Lysine Acetylation Is a Highly Abundant and Evolutionarily Conserved Modification in Escherichia Coli
2008; Elsevier BV; Volume: 8; Issue: 2 Linguagem: Inglês
10.1074/mcp.m800187-mcp200
ISSN1535-9484
AutoresJunmei Zhang, Robert W. Sprung, Jimin Pei, Xiaohong Tan, Sungchan Kim, Heng Zhu, Chuan‐Fa Liu, Nick V. Grishin, Yingming Zhao,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoLysine acetylation and its regulatory enzymes are known to have pivotal roles in mammalian cellular physiology. However, the extent and function of this modification in prokaryotic cells remain largely unexplored, thereby presenting a hurdle to further functional study of this modification in prokaryotic systems. Here we report the first global screening of lysine acetylation, identifying 138 modification sites in 91 proteins from Escherichia coli. None of the proteins has been previously associated with this modification. Among the identified proteins are transcriptional regulators, as well as others with diverse functions. Interestingly, more than 70% of the acetylated proteins are metabolic enzymes and translation regulators, suggesting an intimate link of this modification to energy metabolism. The new dataset suggests that lysine acetylation could be abundant in prokaryotic cells. In addition, these results also imply that functions of lysine acetylation beyond regulation of gene expression are evolutionarily conserved from bacteria to mammals. Furthermore, we demonstrate that bacterial lysine acetylation is regulated in response to stress stimuli. Lysine acetylation and its regulatory enzymes are known to have pivotal roles in mammalian cellular physiology. However, the extent and function of this modification in prokaryotic cells remain largely unexplored, thereby presenting a hurdle to further functional study of this modification in prokaryotic systems. Here we report the first global screening of lysine acetylation, identifying 138 modification sites in 91 proteins from Escherichia coli. None of the proteins has been previously associated with this modification. Among the identified proteins are transcriptional regulators, as well as others with diverse functions. Interestingly, more than 70% of the acetylated proteins are metabolic enzymes and translation regulators, suggesting an intimate link of this modification to energy metabolism. The new dataset suggests that lysine acetylation could be abundant in prokaryotic cells. In addition, these results also imply that functions of lysine acetylation beyond regulation of gene expression are evolutionarily conserved from bacteria to mammals. Furthermore, we demonstrate that bacterial lysine acetylation is regulated in response to stress stimuli. Lysine acetylation is a dynamic, reversible, and regulatory post-translational modification in mammalian cells. Lysine acetylation status and its regulatory enzymes have been shown to influence several fundamental cellular pathways in mammalian cells, including cell survival and apoptosis, cellular differentiation, and metabolism. Dysregulation of the modification is associated with aging (1Haigis M.C. Guarente L.P. 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Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding.J. Biol. Chem. 2003; 278: 26071-26077Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In Salmonella enterica, the lysine acetylation status of acetyl-CoA synthetase is regulated by CobB deacetylase, a Sir2 homolog in bacteria (21Starai 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: 2390-2392Crossref PubMed Scopus (469) Google Scholar, 26Blander G. Guarente L. The Sir2 family of protein deacetylases.Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1299) Google Scholar), as well as Pat acetyltransferase (27Starai V.J. Escalante-Semerena J.C. Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica.J. Mol. 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The proteomics study involves efficient affinity enrichment of lysine-acetylated, tryptic peptides with anti-acetyllysine antibodies and subsequent peptide identification for nano-HPLC/mass spectrometric analysis. The screening identified 138 lysine acetylation sites in 91 proteins in E. coli, of which 25% had mammalian orthologues. Our results suggest that diverse groups of bacterial proteins are the substrates of lysine acetylation, including metabolic enzymes, stress response proteins, and transcription and translation factors. The lysine acetylation substrates in E. coli are highly enriched in metabolic enzymes (∼53%) and proteins involved in translation (∼22%), two processes that are intimately linked to cellular energy status. These data therefore reveal previously unappreciated roles of lysine acetylation in the regulation of prokaryotic biochemical pathways and imply that DNA-independent functions of lysine acetylation are evolutionarily conserved from prokaryotic to eukaryotic cells. The reagents used in this work include Protein A-conjugated-agarose beads from Amersham Biosciences; Luria-Bertani (LB) medium from Invitrogen; iodoacetamide, C18 ziptips from Millipore Corp. (Bedford, MA); Luna C18 resin from Phenomenex (Torrance, CA). E. coli strains MG1655 and JW1106 were both acquired from the Coli Genetic Resource Center at Yale University. MG1655 is the wild type, and JW1106 is a CobB-deficient single-gene knockout of the Keio Collection (29Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.Mol. Syst. Biol. 2006; 2 (2006.0008)Crossref Scopus (5382) Google Scholar). Two anti-acetyllysine antibodies were used: an affinity-purified anti-acetyllysine polyclonal antibody from ImmuneChem Pharmaceuticals Inc. (Burnaby, British Columbia, Canada) and an anti-acetyllysine monoclonal antibody from Cell Signaling Technology (Boston, MA). Because the two antibodies were generated using different antigens (acetyllysine peptide library or acetyllysine residue, please see the vendor information for details) and purified differently (30Qiang L. Xiao H. Campos E.I. Ho V.C. Li G. Development of a PAN-specific, affinity-purified anti-acetylated lysine antibody for detection, identification, isolation, and intracellular localization of acetylated protein.J. Immunoassay Immunochem. 2005; 26: 13-23Crossref PubMed Scopus (14) Google Scholar, 31Zhang H. Zha X. Tan Y. Hornbeck P.V. Mastrangelo A.J. Alessi D.R. Polakiewicz R.D. Comb M.J. Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs.J. Biol. Chem. 2002; 277: 39379-39387Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), they would likely have different binding specificities. Accordingly, a more diverse panel of acetyllyisne substrate peptides would be identified by using two antibodies for the described proteomics screening. E. coli DH5 was grown aerobically in LB medium at 37 °C. The cultured cells were harvested during the exponential growth phase by centrifugation at 4500 × g for 10 min and washed twice by resuspension of the pellet in ice-cold phosphate-buffered saline buffer (0.1 m Na2HPO4, 0.15 m NaCl, pH 7.2). The cells were resuspended in chilled lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 5 mm dithiothreitol) and then sonicated with 12 short bursts of 10 s followed by intervals of 30 s for cooling. Unbroken cells and debris were removed by centrifugation at 4 °C for 30 min at 21,000 × g. The supernatant was divided into aliquots and stored at −80 °C until use. Five milligrams of proteins were precipitated with acetone, followed by centrifugation at 22,000 × g for 10 min. The resulting pellet was digested according to a previously described procedure (32Kim S.C. Chen Y. Mirza S. Xu Y. Lee J. Liu P. Zhao Y. A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea.J. Proteome Res. 2006; 5: 3446-3452Crossref PubMed Scopus (83) Google Scholar). The protein pellet was rinsed twice with cold acetone to remove residual salts, resuspended in 50 mm NH4HCO3 (pH 8.5) (the protein pellet was not completely re-dissolved but rather was suspended as small particles), and digested with trypsin (Promega, Madison, WI) at an enzyme-to-substrate ratio of 1:50 for 16 h at 37 °C to enhance the solubility of the proteins prior to reduction and alkylation. The tryptic peptides were reduced with 5 mm dithiothreitol at 50 °C for 30 min and then alkylated using 15 mm iodoacetamide at ambient temperature for 30 min in darkness. The reaction was terminated with 15 mm cysteine at ambient temperature for 30 min. To ensure complete digestion, additional trypsin at an enzyme-to-substrate ratio of 1:100 was added to the peptide mixture, and the mixture was incubated for an additional 3 h. The anti-acetyllysine antibodies from ImmuneChem Pharmaceuticals Inc. and Cell Signaling Technology were mixed at a ratio of 1:1 and then immobilized on protein A-conjugated-agarose beads at 4–6 mg/ml by incubation at 4 °C for 4 h. The supernatant was removed, and the beads were washed three times with NETN buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40). The tryptic peptides obtained from in-solution digestion were re-dissolved in NETN buffer. Insoluble particles were removed by centrifugation. Affinity purification was carried out by incubating the peptides with 20 μl of anti-acetyllysine antibody protein A-immobilized-agarose beads at 4 °C for 6 h with gentle shaking. The beads were washed three times with 1 ml of NETN buffer and twice with ETN (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA). The bound peptides were eluted from the beads by washing three times with 50 μl of 1% trifluoroacetic acid. The eluates were combined and dried in a SpeedVac. The resulting peptides were cleaned with C18 ZipTips (Millipore Corp.) according to the manufacturer's instructions, prior to nano-HPLC/mass spectrometric analysis. HPLC/MS/MS analysis was performed in an integrated system that includes an Agilent 1100 series nanoflow liquid chromatography system (Agilent, Palo Alto, CA) and an LTQ two-dimensional trap mass spectrometer (Thermo Electron, Waltham, MA) equipped with a nanoelectrospray ionization source. One μl of tryptic peptides in buffer A (97.95% water/2% acetonitrile/0.05% acetic acid) was manually injected and separated in a capillary HPLC column (11 cm length × 75 μm inner diameter) packed in-house with Luna C18 resin (5 μm particle size, 100 Å pore diameter) (Phenomenex). Peptides were eluted from the column with a gradient of 6.0% to 90% buffer B (90% acetonitrile/9.95% water/0.05% acetic acid) in a 2 h LC/MS/MS analysis. The eluted peptides were electrosprayed directly into the LTQ ion trap mass spectrometer. Liquid chromatography tandem mass spectrometry was operated in a data-dependent mode such that the ten strongest ions in each MS scan were subjected to collisionally activated dissociation with a normalized collisionally activated dissociation energy of 35%. Tandem mass spectra were used to search the E. coli entries (51,059 E. coli sequences) of the NCBI-nr database (updated July 31th, 2006 with a total of 3,841,279 sequences). Only the E. coli subset of the database was used for search because we are only interested in E. coli acetylation in the current study and all the cell lysate was from E. coli. The search engine MASCOT (version 2.1, Matrix Science, London, UK) was used for database search, and extract_msn.exe version 4.0 was used for peaklist generation. A low cutoff of peptide score 20 was selected to maximize the identification of lysine-acetylated peptides. Trypsin was specified as the proteolytic enzyme, and up to 6 missed cleavage sites per peptide were allowed. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine and acetylation of lysine as variable modifications. Charge states of +1, +2, or +3 were considered for parent ions. Mass tolerance was set to ±4.0 Da for parent ion masses and ± 0.6 Da for fragment ion masses. Acetylated lysine containing peptides identified with a MASCOT score of 25 were manually verified by the method described previously (33Chen Y. Kwon S.W. Kim S.C. Zhao Y. Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra.J Proteome Res. 2005; 4: 998-1005Crossref PubMed Scopus (158) Google Scholar). E. coli MG1655 and JW1106 were grown, harvested, and lysed as described above. Protein concentration was determined using the Bradford assay (Bio-Rad). Forty μg of proteins from E. coli cells was resolved by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% milk at ambient temperature for 1 h. Then the membrane was incubated with anti-acetyllysine monoclonal antibody (0.8 μg/ml in TBST (25 mm Tris-HCl, pH 8.0 125 mm NaCl, 0.1% Tween 20) with 3% BSA 1The abbreviations used are: BSA, bovine serum albumin; MS, mass spectroscopy; CoA, coenzyme A; HPLC, high performance liquid chromatography; LC/MS/MS, liquid chromatography tandem mass spectrometry. ; from Cell Signaling Technology) overnight at 4 °C. After washing with TBST four times for 5 min each, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse IgG (1 μg/ml in TBST with 3% BSA) at ambient temperature for 2 h. The ECL system (PerkinElmer Life Sciences) was used for signal detection. To carry out the competition experiment, the anti-acetylated lysine antibody was pre-incubated with 3% BSA (acetylated or non-acetylated) at ambient temperature for 2 h before it was incubated with the membrane. Hypoxic treatment of E. coli was carried out as described previously (34Weiss B. Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli.J. Bacteriol. 2006; 188: 829-833Crossref PubMed Scopus (56) Google Scholar). E. coli MG1655 was grown in LB medium in 125-ml flasks. When its optical density (A600) at 600 nm reached 0.3, it was exposed to one of the following conditions: nitrogen only, 5% or 25% air. For these three experiments, nitrogen was bubbled through the cell suspension for 15 min, and the flasks were sealed tightly immediately. For the latter two experiments, 5% or 25% air was then introduced back to the flasks by a syringe needle, and further sealing was applied immediately. The cells were allowed to continue to grow until their A600 reached 0.6. The cells were harvested and prepared for Western blotting analysis as described above. Starvation of E. coli was carried out as described (35Santos J.M. Freire P. Vicente M. Arraiano C.M. The stationary-phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth.Mol. Microbiol. 1999; 32: 789-798Crossref PubMed Scopus (114) Google Scholar). E. coli MG1655 was grown in LB medium as described above. When its A600 reached 0.3, starvation was imposed by sudden depletion of all carbon sources as follows: cells were centrifuged at 4500 × g for 10 min at 4 °C and washed twice with two volumes of sterile ice-cold M9 minimal medium (no carbon source). The cells were resuspended in the same volume of M9 minimal medium and incubated overnight. The cells were harvested and prepared for Western blotting analysis as described above. For each acetylated E. coli protein, BLAST was run against a database of domain sequences with known structures from the SCOP90 representative set of ASTRAL compendium (version 1.71) (36Chandonia J.M. Hon G. Walker N.S. Lo Conte L. Koehl P. Levitt M. Brenner S.E. The ASTRAL compendium in 2004.Nucleic Acids Res. 2004; 32: D189-D192Crossref PubMed Google Scholar, 37Murzin A.G. Brenner S.E. Hubbard T. Chothia C. SCOP: a structural classification of proteins database for the investigation of sequences and structures.J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5589) Google Scholar). Database size of BLAST was set to the size of the protein nr database as of April 6th, 2007 (39,280,211,952 letters) to impose a stringent E-value cutoff. Hits with an E-value less than 0.001 were analyzed. We identified homologous structures for 69 of the 91 acetylated proteins (∼76%). For these proteins, we mapped the positions of acetylated lysines to the model structures using BLAST local alignments and visually inspected the crystal structures to determine the role of the conserved lysine in substrate and protein binding or catalytic activity. Lysine acetylation is more difficult to identify by a candidate approach than protein phosphorylation due to the low radioactivity of [14C]acetyl-CoA and the weak binding affinity of anti-acetyllysine antibody. A lack of protein substrates represents one of the major bottlenecks for characterization of its biological functions. To begin the systematic study of lysine acetylation in prokaryotes, we carried out the first proteomic screening of lysine-acetylated substrates in bacteria. The goals of this study were (i) to determine the spectrum and extent of lysine acetylation in bacteria; (ii) to identify novel lysine acetylation substrates and lysine acetylation sites that could provide candidate proteins for further functional studies; and (iii) to define the molecular pathways that are likely to be affected by lysine acetylation. The proteomics of lysine acetylation was carried out as previously reported (11Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar) and consisted of four steps: (i) the protein lysate of E. coli was proteolytically digested by trypsin; (ii) The resulting tryptic peptides were subjected to affinity purification by anti-acetyllysine antibody, (iii) the isolated, lysine-acetylated peptides were then analyzed by nano-HPLC/MS/MS for peptide identification and precise localization of lysine acetylation sites; and (iv) the peptide candidates were further manually evaluated to ensure the accuracy of the identification (Fig. 1, A and C). The raw spectrum of each acetylated peptide can be found in supplemental Table S1. The strategy described here, integration of immunoisolation with mass spectrometry for characterization of biomolecules, can be traced back more than seventeen years ago (38Suckau D. Kohl J. Karwath G. Schneider K. Casaretto M. Bitter-Suermann D. Przybylski M. Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping.Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9848-9852Crossref PubMed Scopus (151) Google Scholar). Peptides released from immunoisolated complexes have been analyzed by both MALDI-TOF mass spectrometry and electrospray tandem mass spectrometry for epitope mapping (39Zhao Y. Chait B.T. Protein epitope mapping by mass spectrometry.Anal. Chem. 1994; 66: 3723-3726Crossref PubMed Scopus (106) Google Scholar, 40Papac D.I. Hoyes J. Tomer K.B. 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In addition to sequence-specific antibodies, pan-antibodies, such as anti-phosphotyrosine antibody, have also been used to isolate and to identify tyrosine-phosphorylated proteins on a global scale in response to extracellular stimulation (43Pandey A. Podtelejnikov A.V. Blagoev B. Bustelo X.R. Mann M. Lodish H.F. Analysis of receptor signaling pathways by mass spectrometry: identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 179-184Crossref PubMed Scopus (375) Google Scholar, 44Pandey A. Fernandez M.M. Steen H. Blagoev B. Nielsen M.M. Roche S. Mann M. Lodish H.F. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways.J. Biol. Chem. 2000; 275: 38633-38639Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Isolation of modified peptides from tryptic digests by immunoaffinity purification using a pan-antibody is much simpler than that of the corresponding proteins for three obvious reasons. First, the modified residue will not be buried in peptides. In contrast, the modified residue may not be accessible for antibody binding in the context of proteins due to protein-folding or non-covalent interactions (e.g. phosphotyrosine with SH2 domain). Second, a protein typically has more complex domains
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