Global Insights Into Lysine Acylomes Reveal Crosstalk Between Lysine Acetylation and Succinylation in Streptomyces coelicolor Metabolic Pathways

• DOI: 10.1016/j.mcpro.2021.100148
• Publication Date: 2021-09-14T06:25:51Z
• Authors: Yujiao Yang; Hong Zhang; Zhenyang Guo; Siwei Zou; Fei Long; Jiacheng Wu; Peng Li; Guo-ping Zhao; Wei Zhao
• Keywords: Succinylation; Crosstalk; Acetyltransferases

•A highly abundant and dynamic acetylation is discovered in Streptomyces coelicolor.•Quantitative acetylome succinylome analyses coelicolor.•The bimodification proteins are enriched multiple metabolic pathways. Lysine acylations reversible ubiquitous post-translational modifications that play critical roles regulating cellular processes. In the current study, acetylation, besides succinylation, was uncovered a soil bacterium, coelicolor. By affinity enrichment using anti–acetyl-lysine antibody following LC−MS/MS analysis, total of 1298 sites among 601 were identified. Bioinformatics suggested these acetylated have diverse subcellular localization wide range biological functions. Specifically, majority also succinylated tricarboxylic acid cycle protein translation pathways, occurred at same some proteins. The succinylation quantified by knocking out either deacetylase ScCobB1 or desuccinylase ScCobB2, demonstrating possible competitive relationship between two acylations. Moreover, vitro experiments synthetically modified peptides confirmed regulatory crosstalk sirtuins, which may be involved collaborative regulation cell physiology. Collectively, results provided global insights into S. coelicolor acylomes laid foundation for characterizing lysine future. (PTMs) such as function efficient mechanisms fine-tune activity, stability, localization, others (1Walsh C.T. Garneau-Tsodikova Gatto Jr., G.J. Protein posttranslational modifications: chemistry proteome diversifications.Angew. Chem. Int. Ed. Engl. 2005; 44: 7342-7372Crossref PubMed Scopus (939) Google Scholar, 2Wang Z.A. Cole P.A. chemical biology modifications.Cell Biol. 2020; 27: 953-969Abstract Full Text PDF (21) Scholar). Among 20 amino residues proteins, (K) one most frequent targets different PTMs. With rapid development high-resolution MS immunoaffinity purification technology, on lysine, including (3Wang Q. Zhang Y. Yang C. Xiong H. Lin Yao J. Li Xie L. Zhao W. Ning Z.B. Zeng R. 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Our recent work evolutionary divergent (ScCobB2), known deacetylase, providing suitable model study bacteria (22Zhang ScCobB2 other previously (30Mikulik Felsberg Kudrnacova Bezouskova Setinova Stodulkova Zidkova Zidek CobB1 coelicolor.Biochem. 2012; 90: 179-187Crossref (28) shown ParB Here, we presented profile combining methods high-accuracy MS. A large portion found pathways TCA translation. interplay globally explored further tested treatments our metabolism. wildtype M145 cultured tryptic soy broth (TSB) liquid medium (50 mM glucose plus) solid 30 °C. BL21 (DE3) used host strain express pan anti–succinyl-lysine antibodies this obtained Jingjie PTM Biolab (Hangzhou) Co Ltd. unmodified, acetylated, (listed supplemental Table S1) synthesized Guoping Pharmaceutical (Hefei), >98% purity. To determine coelicolor, sample three replicates technical prepared analyzed Q Exactive HF-X (Thermo Fisher Scientific). quantitative succinylome, samples (wildtype, ΔSccobB1, ΔSccobB2) label-free Similar procedures followed culture, preparation, peptide enrichment, LC–MS/MS analysis. paired t tests where p < 0.05 considered significant. Gene Ontology (GO)/Kyoto Encyclopedia Genes Genomes (KEGG) pathway/domains corrected harvested interval 1 day during 6-day cultivation, whole then extracted. Approximately 15 μg separated 12% SDS-PAGE visualized Coomassie brilliant blue staining. For Western blotting assay, similar amounts transferred onto nitrocellulose membranes, standard detection levels. Briefly, 100 Tris–HCl (pH 7.5) 0.5% (v/v) Tween-20 1% peptone blocking, 0.05% 0.1% primary secondary buffer. membranes incubated overnight dilution 1:1000 buffer 4 Nitrocellulose treated ECL Chemiluminescent Substrate reagent ImageQuant LAS 4000 mini (GE Healthcare). levels derivatives ΔSccobB1 ΔSccobB2. 1:2000 studies. ΔSccobB2 °C 3 days. Fresh spores collected cotton swab TSB supplemented 50 glucose. After growing 60 h, (10%) containing log phase after 2 frozen grinded powder nitrogen 50-ml centrifuge tubes sonication lysis (8 M urea, EDTA, 5 DTT, Protease Inhibitor Cocktail III, μM trichostatin A, nicotinamide). debris removed centrifugation 12,000g min. Finally, precipitated cold 15% trichloroacetic h −20 min, supernatant discarded. remaining precipitate washed acetone thrice. dissolved TEAB, pH 8.0), concentration determined 2-D Quant kit Healthcare) according manufacturer's instructions. digestion, slowly make final 20% mixture eddy mixed h. discarded 4500g precooled times. dried, 200 mM, added. dispersed ultrasonication, trypsin mass ratio 1:50 (trypsin protein) overnight. Then, solution reduced DTT min 37 alkylated 11 iodoacetamide room temperature dark. enrich Kac Ksuc peptides, immunoprecipitation Tris–HCl, NaCl, NP-40; 8.0) prewashed beads (PTM Biolabs) gentle shaking, respectively. four times twice double-distilled water. bound eluted trifluoroacetic acid. fractions vacuum resulting desalted C18 ZipTips (Millipore) instructions, mobile LC EASY-nLC 1200 UPLC system. Mobile aqueous formic 2% acetonitrile. B 90% Liquid gradient settings 0 36 9 25% B; 54 25 35% 57 35 80% flow rate maintained 500 nl/min. hybrid quadrupole-Orbitrap spectrometer subjected nanospray ionization MS/MS coupled online UPLC. Intact detected Orbitrap resolution 120,000. Peptides selected normalized collision setting 28, ion fragments 17,500. data-dependent procedure alternated scan scans applied top precursor ions above threshold count 5E4 survey 15-s exclusion. electrospray voltage 2.1 kV. Automatic gain control prevent overfilling trap, 1E5 accumulated generate spectra. scans, m/z 350 1600. fixed set m/z. peak list generated Xcalibur 4.2.47 Fisher). data processed MaxQuant (version 1.6.15.0, Max Planck Institute Biochemistry) integrated Andromeda search engine. Tandem spectra searched against local UniProt database concatenated reverse decoy (Blast_Streptomyces_coelicolor_strain_ATCC_BAA-471_100226_PR_20201113.fasta; release date, November 13, 8038 sequences). Trypsin/P specified cleavage allowing up missing cleavages, five per peptide, charges. error tolerance ppm search, 4.5 main fragment ions. Carbamidomethylation Cys modification, oxidation Met, Lys, N termini variable modifications. false discovery thresholds protein, 1%. minimum length 7. All parameters default values. site probability >0.75. GO derived UniProt-GOA (http://www.ebi.ac.uk/GOA/) annotate functions acetylome. KEGG identify GO/KEGG Functional Annotation Tool DAVID Resources (31Huang D.W. Sherman Kir Bryant Guo Stephens Baseler M.W. Lane H.C. Lempicki R.A. Resources: Expanded annotation algorithms better extract lists.Nucleic 35: W169-175Crossref (1350) InterPro (32Jones Binns Chang H.Y. Fraser McAnulla McWilliam Maslen Mitchell Nuka Pesseat Quinn A.F. Sangrador-Vegas Scheremetjew Yong al.InterProScan 5: Genome-scale classification.Bioinformatics. 30: 1236-1240Crossref (3241) background proteome. two-tailed Fisher's exact test employed enrichments. 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Demchak Bader G.D. exploration 3.Curr. 47: 11-24Crossref (432) graph clustering algorithm, molecular complex (MCODE) (38Bader Hogue C.W. An automated method finding complexes networks.BMC 2003; 4: 2Crossref (3324) utilized densely connected regions. MCODE plug-in kit, Cytoscape. plasmids pET28b-ScCobB1/ScCobB2/CobBL constructed correct checked PCR transformed BL21. single colony picked 5-ml LB kanamycin 1:100 diluted 500-ml When absorbance 600 nm reached about 0.6, 16 induced 0.5 IPTG 6000 rpm suspended 150 10% glycerol, imidazole. disruptor, 13,000g supernatants loaded nickel resin column washing volumes wash (25 imidazole; (elution buffer, 8.0). AKTA system Healthcare), quantified, aliquoted, stored −80 deacylation contained Tris 1.0 6.0 MgCl2, 10.8 3.6 ScCobB2/CobBL, NAD+, 540 peptide. carried 50-μl volume, quenched volume temperature. centrifuged discard sediment, HPLC (Agilent Technologies) Aeris XB-C18 (150 × 4.6 mm, μm; Phenomenex). consisted solvent (0.1% HPLC-grade water) (HPLC-grade acetonitrile). linear 100% ml min−1 over °C, monitored UV light wavelength 215 nm. activities ScCobB1, CobBL calculating area follows:Deacylationpercentage=DeacylationDeacylation+acylation representative group soil-dwelling microorganisms notable lifecycles antibiotic production capacity (39Bentley S.D. Chater K.F. Cerdeno-Tarraga A.M.