculosis. The comparison results showed that 26 lysine glutarylated sites (63.41% of total glutaryl-sites) matched at 14 proteins (58.33% of total glutaryl-proteins), which were acetylated and succinylated at the same position. Among the 14 proteins with overlapping modifications, 3 proteins including only one modification site had exactly the same glutarylation, succinylation and acetylation lysine sites (Table S4). Furthermore, there are two “only acetylated and glutarylated” proteins, and two “only succinylatied and glutarylated” proteins (Table S4). Notably, 60 kDa chaperonin encoded by Rv0440 gene has 11 lysine residues being characterized with glutarylation, succinylation and acetylation (Table S4). The overlap of three important PTMs suggests a crucial role of Rv0440, and also raises question about how these different PTMs are coordinated. Further studies on the coordination of these PTMs in a spatiotemporal effective way are worthwhile. Conserved lysine Glutarylated Proteins Between E. coli and M. tuberculosis 7

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In 2014, Tan et al identified 23 glutarylation sites on 13 glutarylated proteins in E. coli. 14 The size of glutarylproteome found in this study is very similar to that in E. coli, but far less than that in mouse liver (191 glutarylated proteins, 911 glutarylation sites), which may be due to the presence of lysine deglutarylase in bacteria while absent in mouse liver in proteome research.14 Among the identified M. tuberculosis glutarylated proteins, only three proteins including 50S ribosomal protein L7/L12, elongation factor Tu and dihydrolipoamide succinyltransferase have been reported to be glutarylated in E. coli (Figure 3), while the other glutarylated proteins are firstly reported, which expands our understanding of bacterial glutarylation. 50S ribosomal protein L7/L12 encoded by Rv0652 gene in M. tuberculosis, can stimulate powerful serum antibody responses in active TB patients. Recent studies have shown that through the Toll-like receptor 4 (TLR4), Rv0652 protein can induce the generation of monocytes chemoattractant protein-1 (MCP-1) and tumour necrosis factor (TNF) in macrophages. 17 Moreover, Rv0652 obviously increased the expression level of the MHC class II antigen molecules, mannose receptor, CD86, and CD80. 17 Metabolism Process Studies have shown that lysine glutarylation is enriched on metabolic enzymes in mouse liver.

14

In

our data, there are eight glutarylated proteins related with metabolic process including Rv1872c, Rv3389c, Rv2211c, Rv3248c, Rv2215, Rv1347c, Rv0468 and Rv1483. dlaT (Rv2215), E2 component of pyruvate dehydrogenase (PDH) that catalyzes the pyruvate oxidation by nicotinamide adenine dinucleotide (NAD+) to CO2 and acetyl-coenzyme A (acetyl-CoA), was found to be glutarylated at K361. dlaT was demonstrated to be necessary for bacterial optimal growth and for withstanding reactive nitrogen intermediates (RNI).

18

When cultivated in 7H9 medium containing glycerol and

glucose as the source of carbon, H37Rv ∆dlaT strain grown impaired. In addition, H37Rv ∆dlaT mutant strain was easily killed by macrophages and its virulence was reduced in the mouse.18The trans-enoyl hydratases/(R)-specific 3-hydroxyacyl dehydratases are critical proteins in the process of fatty acids biosynthesis. Rv3389c (one member of hydratases 2 family), catalyzing the hydration of enoyl-CoA substrates (C8–C16) in vitro 19, can be glutarylated at K113. Mycobactin siderophores are important for the iron acquisition and transport in M. tuberculosis, 8

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especially during infection or intracellular survival. 20 In the absence of mycobactin siderophores, M. tuberculosis strain was growth defect in iron-depleted medium that is similar to those encountered in the human lung macrophages during infection.20 Rv1347c, one lysine acyltransferase responsible for the acylation of mycobactin,

20

was glutarylated at K3 residue. Rv1347c is involved in the

biosynthesis of mycobactins which is directly associated with M. tuberculosis virulence, and represents an effective target for the discovery of novel antimycobacterial compounds. 21 Stress Response Proteins Previous study identified the heat shock proteins as well as reactive oxygen regulators such as superoxide dismutase as the substrates of lysine glutarylation in eukaryote.13-14Like eukaryotic cell, several stress response proteins in M. tuberculosis are also substrates of lysine glutarylation. These proteins include chaperones and proteins involved in regulation of free radical reduction (such as superoxide dismutase and thiol peroxidase). For example, Rv2031c known as α-crystallin (HspX) can be glutarylated on K114 residue. During latency, HspX is predominantly expressed by the bacterium. In M. tuberculosis, the loss of this protein can weaken the Mtb tolerance to anoxybiosis. 22 In addition, HspX can inhibit the differentiation and maturation of dendritic cells (DCs) which play a critical role in immune evasion. 23 Studies have shown that lysine acetylation can influence the immunogenicity of HspX in M. tuberculosis, which stimulate us to explore whether lysine glutarylation plays similar function. 7 Transcription and Translation Factors Transcription factors and histones are founding members of lysine glutarylation substrates in mammalian cells.

14

Reminiscent of this, one ArsR family transcription factor Rv0576 is lysine

glutarylation substrate. In addition to transcriptional regulators, 3 proteins involved in translational regulation are subjects of lysine glutarylation. These proteins include Elongation factor Tu, 30S ribosomal protein S3 and 50S ribosomal protein L7/L12.

CONCLUSION This study provides the first assessment of the global glutarylome of M. tuberculosis. In total, 42 9

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lysine glutarylation sites occurring on 24 glutarylated proteins were identified. These glutarylated proteins are distributed in different cellular compartments, and are enriched on protein folding and metabolic process. In these identified glutarylated proteins, only three glutarylated proteins are also found in E. coli, and most glutarylated proteins are firstly reported. Furthermore, more than ten glutarylated proteins are found to be acetylated and succinylated. Notably, several proteins related with stress reaction including HspX are found to be glutarylated. However, several limitations exist in our study. For example, due to limited conditions, the obtained profile of M. tuberculosis is not final set of lysine glutarylation and it is highly expected that catalog of glutarylated proteins in the M.tuberculosis will continue to expand greatly with the development of technology. Although performing immunoaffinity in the sample preparation, we do not use western blot method to verify all of the identified proteins. In addition, we just use only a single growth medium and a single time point and it will be very interesting and significant to compare the glutarylation pattern of pathogen grown in different media or different stress conditions. But anyway, our new data provide a novel framework to understand the biology of M. tuberculosis glutarylation.

ASSOCIATED CONTENT Supporting Information Figure S1-Representative spectra of six glutarylated peptides from six proteins; Table S1-All identified glutaryl-lysine peptides and proteins; Table S2-Classification of function and molecular weight; Table S3-GO enrichment analysis of biological process, cellular component, pathway enrichment, and protein domain; Table S4-Overlap modification sites among glutarylation, acetylation and succinylation in M. tuberculosis.

AUTHOR INFORMATION Corresponding Author *E-mail: Jianping Xie, [email protected], Tel.: +862368367108; fax: +86 2368367108. Hairong Huang, [email protected]. Tel.: +861089509159; fax: +861089509159. 10

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Author Contributions ⊥ Longxiang Xie and Guirong Wang are co-first author.

Notes The authors have declared no conflict of interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation [grant numbers 81301394, 81371851, 81511120001, 81071316, 81271882], New Century Excellent Talents in Universities [grant number

NCET-11-0703],

Collaborative

Innovation

Center

of

Infectious

diseases

(PXM2015_014226_000058, 3500-115215), Excellent Ph.D. thesis fellowship of Southwest University [grant numbers kb2010017, ky2011003], National Megaprojects for Key Infectious Diseases [grant numbers 2008ZX10003-006], the Fundamental Research Funds for the Central Universities [grant numbers XDJK2012D007, XDJK2013D003, XDJK2014D040, XDJK2016D025], Graduate research and innovation project of graduate in Chongqing (CYS14044, The Chongqing Municipal Committee of Education for postgraduates excellence program [grant numbers YJG123104], and the undergraduates teaching reform program [grant numbers 2013JY201].

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(2) Zhang, K.; Zheng, S.; Yang, J. S.; Chen, Y.; Cheng, Z. Comprehensive profiling of protein lysine acetylation in Escherichia coli. J Proteome Res 2013, 12, 844-51.

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(5) Liao, G.; Xie, L.; Li, X.; Cheng, Z.; Xie, J. Unexpected extensive lysine acetylation in the trump-card antibiotic producer Streptomyces roseosporus revealed by proteome-wide profiling. J Proteomics 2014, 106, 260-269.

(6) Pan, J.; Ye, Z.; Cheng, Z.; Peng, X.; Wen, L.; Zhao, F. Systematic analysis of the lysine acetylome in Vibrio parahemolyticus. J Proteome Res 2014, 13, 3294-3302.

(7) Liu, F.; Yang, M.; Wang, X.; Yang, S.; Gu, J.; Zhou, J.; Zhang, X.-E.; Deng, J.; Ge, F. Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis. Mol. Cell. Proteomics 2014, 13, 3352-3366.

(8) Weinert, B. T.; Schölz, C.; Wagner, S. A.; Iesmantavicius, V.; Su, D.; Daniel, J. A.; Choudhary, C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell Rep 2013, 4, 842-851.

(9) Pan, J.; Chen, R.; Li, C.; Li, W.; Ye, Z., Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus. J Proteome Res 2015, 14, 4309-4318. (10) Xie, L.; Liu, W.; Li, Q.; Chen, S.; Xu, M.; Huang, Q.; Zeng, J.; Zhou, M.; Xie, J. First Succinyl-Proteome Profiling of Extensively Drug-Resistant Mycobacterium tuberculosis Revealed Involvement of Succinylation in Cellular Physiology. J Proteome Res 2014, 14, 107-119. (11) Singhal, A.; Arora, G.; Virmani, R.; Kundu, P.; Khanna, T.; Sajid, A.; Misra, R.; Joshi, J.; Yadav, V.; Samanta, S. Systematic analysis of mycobacterial acylation reveals first example of acylation-mediated regulation of enzyme activity of a bacterial phosphatase. J Biol Chem 2015, 290, 26218-26234. (12) Pisithkul, T.; Patel, N. M.; Amador-Noguez, D. Post-translational modifications as key regulators of bacterial metabolic fluxes. Curr Opin Microbiol 2015, 24, 29-37. (13) Hirschey, M. D.; Zhao, Y., Metabolic regulation by lysine malonylation, succinylation and glutarylation. Mol. Cell. Proteomics 2015, mcp. R114. 046664. (14) Tan, M.; Peng, C.; Anderson, K. A.; Chhoy, P.; Xie, Z.; Dai, L.; Park, J.; Chen, Y.; Huang, H.; Zhang, Y. Lysine glutarylation is a protein posttranslational modification regulated by SIRT5. Cell Metab. 2014, 19, 605-617.

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(15) Xie, L.; Wang, X.; Zeng, J.; Zhou, M.; Duan, X.; Li, Q.; Zhang, Z.; Luo, H.; Pang, L.; Li, W. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis. Int. J. Biochem. Cell Biol. 2015, 59, 193-202. (16) Yang, M.; Wang, Y.; Chen, Y.; Cheng, Z.; Gu, J.; Deng, J.; Bi, L.; Chen, C.; Mo, R.; Wang, X., Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis. Mol. Cell. Proteomics 2015, 14, 796-811. (17) Kim, K.; Sohn, H.; Kim, J. S.; Choi, H. G.; Byun, E. H.; Lee, K. I.; Shin, S. J.; Song, C. H.; Park, J. K.; Kim, H. J. Mycobacterium tuberculosis Rv0652 stimulates production of tumour necrosis factor and monocytes chemoattractant protein‐1 in macrophages through the Toll‐like receptor 4 pathway. Immunology 2012, 136, 231-240. (18) Shi, S.; Ehrt, S. Dihydrolipoamide acyltransferase is critical for Mycobacterium tuberculosis pathogenesis. Infect. Immun. 2006, 74, 56-63. (19) Sacco, E.; Legendre, V.; Laval, F.; Zerbib, D.; Montrozier, H.; Eynard, N.; Guilhot, C.; Daffé, M.; Quémard,

A.

Rv3389C

from

Mycobacterium

tuberculosis,

a

member

of

the

(R)-specific

hydratase/dehydratase family. BBA-Proteins Proteom 2007, 1774, 303-311. (20) Card, G. L.; Peterson, N. A.; Smith, C. A.; Rupp, B.; Schick, B. M.; Baker, E. N. The crystal structure of Rv1347c, a putative antibiotic resistance protein from Mycobacterium tuberculosis, reveals a GCN5-related fold and suggests an alternative function in siderophore biosynthesis. J Biol Chem 2005, 280, 13978-13986. (21) Frankel, B. A.; Blanchard, J. S. Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine N ε-acyltransferase involved in mycobactin biosynthesis. Arch. Biochem. Biophys. 2008, 477, 259-266. (22) Siddiqui, K. F.; Amir, M.; Gurram, R. K.; Khan, N.; Arora, A.; Rajagopal, K.; Agrewala, J. N. Latency-associated protein Acr1 impairs dendritic cell maturation and functionality: a possible mechanism of immune evasion by Mycobacterium tuberculosis. J Infect Dis 2013, jit595. (23) Cunningham, A. F.; Spreadbury, C. L. Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton α-crystallin homolog. J. Bacteriol. 1998, 180, 801-808.

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Tables Table 1. PROSITE motifs and domain profiles search for lysine- glutarylated proteins. PROSITE Protein function Protein

Gene

position

PROSITE Pattern

Modified sequence

ID FMN-dependent

I6X2I8

Rv1872c

L-lactate

alpha-hydroxy acid

dehydrogenase

dehydrogenase

LldD2 Probable

I6XCV9

Rv1932

_LVVK(kg)GIQTLDDAR_

277

PS51349

thiol

peroxidase

domain profile Thioredoxin domain

_DLPFAQK(kg)R_

90

PS51352

profile

_VEIADIDK(kg)R_

726

PS50126

S1 domain profile

Polyribonucleotide nucleotidyltransferas I6Y1P7

Rv2783c

e

Chaperonins cpn10 I6Y3F9

Rv3418c

10 kDa chaperonin

_VNIK(kg)PLEDK_

7

PS00681

signature Translational

type

guanine nucleotide-binding I6Y4F5

Rv0685

Elongation factor Tu

_VSALK(kg)ALEGDAK_

178

PS51722

(G) domain profile Heat shock hsp20

I6Y869

Rv2031c

Heat shock protein

_TVSLPVGADEDDIK(kg)A

hspX

TYDK_

proteins 114

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PS01031

profile

family

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Figure legend Figure 1. Functional annotation and molecular weight distribution of identified M. tuberculosis lysine glutarylated proteins. Figure 2. The cellular component, biological process of the glutarylated proteins in M. tuberculosis by gene ontology analysis and the list of overrepresented KEGG pathways and protein domains Figure 3. (A) Comparison of glutarylated proteins between E. coli and M. tuberculosis. (B) Annotation of lysine glutarylation sites in Rv0440 protein. Kg, glutarylation; Ac, acetylation; Suc, succinylation; Symbol *, ATP/Mg binding sites.

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Figure 1. Functional annotation and molecular weight distribution of identified M. tuberculosis lysine glutaryl proteins. 109x47mm (300 x 300 DPI)

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Figure 2. The cellular component, biological process of the glutarylated proteins in M.tuberculosis by gene ontology analysis and list of the overrepresented KEGG pathways and domains 109x169mm (300 x 300 DPI)

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Figure 3. (A) Comparison of glutarylated proteins between E. coli and M. tuberculosis. (B) Annotation of lysine glutarylation sites in Rv0440 protein. Kg, glutarylation; Ac, acetylation; Suc, Succinylation; Symbol *, ATP/Mg binding sites. 242x321mm (300 x 300 DPI)

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For TOC only 150x113mm (300 x 300 DPI)

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