Characterization of Protein Lysine Propionylation in Escherichia coli

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Characterization of Protein Lysine Propionylation in Escherichia coli: Global Profiling, Dynamic Change and Enzymatic Regulation. Mingwei Sun, Jun-Yu Xu, Zhixiang Wu, Linhui Zhai, Chengxi Liu, Zhongyi Cheng, Guofeng Xu, Shengce Tao, Bang-Ce Ye, Yingming Zhao, and Minjia Tan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00798 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 3, 2016

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Journal of Proteome Research

Characterization of Protein Lysine Propionylation in Escherichia coli: Global Profiling, Dynamic Change and Enzymatic Regulation.

Mingwei Sun1, 2; Junyu Xu1, 5; Zhixiang Wu4; Linhui Zhai1, 2; Chengxi Liu6; Zhongyi Cheng7 ; Guofeng Xu4; Shengce Tao6; Bang-Ce Ye5; Yingming Zhao1, 3 and Minjia Tan1, 2, *

1

The Chemical Proteomics Center and State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China 2

University of Chinese Academy of Sciences, Beijing 100049, PR China

3

Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois

60637, United States 4

Pediatric Surgery Department, Xinhua Hospital, Shanghai Jiao Tong University

School of Medicine, 1665 Kongjiang Road, Shanghai 200092, PR China 5

Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor

Engineering, East China University of Science and Technology, Shanghai 200237, PR China 6

Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine

(Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, PR China 7

Jingjie PTM BioLab (Hangzhou) Co. Ltd, Hangzhou 310018, PR China

*

Corresponding Author

*Tel.: 86-21-50800172. Fax: 86-21-50806600. E-mail: [email protected]. 1 / 43

ACS Paragon Plus Environment


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ABSTRACT Propionylation at protein lysine residue is characterized to be present in both eukaryotic and prokaryotic species. However, the majority of lysine propionylation substrates still remain largely unknown. Using affinity enrichment and mass spectrometric based proteomics, we identified 1467 lysine propionylation sites in 603 proteins in E. coli. Quantitative propionylome analysis further revealed that global lysine propionylation level was drastically increased in response to propionate treatment, a carbon source for many microorganisms and also a common food preservative. The results indicated that propionylation may play a regulatory role in propionate metabolism and propionyl-CoA degradation. In contrast to lysine acetylation and succinylation, our results revealed that lysine propionylation level of substrates showed an obvious decrease in response to high glucose, suggesting a distinct role of propionylation in bacteria carbohydrate metabolism. This study further showed that bacterial lysine deacetylase CobB and acetyltransferase PatZ could also have regulatory activities for lysine propionylation in E. coli. Our quantitative propionylation substrate analysis between cobB wild-type and cobB knockout strain led to the identification 13 CobB potentially regulated propionylation sites. Together, these findings revealed the broad propionylation substrates in E. coli and suggested new roles of lysine propionylation in bacterial physiology.

Key words: Post-translational modification(s), Lysine propionylation, SILAC, CobB, PatZ 2 / 43


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Abbreviations: PTM(s): Post-translational modification(s); Kac: Lysine acetylation; Kpr: Lysine propionylation; Kbu: Lysine butyrylation; Kmal: Lysine malonylation; Ksucc: Lysine succinylation; Kglu: Lysine glutarylation; Kcr: Lysine crotonylation; Khib: Lysine 2-hydroxyisobutyrylation; LB: Luria–Bertani; TCA: Trihaloacetic acid; HPLC: High Performance Liquid Chromatography; bHPLC: high pH HPLC; IP: Immunoprecipitation; TFA: Trifluoroacetic acid; HCD: Higher-energy collisional dissociation; NCE: Normalized collision energy; PSM: Peptide spectrum match; FDR: False discovery rate; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene annotation; D-PBS: Dulbecco’s Phosphate Buffered Saline.

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INTRODUCTION Recent advancement in mass spectrometry (MS), together with chemistry and biochemistry technologies, has led to the efficient discovery of new types of protein post-translational modifications (PTMs) in the past decade

1, 2

. Many of these new

PTMs have been demonstrated to play vital roles in cellular physiology and pathology in both eukaryotes and prokaryotes. Among them, due to high chemical reactivity of ε-amino group in the lysine side chain, new types of protein lysine ε-N-acylation have continuously been identified, including propionylation (Kpr), butyrylation (Kbu), malonylation (Kmal), succinylation (Ksucc), glutarylation (Kglu), crotonylation (Kcr), and 2-hydroxyisobutyrylation (Khib)

3-6

. Similar to acetyl-CoA as the cofactor for

lysine acetylation, cellular acyl-CoAs (propionyl-CoA, malonyl-CoA, succinyl-CoA, etc.) were utilized as the cofactors for these acyl modifications. Most of these modifications have been demonstrated to be conserved from bacteria to mammals, and were shown to play vital roles in cellular functions, such as energy metabolism and epigenetic regulation. Hundreds to thousands of PTM sites for several of these lysine acylation modifications (such as succinylation, malonylation and glutarylation) were discovered by affinity enrichment based MS analysis

6-8

, which revealed the

broad substrate diversity of these PTMs in different cellular pathways. Of these new types of lysine acylation, little is known on the global substrates of lysine propionylation. Protein lysine propionylation was first identified in histones 3. Further study showed non-histone substrates in mammals could also be propionylated 9. Eukaryotic lysine acetyltransferase p300/CBP and deacylase Sirt1 4 / 43


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showed in vivo propionyltransferase and depropionylase activities 9. In prokaryotes, propionyl-CoA synthetase of Salmonella enterica was first identified as a propionylation substrate, and this study also showed that Gcn-5-related N-acetyltransferase PatZ and sirtuin like deacetylase CobB could regulate lysine propionylation of propionyl-CoA synthetase

10

. A recent proteomic study identified

361 propionylation sites in 183 proteins in an extremely thermophilic bacteria Thermus thermophiles

11

. Despite these efforts, the global lysine propionylation

substrates and their dynamic roles in bacterial physiology still remains poorly understood. In bacteria, the cofactor of lysine propionylation propionyl-CoA is an intermediary metabolite mainly produced from two metabolic pathways (Fig. 1A) 12-14: The beta-oxidation of odd-chain fatty acids and the degradation of isoleucine, threonine and methionine. In addition, propionyl-CoA can also be directly generated by acetyl-CoA synthetase and propionyl-CoA synthetase from propionate, a sole source of carbon utilized by many microorganisms due to its high abundance in nature

12, 14

bacteria:

. For propionyl-CoA degradation, there are also two major pathways in

methylmalonyl-CoA

pathway

and

the

methylcitrate

cycle.

In

methylmalonyl-CoA pathway, propionyl-CoA can be reversibly converted to succinyl-CoA with a requisite of synthesis or the efficient uptake of coenzyme B12 in cells. In methylcitrate cycle, propionyl-CoA is first condensed with oxaloacetate to form 2-methylcitrate by 2-methylcitrate synthase (PrpC), which is further converted into pyruvate and succinate by multiple enzyme-catalyzed chemical reactions 5 / 43


12, 14

,


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similar to those of the citric acid cycle. Via these two pathways, the cellular concentration of propionyl-CoA can be well balanced in low level, as accumulation of propionyl-CoA in cells will cause toxic effects in both microorganisms and mammals. In fact, propionate, the direct precursor of propionyl-CoA, is a powerful antimicrobial inhibitor widely used in food industry. Although antimicrobial activity of propionyl-CoA was reported to be related with the inhibition of pyruvate dehydrogenase

15

and citrate synthase

16, 17

, the molecular mechanisms by which

propionyl-CoA exerts it toxic activity is still poorly understood. Therefore, characterization of the role of propionyl-CoA and protein propionylation in bacterial cellular pathways is likely to provide new insights on the understanding of its toxic mechanism. In this study, we reported the proteome wide analysis of lysine propionylation substrates in the prokaryotic model organism E. coli. Using affinity enrichment and mass spectrometric based proteomics, we identified 1,467 lysine propionylation sites in 603 proteins from E. coli in this study. Our quantitative propionylome analyses revealed that global lysine propionylation level was dynamically regulated in response

to

different

nutritional

environments.

We further

showed

the

bacterial lysine deacetylase CobB and acetyltransferase PatZ could also have general regulatory activities for lysine propionylation in E. coli.

MATERIALS AND METHODS Materials E. coli strain BW25113 and AT713 were obtained from the Coli Genetic Stock Center 6 / 43


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at Yale University (New Haven, CT). M9 minimal salt, isotopically labeled lysine (L-lysine-13C6 hydrochloride), isotopically labeled ariginine (L-ariginine-13C6,

15

N4

hydrochloride), formaldehyde solution, formaldehyde-D2 solution and other reagents were purchased at analytical grade or higher purity from Sigma-Aldrich, Inc. (St. Louis, MO). Sequencing grade trypsin was purchased from Promega (Billerica, WI). The lysine propionylated peptide EIAQDFKprTDLR was commercially synthesized from GL Biochem (Shanghai, China) with 99% purity. C18 Zip-tips were purchased from Millipore Corporation (Billerica, MA). Pan-anti-propionyllysine antibody were purchased from Jingjie PTM BioLab Co. Ltd (Hangzhou, China).

E. coli cell culture Prototrophic E. coli BW25113 cells were cultured in Luria–Bertani (LB) medium for about 12 h until OD600 reached 3.4 for lysine propionylome profiling analysis. For the dynamic analysis of propionylation in response to high sodium propionate and glucose conditions, prototrophic E. coli BW25113 and E. coli AT713 cells were cultured in M9 medium (contained 20 amino acids at 100 mg/l) or M9 medium supplemented with 0.8% propionate, 0.8% glucose, respectively

7, 14, 18

. For dynamic

analysis of propionylation in response to amino acid conditions, E. coli AT713 cells were cultured in M9 medium with or without Ile, Met or Thr

14, 18

. E. coli cells were

harvested, lysed and western blotted with pan-anti-propionyllysine antibody or pan-anti-acetyllysine antibody (Jingjie PTM BioLab Co. Ltd, Hangzhou, China). The lysA knockout (∆lysA) BW25113 E. coli cells were cultured in M9 medium containing light lysine (12C6-lysine) and 0.8% propionate, and the lysA and cobB 7 / 43



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knockout (∆lysA & ∆cobB) BW2511 cells were cultured into M9 medium containing heavy lysine (13C6-lysine) and 0.8% propionate. The cells were cultured at 37°C for about 24 h and harvested at a comparable OD600 value of 1.2. The isotopic amino acid labeling efficiency was more than 98% as validated by mass spectrometry analysis. Three biological replicates were performed to identify the CobB's substrates.

Preparation of protein lysate, TCA precipitation and tryptic digestion E. coli cells were harvested and the whole cell lysate was prepared as the previous report 7. The cell pellet was resuspended in the lysis buffer (8M urea in 100 mM ammonium bicarbonate solution) and sonicated for 10 min. Equal amounts of protein lysates from SILAC samples were mixed. The whole cell lysates were precipitated by trichloroacetic acid (TCA) with a final concentration of 20% (v/v) 19. The dried protein pellets were dissolved in 100 mM ammonium bicarbonate (NH4HCO3) buffer (pH 8.0), and then digested with sequencing grade trypsin.

Dimethylation labeling The labeling method was performed according to the previous report

20

.

Formaldehyde solution (light) and formaldehyde-D2 solution (heavy) were added to the peptide mixture to a final concentration of 40 mM, respectively. Sodium cyanoborohydride (NaBH3CN) was immediately added to a final concentration of 20 mM. The pH value was adjusted to 7.0 and the mixture was incubated at 37 °C overnight. The labeling reaction was quenched by 1 M NH4HCO3 to a final

8 / 43


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concentration of 100 mM and incubated for another 4 h. The dimethylation labeling efficiency was more than 99% as validated by mass spectrometry analysis.

High pH HPLC fractionation and propionyllysine peptide enrichment The tryptic peptides were pre-separated by high pH HPLC (bHPLC) to decrease the sample complexity 19, 21. The tryptic peptides were pre-separated on an Xbridge prep C18 column (5 um, 19X150 cm, Waters Corp., Milford, MA) using a 80 min gradient from 2% to 90% of buffer B (10 mM ammonium formate in 80% ACN, pH 8.5) at a flow rate of 10 ml/min. The peptides were separated, and pooled into 10 fractions for affinity purification. The propionyllysine peptides were enriched using a reported procedure

19, 22

.

The dried peptides were dissolved in immunoprecipitation buffer (IP buffer: 600 mM NaCl, 0.5 mM EDTA and 20 mM Tris-HCl, pH 8.0). Affinity enrichment of propionyllysine peptides was carried out using pan anti-propionyllysine antibody beaded agarose. The beads were washed with IP buffer for three times and incubated with the E. coli peptide mixture overnight at 4°C. The enriched peptides were eluted with 0.1% TFA and dried up in a SpeedVac. The dried peptides were desalted using C18 Zip-tips prior to HPLC/MS/MS analysis.

Nano-HPLC/MS/MS analysis The enriched peptides were dissolved in 4 µl buffer A (0.1% formic acid in water, v/v) and loaded onto a 15 cm capillary C18 reversed phase analytical column (3 um particle size, 90 um pore Å, Dikma Technologies Inc., Lake Forest, CA) via the auto-sampler connected to an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific 9 / 43



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Inc., Waltham, MA). The peptides were eluted with a linear gradient of buffer B (0.1% formic acid in acetonitrile, v/v) from 5% B to 45% B in 60 min followed by a steep increase to 80% B in 10 min at a flow rate of 300 nl/min, and were ionized and sprayed into a Q Exactive or Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) via a nanospray ion source in a positive mode. The peptides with a range of m/z 350-1500 were analyzed with a resolution of 70,000 at m/z 200 and automatic gain control (AGC) target was 1e6. The maximum ion injection time (IT) was 60ms. The mass spectrometric analysis was carried out in a data dependent mode. The top 16 ions were prior to be isolated and subjected to higher-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 28 for Q Exactive and 32 for Orbitrap Fusion. The dynamic exclusion was set to 30s and the charge exclusion was set at 1+ and ≥5+.

Protein sequence database searching and analysis The MS raw data were processed with MaxQuant software (version 1.5.1.2)

23

, and

searched against UniProt E. coli strain K12 database (4305 sequences, 1,356,026 residues). The cleavage enzyme was set as trypsin/P and maximum missed cleavage was set 3. Precursor error tolerance was set as ±10 ppm. Fragment ion was set as ±0.02 Da for Q Exactive and ±0.5 Da for Orbitrap Fusion mass spectrometers, respectively. Protein N-terminal acetylation, methionine oxidation and lysine propionylation were set as variable modifications, and the fixed modification was set to cysteine alkylation by iodoacetamide. For analysis of SILAC raw data, the multiplicity was set to 2 and heavy labeling channel was chosen as L-lysine-13C6 or 10 / 43


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L-lysine-13C6 and L-arginine-13C6, 15N4. For analysis of dimethylation labeling raw data, “DimethLys0” and “DimethNert0” were chosen as light labels, and “DimethLys4” and “DimethNert4” were chosen as heavy labels. Minimal peptide length was 6. The raw data for proteome quantification were similarly processed, except that protein N-terminal acetylation and methionine oxidation were set as variable modifications. Peptide spectrum match (PSM) false discovery rate (FDR) and protein FDR was set to 0.01 for analyzing all data. All the quantitative lysine propionylome data were normalized based on protein expression level.

Data accession Raw data together with the files exported by MaxQuant software were available on the integrated Proteome resources (iProx) (URL: http://www.iprox.org/index) with project ID: IPX00077800.

Motif analysis of propionyllysine modified substrates The propionylated substrates were used to analyze consensus flanking sequence of lysine propionylation sites with iceLogo software (version 1.2) 24. Seven neighboring amino acids residues on each side of the propionylation sites were selected as the positive set for analysis. The embedded Swiss-Prot “Escherichia coli (strain K12)” was used as the negative set.

Functional annotation analysis of lysine propionylome Functional enrichment analysis of the propionylated substrates was carried out by using DAVID (The Database for Annotation, Visualization and Integrated Discovery v6.7) 25, including KEGG pathway 26, Gene Ontology (GO) 27 and Pfam domain 28. The 11 / 43



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Escherichia coli genome was used as the background. P-values were adjusted with a Benjamini using a cutoff of 0.05 to control the FDR.

Protein-protein interaction network analysis The STRING database (version 10)

29

was used for enrichment analysis of lysine

propionylated protein-protein interaction networks in E. coli. All interactions with high confidence (0.7) were visualized in Cytoscape (version 3.2.1)

30

and highly

enriched clusters were identified by MCODE plug-in toolkit 31.

The purification of CobB and HPLC assay for CobB depropionylation activity The cobB overexpression E. coli strain was from the ASKA library

32

. The strain was

cultured in LB medium and induced by 1 mM isopropyl β-D-Thiogalactoside when OD600 reached 0.6. Cells were harvested by centrifugation and washed with pre-cold Dulbecco’s Phosphate Buffered Saline (D-PBS). Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1 CelLytic B, 50 U/ml of Benzonase, 1 mM phenylmethanesulfonyl fluoride, pH 8.0) was added into the cells. The supernatant was collected by centrifugation. The recombinant protein was captured by Ni-NTA beads and incubated at 4°C for 2 h with vigorous shaking. After several rounds of washing, the proteins were eluted and desalted with ZebaTM Spin Desalting Column, 7K MWCO (Thermo Fisher Scientific Inc., Waltham, MA). Protein concentration was determined by BCA Protein Assay. The depropionylation enzymatic activity of CobB was determined with HPLC-MS/MS analysis. Two microgram purified CobB and 0.3 mM propionylated 12 / 43


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peptide (EIAQDFKprTDLR) were incubated in reaction buffer (1 mM NAD+, 1 mM DTT, 20 mM Tris-HCl, pH 7.4) at 37°C for 2 h 6, 33. Then the reaction was stopped with 60 µl 10% TFA.

In vitro acetyl-CoA synthetase (ACS) assays The wild type acs gene was amplified by PCR from prototrophic E. coli BW25113 and was cloned into pET-28a (+) vector. The mutant K609R was cloned into the same vector using a QuckChange mutagenesis kit (Stratagene, La jalla, CA). The mutant was confirmed by DNA sequencing. The proteins were expressed and purified according to a previous report 34. The enzymatic activity of ACS was determined by photometrical ACS assay at 37°C using a microplate reader (BioTek Instruments, Winooski, VT, USA) at 340 nm 34. Equal amounts of wild type and mutant ACS were added to the reaction system (100 mM Tris-HCl, 10 mM L-malate, 0.2 mM coenzyme A, 8 mM ATP, 1 mM NAD+, 10 mM MgCl2, 3 units of malate dehydrogenase, 0.4 unit of citrate synthase, pH 7.5). The reaction was started with 100 mM potassium acetate. Three replicates were performed.

RESULT Landscape of lysine propionylation in E. coli. To systematically identify lysine propionylated substrates in E. coli, we used a pan-anti-propionyllysine antibody approach to enrich propionyllysine peptides from cell lysate digests cultured in LB medium as we previously reported

19, 22, 35-37

. The

tryptic peptides were fractionated by high pH HPLC (bHPLC), further enriched by 13 / 43



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pan-anti-propionyllysine antibody and subjected to nano-HPLC/MS/MS analysis (Fig. 1B). In this experiment, 956 lysine propionylation sites in 461 proteins were identified by MaxQuant software based on criterion of localization probability > 75%, score>40 and 1% FDR on peptide level (Supplemental Table S1). First, we examined the distribution of lysine propionylation site(s) per substrate in E. coli. Of all the 461 propionylated proteins, we found that more than half of all the proteins (258 proteins) were propionylated at one site. Around 20% and 10% proteins were propionylated at two and three sites, respectively. The rest of the proteins (around 15%) were propionylated at 4 sites or more (Fig. 1C). Among the 461 propionylated proteins, the most heavily propionylated proteins were 60 kDa chaperonin (15 sites), tryptophanase (12 sites), chaperone protein DnaK (10 sites) and Glyceraldehyde-3-phosphate dehydrogenase A (10 sites). To determine the sequence preference proximal to propionyllysine sites, we used iceLogo software to analyze their flanking sequence (Fig. 1D). The result indicated that glutamic acid residue was significantly overrepresented surrounding lysine propionylation

sites,

whereas

serine

and

asparagine

residues

were

underrepresented.

Functional annotation analysis of E. coli propionylome To understand the function of lysine propionylated substrates, we used DAVID online bioinformatic tool to perform enrichment analysis 25 against KEGG pathway 26, Gene Ontology (GO)

27

and Pfam domain

28

databases. The result of GO analysis showed

that propionylated substrates were highly enriched in multiple pathways mainly 14 / 43


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associated with energy metabolism and protein processing pathways, such as translation, carbohydrate and amino acid metabolism (Fig. 2A, Supplemental Table S2A, 2B and 2C). In the KEGG pathway analysis, ribosome, glycolysis/gluconeogenesis and pyruvate metabolism were among the most highly enriched pathways (Fig. 2B, Supplemental Table S2D). Interestingly, in consistent with the role of propionyl-CoA in E. coli, we found the enrichment of propionate metabolism, fatty acid metabolism and valine/leucine/isoleucine metabolism pathways (Supplemental Fig. S1). Consistent with GO and KEGG analysis, Pfam domain analysis showed the enrichment of ribosome related S4 domain and S1 RNA binding domain (Fig. 2C, Supplemental Table S2E), suggesting a role of propionylation in ribosome associated protein translation process. We further used the STRING database

29

to identify the

protein-protein interaction network of propionylome. Of all the 461 substrates, we identified 15 protein-protein highly connected interaction networks (Fig. 2D, Supplemental Fig. S2 and Supplemental Table S3). We found that top five clusters were mainly associated with ribosome, glycolysis/gluconeogenesis, aminoacyl-tRNA biosynthesis, pyruvate metabolism.

The dynamics of lysine propionylation in response to propionate. We next carried out quantitative lysine propionylome analysis in response to propionate to investigate its role as a sole carbon source in E. coli metabolism. We used prototrophic E. coli strain BW25113 to performed quantitative propionylome analysis according to the previous reports

38, 39

. The prototrophic E. coli strain

BW25113 cells were cultured in regular light amino acid M9 medium (light) 15 / 43



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supplemented with 0.8% sodium propionate, or M9 medium supplemented with 13

C6-lysine and

13

C6,

15

N4-arginine (heavy) (Fig. 3A). We then carried out affinity

enrichment and mass spectrometry analysis to quantify the change of lysine propionylated peptides. The propionylation quantification results were further normalized based on protein expression level. In this experiment, we identified 713 Kpr sites in 371 proteins and 63 sites can be quantified with a normalized median H/L SILAC ratio of 0.08 and 591 Kpr sites were only identified in propionate treated sample (Fig. 3B, Supplemental Table S4A, 4B and 4C). Our data showed that propionate could significantly increase the level of propionylation. Similarly, we used DAVID online bioinformatic tool to perform enrichment analysis of the Kpr sites significantly induced (H/L ratio < 0.5 or light only form) by propionate

25

(Supplemental Table S4D~4G). In contrast to the propionylome of E.

coli cultured in regular LB medium, we found glucogenic amino acid metabolism pathways were enriched in propionate treatment, including glycine, serine and threonine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis pathway (Fig. 3C and Supplemental Table S5). The result suggested that when E. coli utilized propionate as a sole carbon source, lysine propionylation was likely to have an impact on the glucogenesis associated amino acid metabolism. Interestingly, we found several enzymes involved in propionate degradation and methylcitrate cycle were propionylated only in propionate treated sample, including propionyl-CoA synthetase (PrpE, 1 Kpr site), acetyl-CoA synthetase (ACS, 3 Kpr sites), 2-methylcitrate synthase (PrpC, 4 Kpr sites), 2-methylcitrate dehydratase (PrpD, 3 16 / 43


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Kpr sites) and 2-methylisocitrate lyase (PrpB, 3 Kpr sites) (Fig. 3D, Supplemental Fig. S3). In E. coli, propionate can be converted into propionyl-CoA for further utilization by two enzymes, propionyl-CoA synthetase and acetyl-CoA synthetase (Fig. 1 A). Propionylation at Lys592 of propionyl-CoA synthetase was previously characterized to inactivate its enzymatic activity in Salmonella enterica

10

. Similarly, our study

identified propionylation at Lys592 of propionyl-CoA synthetase in E. coli. We also found that Lys111, Lys221 and Lys609 of acetyl-CoA synthetase can be propionylated under propionate condition (Fig. 3D). This result suggested propionylation may play an important role in propionate metabolism. In methylcitrate cycle metabolism, we found that Lys80 and Lys234 of the rate limiting enzyme PrpC were propionylated. These two sites are very close to its substrate binding (Lys82) and active (His235) sites

40

(Supplemental Fig. S4), suggesting a potential role of propionylation in

regulation of its enzymatic activity. Similarly, PrpB and PrpD were also propionylated in this analysis. In contrast, in the propionylome of E. coli grown in LB medium, no propionylation sites of these methylcitrate cycle enzymes were identified (Supplemental Fig. S3). The results suggested a role of lysine propionylation in propionate utilization. To further investigate the functional consequence of lysine propionylation in propionate metabolism, we carried out sequence alignment analysis of propionylated lysine residues, Lys111, Lys221, Lys609 of ACS, a rate limiting enzyme in propionate metabolism. The result showed that Lys609 is evolutionarily conserved among all five species we examined from bacteria to human, suggesting a vital role of this residue in 17 / 43



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its enzymatic activity

41

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(Fig. 4A, 4B and 4C). To further analyze the functional

consequence of propionylation at Lys609 of ACS, we carried out mutagenesis experiments. The Lys609 was mutated to arginine, which mimics its positive charge state in vivo. The purified ACS proteins were used to evaluate the enzymatic activity. The result illustrated that mutation of Lys609 to arginine led to decrease of the enzymatic activity (Fig. 4D and 4E), further suggesting that lysine propionylation in 609 lysine residue may play an important role in regulating ACS enzymatic activity. Together, these data suggested a new role of propionylation in bacterial propionate metabolism.

The dynamics of lysine propionylation in response to nutritional conditions involved in propionyl-CoA metabolism. Our bioinformatic analysis revealed that lysine propionylated substrates are enriched in carbohydrate and amino acid metabolism. We hypothesized that disturbance of the carbon sources and the amino acids involved in propionyl-CoA metabolic pathway would affect the level of lysine propionylation in vivo

7, 14, 18

(Fig. 1A).

Toward this goal, we evaluated the dynamics of E. coli lysine propionylation in response to glucose using SILAC based quantitative proteomics approach (Fig. 5A). We cultured auxotrophic E. coli AT713 strain, which is incapable of lysine and arginine self-biosynthesis, in M9 medium containing natural amino acids (light labeled) and M9 medium supplemented with 0.8% glucose containing L-lysine-13C6 and L-arginine-13C6,

15

N4 (heavy labeled). In this analysis, we identified 126 Kpr sites.

Among the 126 Kpr sites, 84 Kpr sites could be quantified with a normalized median 18 / 43


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H/L ratio of 0.78 and 40 sites were identified only in light form (Fig. 5B, Supplemental Table S6A, 6B and 6C). Strikingly, in contrast to our previous finding that lysine acetylation and succinylation level were increased 7, the level of lysine propionylation was mostly decreased under glucose treatment condition. To further validate our finding, we next carried out the quantitative propionylome analysis in the prototrophic E. coli BW25113 strain cultured in LB medium as a biological replicate. BW25113 cells were cultured in regular LB medium with or without 0.8% glucose treatment. As SILAC based approach cannot be used under such condition, we used a previously reported isotopic dimethylation labeling method 20 to quantify the Kpr change under the two nutritional conditions (Fig. 5C). The free primary amino groups of tryptic peptides from regular LB medium were labeled with light dimethyl group, whereas those from LB medium supplemented with 0.8% glucose were labeled with D2-dimethyl group (+4 Da mass shift compared to a light dimethyl group). This analysis led to the identification of 166 Kpr sites. In consistency, most of the Kpr sites (126 sites) were only identified under regular LB medium culture condition (Fig. 5D, Supplemental Table S7A). Among the 18 quantifiable Kpr sites, they also showed an obvious decrease in response to high glucose condition, with a normalized median SILAC H/L ratio of 0.06 (Fig. 5D, Supplemental Table S7B and 7C). In parallel, we carried out the lysine acetylome analysis using the same batch of sample, and identified 3,065 Kac sites. In consistent with our previous study 7, 2,202 Kac sites were only identified under high glucose condition, and the 531 quantifiable sites showed a normalized median SILAC H/L 19 / 43



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ratio of 16.39 (Fig 5D, Supplemental Table S7D and 7E). Together, our data showed that there was an obvious seesaw effect between lysine propionylation and acetylation in response to glucose condition, suggesting different roles of these two modifications in energy metabolism. To further gain an insight on this finding, we compared the Kpr sites and Kac sites identified from the same sample. We found that 119 Kpr sites and 119 Kac sites shared the same lysine residues (Fig. 5E, Supplemental Table S7F). Among these modification sites, 73 acetylation sites could be quantified with a median H/L ratio of 69.27 and 31 acetylation sites were only identified under heavy medium condition. In stark contrast, the median H/L ratio of 13 quantifiable propionylation sites were 0.07 and 94 propionylation sites were only identified under light medium condition (Fig. 5F, Supplemental Fig. S5A). Bioinformatic analysis showed that most of these proteins were enriched in glycolysis and ribosome related translation activity (Supplemental Fig. S5B, Supplemental Table S7G). We found that lysine propionylation and acetylation of the three rate-limiting enzymes in glycolysis and TCA cycle, fructose-bisphosphate aldolase class 2 (Lys91), pyruvate kinase (Lys68 in KPY1, and Lys82 and Lys 351 in KPYK2) and isocitrate dehydrogenase (Lys235 and Lys 242 in KPYK2) were changed in the opposite way (Fig. 5F). Therefore, our propionylome and acetylome analysis suggested that propionylation and acetylation are likely to play an opposite (possibly competitive) role in bacterial glucose metabolism and protein translation under different nutritional conditions.

20 / 43


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In addition to glucose metabolism, the metabolism of isoleucine, methionine and threonine plays an important role in propionyl-CoA metabolism. We thus further evaluated the dynamics of E. coli lysine propionylation in response to the three amino acids. We cultured auxotrophic E. coli AT713 in M9 medium supplemented with or without isoleucine, methionine and threonine. Interestingly, increasing the metabolism flux of isoleucine, methionine and threonine led to the increase of global lysine propionylation level in E. coli, whereas the level of lysine acetylation was decreased (Supplemental Fig. S6A). As a control, we randomly chose three amino acids (Ala, Gly and Ser) which are not involved in propionyl-CoA metabolic pathway for further investigation. The western blotting showed that lysine propionylation and acetylation were largely unchanged in this case (Supplemental Fig. S6B). The results indicated that metabolic disturbance of alanine, methionine and threonine may affect physiological processes via lysine propionylation.

Characterization of the enzymatic activities of PatZ and CobB in lysine propionylation PatZ and CobB are known to be the bacterial lysine acetylatransferase and deacetylase, respectively

42, 43

. A study reported that PatZ and CobB could regulate

propionylation of propionyl-CoA synthetase in Salmonella enterica 10, suggesting PatZ and CobB could also play a general regulatory role in bacterial lysine propionylation. To test this hypothesis, we carried out Western blotting in patZ overexpression (patZ OE) E. coli strain. The result showed that the global propionylation level was increased as compared to the wide type strain under different carbon resource 21 / 43



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conditions. Similarly, knockdown of cobB could also lead to the increase of global lysine propionylation level (Fig. 6A). These results suggested that PatZ and CobB were likely to have lysine propionylation regulation activities in vivo. We next used a synthetic propionylated peptide (EIAQDFKprTDLR) to test CobB’s depropionylation activity in vitro similar to our previous study 7. The result illustrated that CobB can depropionylate the propionyllysine peptide in vitro (Fig. 6B). To further identify the potential lysine propionylated substrates regulated by CobB, we used a SILAC-based proteomic analysis in cobB knockout versus control E. coli strains (Fig. 6C) 44. The isotopic amino acid labeling efficiency was more than 98% as validated by mass spectrometry analysis (Supplemental Fig. S7). Three biological replicates were performed for Kpr quantification. In total, we identified 313 lysine propionylation sites in 178 proteins (Supplemental Table S8). Of the 313 lysine propionylation sites, 303 sites in 169 proteins could be quantified (Fig. 6D, Supplemental Table S8C). Among them, 13 lysine propionylation sites were identified in at least 2 replicates with more than two fold of increase in cobB knockout strain (Table 1). These sites could be potentially regulated by CobB. The 13 potential CobB substrates are associated with various biological processes, such as TCA cycle, protein expression, L-tryptophan degradation, DNA metabolism, etc.

DISCUSSION Lysine propionylation belongs to the expanded landscape of lysine acylation modifications in bacteria. This study lead to the most comprehensive propionylome 22 / 43


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dataset in bacteria up to date. In this study, we identified 956 Kpr sites in LB medium culture experiment (Fig. 1), 713 Kpr sites in propionate supplemented experiment (Fig. 3), 126 Kpr sites in glucose supplemented M9 medium experiment (Fig. 5A and 5B), 166 Kpr sites in supplemented LB medium experiment (Fig. 5C and 5D) and 313 Kpr sites in the CobB substrate identification experiment (Fig. 6). Taking together, we identified a total of 1467 nonredundant propionylation sites in 604 proteins (Supplemental Table S9), which significantly increased the reported bacterial propionylome. Our results suggested the broad substrate diversity in bacteria. In nature, propionate is a high abundant carbon source, which can be utilized by E. coli and many bacteria as a sole carbon source. On the other hand, probably due to the accumulation of toxic propionyl-CoA, high concentration of propionate is widely used as an antimicrobial agent in food and agriculture industry. Our quantitative propionylome analysis revealed that propionate can drastically increase the global level of lysine propionylation, especially enriched in propionate metabolism and glucogenic amino acid metabolism pathways. Of particular note, we found the two enzymes involved in propionate metabolism, ACS and PrpE, as well as the enzymes involved in methylcitrate cycle, PrpC, PrpB and PrpD, were propionylated in response to propionate treatment 10. Our mutagenesis experiment of the propionylation site at Lys609 of ACS, suggested that propionylation is likely to inhibit its activity, and have an impact on methylcitrate cycle metabolism. Together, our data suggested protein lysine propionylation is likely to play an important role in bacterial propionate metabolism and suggested a new mechanism of the 23 / 43



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Page 24 of 43

anti-microbial activity of propionate as a food preservative. Our previous data showed the dramatic increase of lysine acetylation and succinylation in response to glucose stimulation 7. In stark contrast to these lysine acylation modifications, we found that propionylation substrates predominantly existed under lower glucose condition. Particularly, acetylation and propionylation level of the same sites of the enzymes involved glycolysis were oppositely regulated in response to glucose. A recent study reported that histone butyrylation at H4 could play a competing role against acetylation in spermatogenesis

45

. It is likely that

propionylation and acetylation could also play a competing role in bacteria energy metabolism regulation. A previous study showed that bacterial lysine acetylation regulatory enzymes, PatZ and CobB, could also regulate propionylation at Lys592 of propionyl-CoA synthetase 10. Our Western blotting data suggested that PatZ and CobB could be the lysine propionyltransferase and depropoinylase for various substrates in bacteria. In addition, our previous data also showed that CobB could act as a desuccinylase in bacteria 7. Together, these results suggested that PatZ and CobB could possibly act as a general lysine deacylase instead of deacetylation activity in bacteria, suggesting broader activities of these two enzymes. To further identify the depropionylation substrates, we carried out the quantitative propionylome analysis in cobB knockout and prototrophic E. coli cells. We identified 13 significantly changed lysine propionylation sites, which may be regulated by CobB. In fact, only a pretty small percentage (13 out of 303 sites) in this study were showed obvious change in 24 / 43


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response to cobB knockdown, suggested that CobB could have substrate preference towards the regulation of lysine depropionylation. We next compared our data with a recent study by Castano-Cerezo et al. on system wide analysis of deacetylation substrates of CobB

46

. We found 8 overlapping upregulated propionylated and

acetylated sitesin cobB knockout strain (Supplemental Table S8D) between our propionylome data and their acetylome data. In addition, we identified 5 up-regulated (increase more than two fold) propionylation sites in cobB knockout strain that were not reported in the acetylation dataset

46

. For example, the KH

type-2 domain (position: 39~107) of 30s ribosomal protein S3 was propionylated at Lys79 and the propionylation site was regulated by CobB. Because KH domain binds RNA and play a function in RNA recognition, CobB may affect protein expression by regulating the Lys79 propionylation of 30s ribosomal protein. The result suggested that CobB could possibly regulate the lysine propionylation to affect the functions of its various substrates. It was reported that acetyl-CoA can chemically acetylate lysine residues in vitro, and acetyl-phosphate (AcP) produced from acetyl-CoA in bacteria can also chemically acetylate lysine residues without enzymatic catalyzation

47-49

.

Similarly, it is also possible that lysine propionylation could also occur in a non-enzymatically manner in E. coli. Together, our results revealed the broad propionylation substrates, and its dynamics in response to different nutritional conditions and its potential regulatory enzymes in E. coli, suggesting new roles of lysine propionylation in bacterial physiology. 25 / 43



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Supplemental Information List Supplemental Fig. S1. Representative enriched KEGG pathways of lysine propionylated proteins. Supplemental Fig. S2. The complete Kpr interaction network. Supplemental Fig. S3. The propionylated proteins involved in propionate metabolism pathway. Supplemental Fig. S4. The spatial position of lysine propionylation within three dimensional structure of PrpC. Supplemental Fig. S5. The information of overlapping sites between the Kpr and Kac in response to high glucose. Supplemental Fig. S6. Western blotting analysis of the dynamics of E. coli lysine propionylation in response to the amino acids involved in propionyl-CoA metabolic pathway and uninvolved in propionyl-CoA metabolic pathway as a control. Supplemental Fig. S7. The SILAC labeling efficiency test result. Supplemental Table S1: The list of lysine propionylation (Kpr) sites identified in LB medium experiment. Supplemental Table S2: Functional annotation analysis of Kpr sites identified in LB medium experiment. Supplemental Table S3: Protein network analysis of propionylated proteins by using STRING database by MCODE for Kpr sites identified in LB medium experiment. Supplemental Table S4: The lysine propionylome data and functional analysis in response to propionate treatment in SILAC experiment. 26 / 43


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Supplemental Table S5: Functional annotation comparation between Kpr sites identified in LB medium and in propionate condition experiment. Supplemental Table S6: The lysine propionylome data got in SILAC medium with or without glucose experiment. Supplemental Table S7: The lysine propionylome data and acetylome data got in LB medium with or without glucose experiment. Supplemental Table S8: The lysine propionylome data for identifying CobB regulated substrate experiment. Supplemental Table S9: The total Kpr sites identified in this study.

Acknowledgments This work was supported by the National Basic Research Program of China (973 Program) (No. 2014CBA02004), the Natural Science Foundation of China (No. 31670066 and No. 31370813), the National Science & Technology Major Project (No. 2014ZX09507-002), the Shanghai Municipal Science and Technology Commission (No. 14DZ2261100, No. 16YF1414000 and No. 15410723100) , the Strategic Priority Research

Program

of

the

Chinese

Academy

of

Sciences,

"Personalized

Medicines—Molecular Signature-based Drug Discovery and Development" (No. XDA12020314), and the National Institute of Health (NIH) of the United States 27 / 43



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interactions in 630 organisms. Nucleic Acids Res 2009, 37, (Database issue), D412-6. 30. Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.; Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T., Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 2003, 13, (11), 2498-504. 31. Bader, G. D.; Hogue, C. W., An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics 2003, 4, 2. 32. Kitagawa, M.; Ara, T.; Arifuzzaman, M.; Ioka-Nakamichi, T.; Inamoto, E.; Toyonaga, H.; Mori, H., Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 2005, 12, (5), 291-9. 33. Rardin, M. J.; He, W.; Nishida, Y.; Newman, J. C.; Carrico, C.; Danielson, S. R.; Guo, A.; Gut, P.; Sahu, A. K.; Li, B.; Uppala, R.; Fitch, M.; Riiff, T.; Zhu, L.; Zhou, J.; Mulhern, D.; Stevens, R. D.; Ilkayeva, O. R.; Newgard, C. B.; Jacobson, M. P.; Hellerstein, M.; Goetzman, E. S.; Gibson, B. W.; Verdin, E., SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks. Cell Metab 2013, 18, (6), 920-33. 34. You, D.; Yao, L. L.; Huang, D.; Escalante-Semerena, J. C.; Ye, B. C., Acetyl coenzyme A synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single Gcn5-type N-acetyltransferase (GNAT) domain in Saccharopolyspora erythraea. J Bacteriol 2014, 196, (17), 3169-78. 35. Choudhary, C.; Weinert, B. T.; Nishida, Y.; Verdin, E.; Mann, M., The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 2014, 15, (8), 536-50. 36. Colak, G.; Pougovkina, O.; Dai, L.; Tan, M.; Te Brinke, H.; Huang, H.; Cheng, Z.; Park, J.; Wan, X.; Liu, X.; Yue, W. W.; Wanders, R. J.; Locasale, J. W.; Lombard, D. B.; de Boer, V. C.; Zhao, Y., Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation. Mol Cell Proteomics 2015, 14, (11), 3056-71. 37. Mertins, P.; Qiao, J. W.; Patel, J.; Udeshi, N. D.; Clauser, K. R.; Mani, D. R.; Burgess, M. W.; Gillette, M. A.; Jaffe, J. D.; Carr, S. A., Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 2013, 10, (7), 634-7. 38. Frohlich, F.; Christiano, R.; Walther, T. C., Native SILAC: metabolic labeling of proteins in prototroph microorganisms based on lysine synthesis regulation. Mol Cell Proteomics 2013, 12, (7), 1995-2005. 39. Ping, L.; Zhang, H.; Zhai, L.; Dammer, E. B.; Duong, D. M.; Li, N.; Yan, Z.; Wu, J.; Xu, P., Quantitative proteomics reveals significant changes in cell shape and an energy shift after IPTG induction via an optimized SILAC approach for Escherichia coli. J Proteome Res 2013, 12, (12), 5978-88. 40. Chittori, S.; Savithri, H. S.; Murthy, M. R., Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity. J Struct Biol 2011, 174, (1), 58-68. 41. Reger, A. S.; Carney, J. M.; Gulick, A. M., Biochemical and crystallographic analysis of substrate binding and conformational changes in acetyl-CoA synthetase. Biochemistry 2007, 46, (22), 6536-46. 42. Starai, V. J.; Celic, I.; Cole, R. N.; Boeke, J. D.; Escalante-Semerena, J. C., Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 2002, 298, (5602), 2390-2. 43. Liang, W.; Malhotra, A.; Deutscher, M. P., Acetylation regulates the stability of a bacterial protein: growth stage-dependent modification of RNase R. Mol Cell 2011, 44, (1), 160-6. 44. Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression 30 / 43


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proteomics. Mol Cell Proteomics 2002, 1, (5), 376-86. 45. Goudarzi, A.; Zhang, D.; Huang, H.; Barral, S.; Kwon, O. K.; Qi, S.; Tang, Z.; Buchou, T.; Vitte, A. L.; He, T.; Cheng, Z.; Montellier, E.; Gaucher, J.; Curtet, S.; Debernardi, A.; Charbonnier, G.; Puthier, D.; Petosa, C.; Panne, D.; Rousseaux, S.; Roeder, R. G.; Zhao, Y.; Khochbin, S., Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters. Mol Cell 2016, 62, (2), 169-80. 46. Castano-Cerezo, S.; Bernal, V.; Post, H.; Fuhrer, T.; Cappadona, S.; Sanchez-Diaz, N. C.; Sauer, U.; Heck, A. J.; Altelaar, A. F.; Canovas, M., Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli. Mol Syst Biol 2014, 10, 762. 47. Weinert, B. T.; Iesmantavicius, V.; Wagner, S. A.; Scholz, C.; Gummesson, B.; Beli, P.; Nystrom, T.; Choudhary, C., Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli. Mol Cell 2013, 51, (2), 265-72. 48. Weinert, B. T.; Iesmantavicius, V.; Moustafa, T.; Scholz, C.; Wagner, S. A.; Magnes, C.; Zechner, R.; Choudhary, C., Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. Mol Syst Biol 2014, 10, 716. 49. Baeza, J.; Smallegan, M. J.; Denu, J. M., Site-specific reactivity of nonenzymatic lysine acetylation. ACS Chem Biol 2015, 10, (1), 122-8.

31 / 43



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure Legends Fig. 1. Landscape of lysine propionylome in E. coli. (A) Metabolic pathways for propionyl-CoA biosynthesis and metabolism. (B) Schematic overview showing the procedure for lysine propionylome profiling. (C) Pie chart showing the distribution of the number of Kpr sites per protein. (D) Sequence motifs of Kpr sites generated by iceLogo software. Fig. 2. Functional annotation and protein-protein interaction analysis of lysine propionylome in E. coli. (A) Representative GO annotations of lysine propionylated proteins for biological process. (B) Representative pathways of lysine propionylated proteins for KEGG. (C) Representative Pfam domain annotations of lysine propionylated proteins. (D) The top five clusters of highly interconnected lysine propionylated protein (listed in gene names) networks from STRING database. The lysine propionylated proteins in the top five clusters are shown in pink, blue, purple, green and yellow, respectively. Protein(s) without lysine propionylation is indicated in gray in each cluster. Fig. 3. Quantitative proteomic analysis of E. coli lysine propionylome in response to sodium propionate. (A) The workflow showing the procedure for quantitative proteomic analysis of E. coli lysine propionylome in response to sodium propionate. (B) The quantitive lysine propionylome in response to propionate. Scatter plot shows the distribution of lysine propionylation dynamics in response to propionate treatment. Kpr ratio (H/L): MS signal intensity from M9 only medium (heavy) divided by that from high sodium propionate medium (light). (C) Representative enriched 32 / 43


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Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


KEGG pathways of upregulated lysine propionylated proteins in response to propionate. Substrates with a ratio (H/L) < 0.5 of the and substrates identified in light only form were used for KEGG enrichment analysis. (D) The identified propionylated substrates involved in propionate degradation and methycitrate cycle. Fig. 4. Mutagenesis and enzymatic activity analysis of acetyl-coenzyme A synthetase (ACS). (A) MS/MS spectrum of a propionylated peptide (SGKprIMR) identified from ACS around Lys609 residue. (B) The three dimensional structure of ACS. Structural data was obtained from the Molecular Modeling Database (MMDB ID 46488) and visualized in Cn3D. Lys609 was modified by propionylation and indicated in red. Val537, His539 and Ile542 are metal binding residues and Arg584 is coenzyme A binding site, which are indicated in yellow. (C) Sequence alignment analysis of Lys609 of ACS by ClustalW. ACS homology sequence were retrieved from UniProt database: H. sapiens (accession ID: Q9NR19), M. musculus (accession ID: A2AQN4), C. elegans (accession ID: Q18496), E. coli (accession ID: P27550) and S. cerevisiae (accession ID: A7A0B4). “*” indicates positions which have a single, fully conserved residue; “:” indicates that one of the following "strong" groups is fully conserved; “.” denotes that one of the following “weaker” groups is fully conserved. The conserved propionyllysine residue of interest is indicated in red. (D) Purity of the wild-type and mutated proteins shown by SDS-PAGE gel. (E) Enzymatic activities of wild-type and K609R ACS mutant. Fig. 5. Quantitative analysis of lysine propionylome in response to high glucose in E. coli. (A) Flowchart showing the experimental procedure for the quantitative analysis 33 / 43



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of lysine propionylome in response to high glucose in AT713 E. coli strain, which is incapable of lysine and arginine self-biosynthesis. (B) Scatter plot showing the distribution of the quantifiable lysine propionylated peptides in response to high glucose. Kpr ratio (H/L): MS signal intensity from M9 medium supplemented with 0.8% glucose (heavy) divided by that from M9 only medium (light). (C) The workflow showing the experimental procedure for the quantitative propionylome analysis in response to high glucose by isotopic dimethylation labeling in prototrophic E. coli BW25113 strain. (D) Scatter plot showing the distribution of the quantifiable lysine propionylated peptides and acetylated peptides in response to high glucose. The red " " represents lysine propionylation and the blue "

" represents lysine acetylation.

Kpr or Kac ratio (H/L): MS signal intensity from LB medium supplemented with 0.8% glucose (heavy) divided by that from LB medium (light). (E) Venn diagram showing the number of propionylation only, acetylation only, and their overlapping sites from the same batch of sample prepared in LB medium with or without 0.8% glucose in prototrophic E. coli BW25113 strain. (F) The distribution diagram showing the intensities of Kpr and Kac in heavy form and light form, which were detected in the same E .coli sample prepared in LB medium with 0.8% glucose (heavy) or without 0.8% glucose (light). (G) The dynamics of Kpr and Kac substrates in glycolysis and TCA cycle in response to high glucose. Fig. 6. Characterization of the enzymatic activities of PatZ and CobB for lysine propionylation. (A) Western blotting analysis of lysine propionylation level in cobB knockout and patZ overexpression (patZ OE) strains. (B) HPLC analysis of the products 34 / 43


Page 34 of 43

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from a synthetic lysine propionylated peptide (EIAQDFKprTDLR) incubated with CobB in vitro. (C) The workflow showing the SILAC-based experimental procedure for CobB potential substrate identification. The lysA knockout (∆lysA) BW25113 E. coli cells were cultured in M9 medium containing light lysine (12C6-lysine) and 0.8% propionate, and the lysA and cobB knockout (∆lysA & ∆cobB) BW2511 cells were cultured into M9 medium containing heavy lysine (13C6-lysine) and 0.8% propionate. (D) Scatter plot showing the distribution of the quantifiable lysine propionylated peptides in response to CobB knockout. Table 1. The list of 13 propionylation sites with more than two fold of increase in cobB knockout strain.

Fig. 1 35 / 43



A

Glycolysis

Pyruvate Acetyl-CoA Oxaloacetate

L-Thr L-Met L-Ile

Glucose

Succinate SucCD

Succinyl-CoA

Protein extraction

ScpA

(R)-Methylmalonyl-CoA ScpC

3-Methyl-2-oxopentanoate

(S)-Methylmalonyl-CoA

Trypsin digestion 2-Methylacetoacetyl-CoA PrpD

Methylcitrate

PrpC

ScpB

Propionyl-CoA

2-Methyl-cis-aconitic acid Oxaloacetate AcnB 2-Methylisocitrate

ACS, PrpE

Propionate bHPLC fractionation

Malate

Odd chain fatty acid

PrpB

Fumarate

Immunoprecipitation

Succinate

C

D

HPLC-MS/MS

≥5 sites 7.6% 7.2%

4 sites 3 sites

P value =0.05 of difference

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 43

9.8% 1 site

56.0% 19.5% 2 sites

Fig. 2 36 / 43


Page 37 of 43

A

B

0

D Cluster 1 Score =40.86

C

KEGG pathway

Ribosome Glycolysis / Gluconeogenesis Pyruvate metabolism Citrate cycle (TCA cycle) Propanoate metabolism RNA degradation beta-Alanine metabolism Fatty acid biosynthesis Val, Leu and Ile degradation 0

10 20 30 -log10 (Adjusted p value)

2.6

2.8 3 3.2 3.4 -log10 (Adjusted p value)

Aldehyde dehydrogenase family S1 RNA binding domain S1 S4 domain 10 20 30 -log10 (Adjusted p value)

40

50

Cluster 3 Score =8.92



Cluster 5 Score =5

Fig. 3 37 / 43


40

Pfam domain

Translation Carbohydrate catabolic process Glucose metabolic process tRNA aminoacylation Amino acid activation tRNA aminoacylation for protein… Glycolysis Alcohol catabolic process Coenzyme metabolic process Hexose metabolic process Aerobic respiration Pyruvate metabolic process Acetyl-CoA metabolic process Tricarboxylic acid cycle Gluconeogenesis Cellular amino acid derivative… Glyoxylate metabolic process

Biological process

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.6


A

B # of sites identified

# of quantifiable sites

713

63

median of ratio (H/L)

# of light only form

# of heavy only form

591

0

0.08

Log10 peptide intensity

Light: M9+0.8% propionate Heavy: M9 only

Protein extraction

Trypsin digestion

9 8 7 6 5 4 -7

-5

-3

-1

1

Log2 Kpr ratio (H/L) Immunoprecipitation

D Pathway

HPLC-MS/MS Propionate degradation

C

Ribosome Glycolysis / Gluconeogenesis Pyruvate metabolism Propanoate metabolism Citrate cycle (TCA cycle) RNA degradation beta-Alanine metabolism Gly, Ser and Thr metabolism Fatty acid biosynthesis Phe, Tyr and Try biosynthesis

KEGG pathway

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 43

0

10

20

30

Methylcitrate cylcle

Protein name

Kpr sites (light only)

Acetyl-Co A synthetase

K111, K221 and K609

Propionyl-CoA synthetase

K592

2-Methylcitrate synthase

K15, K80,K234 and K318

2-Methylcitrate dehydratase

K374, K382 and K444

Aconitate hydratase Methylisocitrate lyase

40

-log10 (Adjusted p value)

Fig. 4 38 / 43


K71, K342, K482, K559 and K610 K8, K121 and K129

Page 39 of 43

B

A y5 y4 y3 y2 y1

S G Kpr I M R

y3

100

b3

419.2443 90

374.2141 Z=2 374.7157 Z=2 375.2156 Z=2

80

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


70

y1 175.1193

60

y2

H539 V537

306.1599 50

b3

I542

329.1825

40

y4

30

y5

603.3659 660.3875

R584

K609

20 10 0 100

C

D

200

300

400

500

600

700

m/z

H.sapiens M.musculus C.elegans E.coli S.cerevisiae

YIQNAPGLPKTRSGKIMRRVLRKIAQN-DHDLGDMSTVADPSVISHLFSH YIQNAPGLPKTRSGKIMRRVLRKIAQN-DHDLGDTSTVADPSVINHLFSH VIQEAPGLPKTRSGKVTRRILRKIAEGSESGIGDTTTLVDESVIKQLISG VLHWTDSLPKTRSGKIMRRILRKIAAGDTSNLGDTSTLADPGVVEKLLEE LIILVDDLPKTRSGKIMRRILRKILAGESDQLGDVSTLSNPGIVRHLIDS : . .********: **:**** . :** :*: : .:: :*:.

E

Mark WT K609R 170kDa 130kDa 95kDa 72kDa 55kDa 43kDa 34kDa 26kDa 17kDa 10kDa

Fig. 5 39 / 43



B

A Light W/O glucose Heavy+0.8% glucose

# of sites identified


126

84

Kpr




40

2

0.78


10 Protein extraction

Trypsin digestion

9 8 7 6 5

-6

-4

-2

0

Log2 Kpr ratio (H/L)

Immunoprecipitation

D # of sites identified

HPLC-MS/MS

MaxQuant

LB medium+0.8% glucose

Protein extraction

Trypsin digestion




166

18

0.06

126

1

Kac

3065

568

16.39

46

2202

10

Kpr ratio (H/L) Kac ratio (H/L)


LB medium


Kpr

C

9 8 7 6 5

light

heavy

Dimethylation labelling

E

-10

-5

0

Log2 Kpr or Kac ratio (H/L)

5

10

Immunoprecipitation

Kpr

HPLC-MS/MS

47 119

2945

Kac

MaxQuant

G

F

Glucose

Kac

Glucose-6-phosphate Lysine propionylation

Heavy Light

Peptide intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 43

Kpr Succinyl-CoA

Frutose-6-phosphate PFKA

SUCC

Succinate

Frutose-1,6-biphosphate

a-ketoglutarate

ALF

Dihydroxyacetone phosphate

IDH

Fumarate

TPIS FUMB

Glyceraldedehyde 3-phophate

isocitrate

Malate

G3P1

ACON1

1,3-bis-posphoglycerate PGK

Lysine acetylation

Modified peptides

Heavy

3-posphoglycerate

Light

2-posphoglycerate

oxaloacetate Acetyl-CoA

GPMA

ODP1

ENO

Phosphoenolpyruvate

Pyruvate KPYK1/2

Fig. 6 40 / 43


Citrate

Page 41 of 43

A

M9 +0.8% propionate M9 +0.8% propionate WT ΔcobB

WT

WT patZ OE WT patZ OE

ΔcobB

WT patZ OE

Marker WT patZ OE WT patZ OE WT patZ OE

165kD 125kD 93kD 72kD 57kD 42kD 31kD 24kD

Anti-Kpr

Loading control

Anti-Kpr

Propionyl peptide: ○

B 100

○

0 100

○

Loading control

C

Depropionylated peptide: ◊

Light & ΔlysA

Heavy & ΔlysAΔcobB

696.3622 m/z 696

698

◊

+ CobB: 10min

0 100

Protein extraction

◊

+ CobB: 30min 0 100

○ ◊

668.3488 m/z

+ CobB: 60min

668

0 0

5

10

Trypsin digestion

670

15

20

Time (min)

D Log10 peptide intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-4

9 8

Immunoprecipitation

7

HPLC-MS/MS

6 5 4 -3

-2

-1

0

1

2

3

4

5

6

Log2 Kpr ratio (H/L)

Table 1 41 / 43



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Positions

Protein name

Page 42 of 43

Gene name

Kpr ratio H/L

164

UPF0227 protein YcfP

ycfP

21.80

120

DNA-binding protein H-NS

hns

13.32

584

Succinate dehydrogenase flavoprotein subunit

sdhA

9.84

459

Tryptophanase

tnaA

9.47

gpmA

7.95

2,3-bisphosphoglycerate-dependent 113 phosphoglycerate mutase 79

30S ribosomal protein S3

rpsC

7.68

85

Protein GrpE

grpE

5.25

412

Pyruvate dehydrogenase E1 component

aceE

5.10

467

Tryptophanase

tnaA

4.35

24

Elongation factor Ts

tsf

4.02

169

Ribosome-recycling factor

frr

3.96

108

50S ribosomal protein L7/L12

rplL

3.46

88

Single-stranded DNA-binding protein

ssb

2.30

For TOC only 42 / 43


Page 43 of 43

≥5 sites 7.6% 7.2% P value =0.05 of difference

4 sites 3 sites

9.8% 1 site

56.0% 19.5% 2 sites

Propionyl peptide: ○ Depropionylated peptide: ◊ ○

100

Translation Carbohydrate catabolic process Glucose metabolic process tRNA aminoacylation Amino acid activation tRNA aminoacylation for protein… Glycolysis Alcohol catabolic process Coenzyme metabolic process Hexose metabolic process Aerobic respiration Pyruvate metabolic process Acetyl-CoA metabolic process Tricarboxylic acid cycle Gluconeogenesis Cellular amino acid derivative… Glyoxylate metabolic process

696.3622 m/z 696

0 100

698

○

+ CobB: 10min

0 100

◊ ◊

+ CobB: 30min 0 100

○ ◊

+ CobB: 60min

668.3488 m/z 668

0 0

5

10

15

670

20

Time (min)

0

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Biological process

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10 20 30 -log10 (Adjusted p value)

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Characterization of Protein Lysine Propionylation in Escherichia coli

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