Characterization of Protein Lysine Propionylation in Escherichia coli

Article pubs.acs.org/jpr

Characterization of Protein Lysine Propionylation in Escherichia coli: Global Profiling, Dynamic Change, and Enzymatic Regulation Mingwei Sun,†,‡ Junyu Xu,†,⊥ Zhixiang Wu,∥ Linhui Zhai,†,‡ Chengxi Liu,# Zhongyi Cheng,▽ Guofeng Xu,∥ Shengce Tao,# Bang-Ce Ye,⊥ Yingming Zhao,†,§ and Minjia Tan*,†,‡

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†

The Chemical Proteomics Center and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China § Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois 60637, United States ∥ Pediatric Surgery Department, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, P. R. China ⊥ Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China # Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, P. R. China ▽ Jingjie PTM BioLab (Hangzhou) Co. Ltd, Hangzhou 310018, P. R. China S Supporting Information *

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 massspectrometric-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 with lysine acetylation and succinylation, our results revealed that the 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 of 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. KEYWORDS: post-translational modification(s), lysine propionylation, SILAC, CobB, PatZ

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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, because of high chemical reactivity of ε-amino group in the lysine side chain, new types of protein lysine ε-Nacylation 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 acylCoA’s (propionyl-CoA, malonyl-CoA, succinyl-CoA, etc.) were © 2016 American Chemical Society

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 affinityenrichment-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 about the global substrates of lysine propionylation. Protein lysine propionylation was first identified in histones.3 Further study Received: September 1, 2016 Published: November 2, 2016 4696

DOI: 10.1021/acs.jproteome.6b00798 J. Proteome Res. 2016, 15, 4696−4708

Article

Journal of Proteome Research

Figure 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.

uptake of coenzyme B12 in cells. In methylcitrate cycle, propionyl-CoA is first condensed with oxaloacetate to form 2methylcitrate by 2-methylcitrate synthase (PrpC), which is further converted to pyruvate and succinate by multiple enzyme-catalyzed chemical reactions,12,14 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 to the inhibition of pyruvate dehydrogenase15 and citrate synthase,16,17 the molecular mechanisms by which propionyl-CoA exerts it toxic activity are still poorly understood. Therefore, characterization of the role of propionyl-CoA and protein propionylation in bacterial cellular pathways is likely to provide new insights into 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 massspectrometric-based proteomics, we identified 1467 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 that nonhistone substrates in mammals could also be propionylated.9 Eukaryotic lysine acetyltransferase p300/CBP and deacylase Sirt1 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 Gcn5-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 remain poorly understood. In bacteria, the cofactor of lysine propionylation propionylCoA is an intermediary metabolite mainly produced from two metabolic pathways (Figure 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 For propionyl-CoA degradation, there are also two major pathways in bacteria: 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 4697


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dride (NaBH 3CN) 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 concentration of 100 mM and incubated for another 4 h. The dimethylation labeling efficiency was >99%, as validated by MS analysis.

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

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MATERIALS AND METHODS

Materials

E. coli strain BW25113 and AT713 were obtained from the Coli Genetic Stock Center at Yale University (New Haven, CT). M9 minimal salt, isotopically labeled lysine (L-lysine-13C6 hydrochloride), isotopically labeled ariginine (L-ariginine-13C6, 15N4 hydrochloride), formaldehyde solution, formaldehyde-D2 solution, and other reagents were purchased at analytical grade or higher purity from Sigma-Aldrich. (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 (Billerica, MA). Pan-antipropionyllysine antibody was purchased from Jingjie PTM BioLab (Hangzhou, China).

High-pH HPLC Fractionation and Propionyllysine Peptide Enrichment

The tryptic peptides were preseparated by high-pH HPLC (bHPLC) to decrease the sample complexity.19,21 The tryptic peptides were preseparated on an Xbridge prep C18 column (5 μm, 19 × 150 cm, Waters, 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 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.

E. coli Cell Culture

Prototrophic E. coli BW25113 cells were cultured in Luria− Bertani (LB) medium for ∼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-antiacetyllysine antibody (Jingjie PTM BioLab, Hangzhou, China). The lysA knockout (ΔlysA) BW25113 E. coli cells were cultured in M9 medium containing light lysine (12C6-lysine) and 0.8% propionate, and the lysA and cobB knockout (ΔlysA and ΔcobB) BW2511 cells were cultured into M9 medium containing heavy lysine (13C6-lysine) and 0.8% propionate. The cells were cultured at 37 °C for ∼24 h and harvested at a comparable OD600 value of 1.2. The isotopic amino acid labeling efficiency was >98%, as validated by MS analysis. Three biological replicates were performed to identify the CobB’s substrates.

Nano-HPLC−MS/MS Analysis

The enriched peptides were dissolved in 4 μL of buffer A (0.1% formic acid in water, v/v) and loaded onto a 15 cm capillary C18 reversed-phase analytical column (3 μm particle size, 90 μm pore Å, Dikma Technologies, Lake Forest, CA) via the autosampler connected to an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific, 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, 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 1 × 106. The maximum ion injection time (IT) was 60 ms. 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 30 s, and the charge exclusion was set at 1+ and ⩾5+.

Preparation of Protein Lysate, TCA Precipitation, and Tryptic Digestion

E. coli cells were harvested and the whole cell lysate was prepared as described in the previous report.7 The cell pellet was resuspended in the lysis buffer (8 M 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.

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 maxima missed cleavage was set to 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

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 cyanoborohy4698


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Figure 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) 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.

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 Llysine-13C6 or L-lysine-13C6 and L-arginine-13C6,15N4. For the 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 were set to 0.01 for analyzing all data. All 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. 4699



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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.

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RESULTS

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 pan-anti-propionyllysine antibody and subjected to nano-HPLC−MS/MS analysis (Figure 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 461 propionylated proteins, we found that more than half of all of 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 (∼15%) were propionylated at four sites or more (Figure 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 glyceraldehyde3-phosphate dehydrogenase A (10 sites). To determine the sequence preference proximal to propionyllysine sites, we used iceLogo software to analyze their flanking sequence (Figure 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 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 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 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 precold 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 to 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 Zeba Spin Desalting Column, 7K MWCO (Thermo Fisher Scientific, Waltham, MA). Protein concentration was determined by BCA Protein Assay. The depropionylation enzymatic activity of CobB was determined with HPLC−MS/MS analysis. 2 μg purified CobB and 0.3 mM propionylated 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 of 10% TFA.

Functional Annotation Analysis of E. coli Propionylome

To understand the function of lysine propionylated substrates, we used DAVID online bioinformatic tool to perform enrichment analysis25 against KEGG pathway,26 Gene Ontology (GO),27 and Pfam domain28 databases. The result of GO analysis showed that propionylated substrates were highly enriched in multiple pathways mainly associated with energy metabolism and protein-processing pathways, such as translation and carbohydrate and amino acid metabolism (Figure 2A, Supplemental Table S2A−C). In the KEGG pathway analysis, ribosome, glycolysis/gluconeogenesis, and pyruvate metabolism were among the most highly enriched pathways (Figure 2B, Supplemental Table S2D). Interestingly, 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 Figure S1). Consistent with GO and KEGG analysis, Pfam domain analysis showed the enrichment of ribosomerelated S4 domain and S1 RNA binding domain (Figure 2C, Supplemental Table S2E), suggesting a role of propionylation in ribosome-associated protein translation process. We further used the STRING database29 to identify the protein−protein interaction network of propionylome. Of all 461 substrates, we identified 15 protein−protein highly connected interaction networks (Figure 2D, Supplemental Figure S2 and Supplemental Table S3). We found that the top five clusters were mainly associated with ribosome, glycolysis/gluconeogenesis, aminoacyl-tRNA biosynthesis, and pyruvate metabolism.

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 Jolla, 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) 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. 4700


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Figure 3. Quantitative proteomic analysis of E. coli lysine propionylome in response to sodium propionate. (A) Workflow showing the procedure for quantitative proteomic analysis of E. coli lysine propionylome in response to sodium propionate. (B) 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 KEGG pathways of upregulated lysine propionylated proteins in response to propionate. Substrates with a ratio of (H/L) < 0.5 and substrates identified in light-only form were used for KEGG enrichment analysis. (D) Identified propionylated substrates involved in propionate degradation and methylcitrate cycle.

Dynamics of Lysine Propionylation in Response to Propionate

lome 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, and tyrosine and tryptophan biosynthesis pathway (Figure 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 propionylCoA synthetase (PrpE, 1 Kpr site), acetyl-CoA synthetase (ACS, 3 Kpr sites), 2-methylcitrate synthase (PrpC, 4 Kpr sites), 2-methylcitrate dehydratase (PrpD, 3 Kpr sites), and 2methylisocitrate lyase (PrpB, 3 Kpr sites) (Figure 3D, Supplemental Figure S3). In E. coli, propionate can be converted to propionyl-CoA for further utilization by two enzymes, propionyl-CoA synthetase and acetyl-CoA synthetase (Figure 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

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 perform 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) supplemented with 0.8% sodium propionate or M9 medium supplemented with 13C6-lysine and 13 C6, 15N4-arginine (heavy) (Figure 3A). We then carried out affinity enrichment and MS 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 (Figure 3B, Supplemental Table S4A−C). 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 98%, as validated by MS analysis (Supplemental Figure 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 (Figure 6D, Supplemental Table S8C). Among them, 13 lysine propionylation sites were identified in at least two replicates with more than 2-fold 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, and so on.

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DISCUSSION Lysine propionylation belongs to the expanded landscape of lysine acylation modifications in bacteria. This study leads to the most comprehensive propionylome data set in bacteria to date. In this study, we identified 956 Kpr sites in LB medium culture experiment (Figure 1), 713 Kpr sites in propionatesupplemented experiment (Figure 3), 126 Kpr sites in glucosesupplemented M9 medium experiment (Figure 5A,B), 166 Kpr sites in supplemented LB medium experiment (Figure 5C,D), and 313 Kpr sites in the CobB substrate identification experiment (Figure 6). Taken together, we identified a total 4705


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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 showed obvious change in response to cobB knockdown, suggesting that CobB could have substrate preference toward 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 eight overlapping upregulated propionylated and acetylated sites in cobB knockout strain (Supplemental Table S8D) between our propionylome data and their acetylome data. In addition, we identified five up-regulated (increase of more than 2-fold) propionylation sites in cobB knockout strain that were not reported in the acetylation data set.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 plays 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 acetylCoA 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 nonenzymatically manner in E. coli. Together, our results revealed the broad propionylation substrates, their dynamics in response to different nutritional conditions, and their potential regulatory enzymes in E. coli, suggesting new roles of lysine propionylation in bacterial physiology.

Table 1. List of 13 Propionylation Sites with More than Two Fold Increase in cobB Knockout Strain positions

protein name

164 120 584

UPF0227 protein YcfP DNA-binding protein H-NS succinatedehydrogenase flavoprotein subunit tryptophanase 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 30S ribosomal protein S3 protein GrpE pyruvatedehydrogenase E1 component tryptophanase elongation factor Ts ribosome-recycling factor 50S ribosomal protein L7/L12 single-stranded DNA-binding protein

459 113 79 85 412 467 24 169 108 88

gene name

Kpr ratio H/L

ycf P hns sdhA

21.80 3.32 9.84

tnaA gpmA

9.47 7.95

rpsC grpE aceE tnaA tsf f rr rplL ssb

7.68 5.25 5.10 4.35 4.02 3.96 3.46 2.30

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 contrary, 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 that 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 that protein lysine propionylation is likely to play an important role in bacterial propionate metabolism and suggested a new mechanism of the antimicrobial activity of propionate as a food preservative. Our previous data showed the dramatic increase in lysine acetylation and succinylation in response to glucose stimulation.7 In stark contrast with these lysine acylation modifications, we found that propionylation substrates predominantly existed under lower glucose condition. In particular, acetylation and propionylation level of the same sites of the enzyme-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

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00798. Supplemental Figure S1. Representative enriched KEGG pathways of lysine propionylated proteins. Supplemental Figure S2. Complete Kpr interaction network. Supplemental Figure S3. Propionylated proteins involved in propionate metabolism pathway. Supplemental Figure S4. Spatial position of lysine propionylation within threedimensional structure of PrpC. Supplemental Figure S5. Information on overlapping sites between the Kpr and Kac in response to high glucose. Supplemental Figure 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 Figure S7. SILAC labeling efficiency test result. Supplemental Table S1. List of lysine propionylation (Kpr) sites identified in LB medium experiment. Supplemental Table S2. Functional annotation analysis 4706


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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. Supplemental Table S5. Functional annotation comparison between Kpr sites identified in LB medium and in propionate condition experiment. Supplemental Table S6. Lysine propionylome data got in SILAC medium with or without glucose experiment. Supplemental Table S7. Lysine propionylome data and acetylome data got in LB medium with or without glucose experiment. Supplemental Table S8. Lysine propionylome data for identifying CobB regulated substrate experiment. Supplemental Table S9. Total Kpr sites identified in this study. (ZIP)

AUTHOR INFORMATION

Corresponding Author

*Tel: 86-21-50800172. Fax: 86-21-50806600. E-mail: mjtan@ simm.ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program) (No. 2014CBA02004), the Natural Science Foundation of China (Nos. 31670066 and 31370813), the National Science & Technology Major Project (No. 2014ZX09507-002), the Shanghai Municipal Science and Technology Commission (Nos. 14DZ2261100, 16YF1414000, and 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 Institutes of Health (NIH) of the United States (GM105933 and CA160036).

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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 2hydroxyisobutyrylation; 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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Characterization of Protein Lysine Propionylation in Escherichia coli

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