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• W2766171218 abstract "Nε-lysine acetylation represents a highly dynamic and reversibly regulated post-translational modification widespread in almost all organisms, and plays important roles for regulation of protein function in diverse metabolic pathways. However, little is known about the role of lysine acetylation in photosynthetic eukaryotic microalgae. We integrated proteomic approaches to comprehensively characterize the lysine acetylome in the model diatom Phaeodactylum tricornutum. In total, 2324 acetylation sites from 1220 acetylated proteins were identified, representing the largest data set of the lysine acetylome in plants to date. Almost all enzymes involved in fatty acid synthesis were found to be lysine acetylated. Six putative lysine acetylation sites were identified in a plastid-localized long-chain acyl-CoA synthetase. Site-directed mutagenesis and site-specific incorporation of N-acetyllysine in acyl-CoA synthetase show that acetylation at K407 and K425 increases its enzyme activity. Moreover, the nonenzymatically catalyzed overall hyperacetylation of acyl-CoA synthetase by acetyl-phosphate can be effectively deacetylated and reversed by a sirtuin-type NAD+-dependent deacetylase with subcellular localization of both the plastid and nucleus in Phaeodactylum. This work indicates the regulation of acyl-CoA synthetase activity by site-specific lysine acetylation and highlights the potential regulation of fatty acid metabolism by lysine actetylation in the plastid of the diatom Phaeodactylum. Nε-lysine acetylation represents a highly dynamic and reversibly regulated post-translational modification widespread in almost all organisms, and plays important roles for regulation of protein function in diverse metabolic pathways. However, little is known about the role of lysine acetylation in photosynthetic eukaryotic microalgae. We integrated proteomic approaches to comprehensively characterize the lysine acetylome in the model diatom Phaeodactylum tricornutum. In total, 2324 acetylation sites from 1220 acetylated proteins were identified, representing the largest data set of the lysine acetylome in plants to date. Almost all enzymes involved in fatty acid synthesis were found to be lysine acetylated. Six putative lysine acetylation sites were identified in a plastid-localized long-chain acyl-CoA synthetase. Site-directed mutagenesis and site-specific incorporation of N-acetyllysine in acyl-CoA synthetase show that acetylation at K407 and K425 increases its enzyme activity. Moreover, the nonenzymatically catalyzed overall hyperacetylation of acyl-CoA synthetase by acetyl-phosphate can be effectively deacetylated and reversed by a sirtuin-type NAD+-dependent deacetylase with subcellular localization of both the plastid and nucleus in Phaeodactylum. This work indicates the regulation of acyl-CoA synthetase activity by site-specific lysine acetylation and highlights the potential regulation of fatty acid metabolism by lysine actetylation in the plastid of the diatom Phaeodactylum. Diatoms are a highly diverse group of eukaryotic unicellular microalgae living in marine and freshwater environments and are considered to be responsible for one-fourth of global primary productivity (1.Falkowski P.G. Barber R.T. Smetacek V.V. Biogeochemical controls and feedbacks on ocean primary production.Science. 1998; 281: 200-207Crossref PubMed Scopus (1835) Google Scholar, 2.Field C.B. Behrenfeld M.J. Randerson J.T. Falkowski P. Primary production of the biosphere: integrating terrestrial and oceanic components.Science. 1998; 281: 237-240Crossref PubMed Scopus (3800) Google Scholar). Photosynthetic diatoms are of ecological significance in aquatic ecosystems and the global carbon cycle, and some diatoms are considered as promising sources of renewable and sustainable biodiesel (3.Courchesne N.M. Parisien A. Wang B. Lan C.Q. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches.J. Biotechnol. 2009; 141: 31-41Crossref PubMed Scopus (431) Google Scholar) as they can accumulate large amounts of triacylglycerols when their growth is limited by nutrients. The pennate diatom Phaeodactylum, a model diatom species with a sequenced genome (4.Bowler C. Allen A.E. Badger J.H. Grimwood J. Jabbari K. Kuo A. Maheswari U. Martens C. Maumus F. Otillar R.P. Rayko E. Salamov A. Vandepoele K. Beszteri B. Gruber A. Heijde M. Katinka M. Mock T. Valentin K. Verret F. Berges J.A. Brownlee C. Cadoret J.P. Chiovitti A. Choi C.J. Coesel S. De Martino A. Detter J.C. Durkin C. Falciatore A. Fournet J. Haruta M. Huysman M.J. Jenkins B.D. Jiroutova K. Jorgensen R.E. Joubert Y. Kaplan A. Kroger N. Kroth P.G. La Roche J. Lindquist E. Lommer M. Martin-Jezequel V. Lopez P.J. Lucas S. Mangogna M. McGinnis K. Medlin L.K. Montsant A. Oudot-Le Secq M.P. Napoli C. Obornik M. Parker M.S. Petit J.L. Porcel B.M. Poulsen N. Robison M. Rychlewski L. Rynearson T.A. Schmutz J. Shapiro H. Siaut M. Stanley M. Sussman M.R. Taylor A.R. Vardi A. von Dassow P. Vyverman W. Willis A. Wyrwicz L.S. Rokhsar D.S. Weissenbach J. Armbrust E.V. Green B.R. Van de Peer Y. Grigoriev I.V. The Phaeodactylum genome reveals the evolutionary history of diatom genomes.Nature. 2008; 456: 239-244Crossref PubMed Scopus (1214) Google Scholar), has evolved a set of sophisticated cellular and molecular mechanisms to cope with a wide variety of nutrient stresses. Over the past 5 years, metabolic change triggered by nutrient stress has been examined using omics-based approaches, which show that Phaeodactylum can globally regulate metabolic pathways at both transcriptional and translational levels, enabling their survival under nutrient stress (5.Alipanah L. Rohloff J. Winge P. Bones A.M. Brembu T. Whole-cell response to nitrogen deprivation in the diatom Phaeodactylum tricornutum.J. Exp. Bot. 2015; 66: 6281-6296Crossref PubMed Scopus (167) Google Scholar, 6.Yang Z.K. Niu Y.F. Ma Y.H. Xue J. Zhang M.H. Yang W.D. Liu J.S. Lu S.H. Guan Y. Li H.Y. Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation.Biotechnol. Biofuels. 2013; 6: 67Crossref PubMed Scopus (265) Google Scholar, 7.Levitan O. Dinamarca J. Zelzion E. Lun D.S. Guerra L.T. Kim M.K. Kim J. Van Mooy B.A. Bhattacharya D. Falkowski P.G. Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 412-417Crossref PubMed Scopus (159) Google Scholar, 8.Ge F. Huang W. Chen Z. Zhang C. Xiong Q. Bowler C. Yang J. Xu J. Hu H. Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum.Plant Cell. 2014; 26: 1681-1697Crossref PubMed Scopus (88) Google Scholar, 9.Cruz de Carvalho M.H. Sun H.X. Bowler C. Chua N.H. Noncoding and coding transcriptome responses of a marine diatom to phosphate fluctuations.New Phytol. 2016; 210: 497-510Crossref PubMed Scopus (65) Google Scholar). However, post-translational control of gene regulation and potentially protein stability and activity in Phaeodactylum is only beginning to be understood. A recent study reported the identification of multiple post-translational modifications (PTMs) 1The abbreviations used are: PTM, post-translational modification;ACCase, acetyl-CoA carboxylase;AcP, acetyl-phosphate;CoA, acetyl Coenzyme A;E1, ubiquitin activating enzyme;E2, ubiquitin conjugating enzyme;E3, ubiquitin ligase;FabI, enoyl-acp reductase;FabD, malonyl-CoA: ACP transacylase;FabZ, 3R-hydroxyacyl-ACP dehydrase;FabF, 3-oxoacyl-ACP synthase;FabG, 3-oxoacyl-ACP reductase;FDR, false discovery rate;GO, Gene Ontology;KATs, lysine acetyltransferases;KDACs, lysine deacetylases;LACS, long chain acyl-CoA synthase;Lys, Nε-lysine;MS, mass spectrometry;SIR2, Silent Information Regulator 2;SRT, sirtuin-type deactylases;Ub, ubiquitin;WT, wild-type. 1The abbreviations used are: PTM, post-translational modification;ACCase, acetyl-CoA carboxylase;AcP, acetyl-phosphate;CoA, acetyl Coenzyme A;E1, ubiquitin activating enzyme;E2, ubiquitin conjugating enzyme;E3, ubiquitin ligase;FabI, enoyl-acp reductase;FabD, malonyl-CoA: ACP transacylase;FabZ, 3R-hydroxyacyl-ACP dehydrase;FabF, 3-oxoacyl-ACP synthase;FabG, 3-oxoacyl-ACP reductase;FDR, false discovery rate;GO, Gene Ontology;KATs, lysine acetyltransferases;KDACs, lysine deacetylases;LACS, long chain acyl-CoA synthase;Lys, Nε-lysine;MS, mass spectrometry;SIR2, Silent Information Regulator 2;SRT, sirtuin-type deactylases;Ub, ubiquitin;WT, wild-type.on histones of Phaeodactylum, revealing the dynamic feature of chromatin modifications in regulating target genes in response to nitrogen limitation (10.Veluchamy A. Rastogi A. Lin X. Lombard B. Murik O. Thomas Y. Dingli F. Rivarola M. Ott S. Liu X. Sun Y. Rabinowicz P.D. McCarthy J. Allen A.E. Loew D. Bowler C. Tirichine L. An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum.Genome Biol. 2015; 16: 102Crossref PubMed Scopus (47) Google Scholar). These histone modifications demonstrate the importance of PTMs in defining chromatin states and regulating gene expression in response to nutrient stress. Of more than 200 reported PTMs, Nε-lysine (Lys/K) acetylation is one of the most abundant and extensively studied PTMs with an evolutionary conservation from prokaryotes to eukaryotes (11.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. Bio. 2014; 15: 536-550Crossref PubMed Scopus (892) Google Scholar). The best-known function of Lys acetylation is the regulation of histone proteins by affecting chromatin structure and gene expression (12.Eberharter A. Becker P.B. Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics.EMBO Rep. 2002; 3: 224-229Crossref PubMed Scopus (682) Google Scholar). In contrast to irreversible Nα-acetylation, the reversible Lys acetylation is catalyzed by Lys acetyltransferases (KATs) and reversed by Lys deacetylases (KDACs) (13.Kim G.W. Yang X.J. Comprehensive lysine acetylomes emerging from bacteria to humans.Trends Biochem. Sci. 2011; 36: 211-220Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), making it an important mechanism to reversibly regulate protein function. In enzymatically catalyzed Lys acetylation reactions, KATs catalyze the transfer of an acetyl group from acetyl Coenzyme A (CoA) to the ε-amino group of protein lysine residues; whereas the KDAC activity removes the acetyl group from the modified lysine residue. In addition to enzymatic catalysis, Lys acetylation can also occur by chemical catalysis (14.Kuhn M.L. Zemaitaitis B. Hu L.I. Sahu A. Sorensen D. Minasov G. Lima B.P. Scholle M. Mrksich M. Anderson W.F. Gibson B.W. Schilling B. Wolfe A.J. Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.PloS One. 2014; 9: e94816Crossref PubMed Scopus (187) Google Scholar). Over the past decade, the development of new approaches to identify acetylated proteins, especially mass spectrometry-based proteomics, has advanced the research of Lys acetylation beyond histone protein modification. Substrates of Lys acetylation have been extended from histone proteins to a wide variety of non-histone proteins/enzymes involved in diverse biological processes, coupling Lys acetylation to cellular metabolism beyond epigenetic control of gene expression and chromatin dynamics that is associated with histones. Analyses of the overall picture of Lys acetylomes in yeast and humans greatly broaden its regulatory scope and demonstrate that it contributes not only to the regulation of nuclear function but also to the control of cytoplasmic and mitochondrial functions (15.Henriksen P. Wagner S.A. Weinert B.T. Sharma S. Bacinskaja G. Rehman M. Juffer A.H. Walther T.C. Lisby M. Choudhary C. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2012; 11: 1510-1522Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 16.Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions.Science. 2009; 325: 834-840Crossref PubMed Scopus (3152) Google Scholar, 17.Zhao S. Xu W. Jiang W. Yu W. Lin Y. Zhang T. Yao J. Zhou L. Zeng Y. Li H. Li Y. Shi J. An W. Hancock S.M. He F. Qin L. Chin J. Yang P. Chen X. Lei Q. Xiong Y. Guan K.L. Regulation of cellular metabolism by protein lysine acetylation.Science. 2010; 327: 1000-1004Crossref PubMed Scopus (1458) Google Scholar). Although proteome-wide analyses of acetylated proteins have been reported in several organisms, including bacteria (18.Zhang J. Sprung R. Pei J. Tan X. Kim S. Zhu H. Liu C.F. Grishin N.V. Zhao Y. Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli.Mol. Cell. Proteomics. 2009; 8: 215-225Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 19.Wang Q. Zhang Y. Yang C. Xiong H. Lin Y. Yao J. Li H. Xie L. Zhao W. Yao Y. Ning Z.B. Zeng R. Xiong Y. Guan K.L. Zhao S. Zhao G.P. Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux.Science. 2010; 327: 1004-1007Crossref PubMed Scopus (791) Google Scholar, 20.Mo R. Yang M. Chen Z. Cheng Z. Yi X. Li C. He C. Xiong Q. Chen H. Wang Q. Ge F. Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803.J. Proteome Res. 2015; 14: 1275-1286Crossref PubMed Scopus (75) Google Scholar), yeast (15.Henriksen P. Wagner S.A. Weinert B.T. Sharma S. Bacinskaja G. Rehman M. Juffer A.H. Walther T.C. Lisby M. Choudhary C. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2012; 11: 1510-1522Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), plants (21.Finkemeier I. Laxa M. Miguet L. Howden A.J. Sweetlove L.J. Proteins of diverse function and subcellular location are lysine acetylated in Arabidopsis.Plant Physiol. 2011; 155: 1779-1790Crossref PubMed Scopus (177) Google Scholar, 22.Wu X. Oh M.H. Schwarz E.M. Larue C.T. Sivaguru M. Imai B.S. Yau P.M. Ort D.R. Huber S.C. Lysine acetylation is a widespread protein modification for diverse proteins in Arabidopsis.Plant Physiol. 2011; 155: 1769-1778Crossref PubMed Scopus (155) Google Scholar) and human (16.Choudhary C. Kumar C. Gnad F. Nielsen M.L. Rehman M. Walther T.C. Olsen J.V. Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions.Science. 2009; 325: 834-840Crossref PubMed Scopus (3152) Google Scholar, 17.Zhao S. Xu W. Jiang W. Yu W. Lin Y. Zhang T. Yao J. Zhou L. Zeng Y. Li H. Li Y. Shi J. An W. Hancock S.M. He F. Qin L. Chin J. Yang P. Chen X. Lei Q. Xiong Y. Guan K.L. Regulation of cellular metabolism by protein lysine acetylation.Science. 2010; 327: 1000-1004Crossref PubMed Scopus (1458) Google Scholar, 23.Guan K.L. Yu W. Lin Y. Xiong Y. Zhao S.M. Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides.Nat. Protoc. 2010; 5: 1583-1595Crossref PubMed Scopus (82) Google Scholar, 24.Kim 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), little is known about the acetylomes in photosynthetic microalgae. In this study, we integrated anti acetyllysine-based enrichments, high accuracy MS techniques and bioinformatic analyses to profile the Lys acetylome in the model diatom Phaeodactylum, and we identified 2324 unique acetylation sites from the peptides of 1220 proteins. We performed proteome-wide analysis of Lys acetylated proteins in Phaeodactylum, and characterized the effect of Lys acetylation on enzyme activity of a plastid-localized long-chain acyl-CoA synthetase, which shows that acetylation enhances its enzyme activity in vitro. Our results provide a comprehensive view of the acetylome in Phaeodactylum and pave the way to understand the functional importance of Lys acetylation for cellular metabolic enzymes in this model diatom. Axenic cells of Phaeodactylum tricornutum Bohlin (CCMP 2561) from the culture collection of the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (Bigelow Laboratory for Ocean Sciences, East Boothbay, ME) were grown as previously described (8.Ge F. Huang W. Chen Z. Zhang C. Xiong Q. Bowler C. Yang J. Xu J. Hu H. Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum.Plant Cell. 2014; 26: 1681-1697Crossref PubMed Scopus (88) Google Scholar). For proteomic analysis, cells (4 × 105 cells/ml) from mid-logarithmic phase cultures were inoculated in artificial seawater enriched with f/2 (25.Guillard R.R.L. Culture of phytoplankton for feeding marine invertebrates.in: Smith W. Chanley M. Culture of Marine Invertebrate Animals. Springer, New York1975: 29-60Crossref Google Scholar) at 22 °C, bubbled with filtered air and continuously irradiated with 100 μmol photons/m2/s. To obtain a large amount of the Lys acetylation proteome in this model diatom, cells at mid-logarithmic phase were exposed to different stresses for 12 h, including nitrogen deficiency (-N), iron deficiency (-Fe), and phosphate deficiency (-P). To inhibit endogenous protein deacetylase activity, nicotinamide was added to the cell cultures at a final concentration of 10 mm and incubated for an additional 30 min. The cells were harvested by centrifugation (6000 × g for 5 min at 10 °C), washed with f/2 medium twice and frozen by liquid nitrogen. Then, the sample was grinded with liquid nitrogen, transferred to 15 ml centrifuge tube and sonicated three times with an output of 135 W (JY92-IIN; Ningbo Scientz Bio-technology Co., Ltd., Ningbo, Zhejiang, China) on ice-water (2 s on/2 s off) for about 30 min in lysis buffer (8 m urea, 10 mm DTT, 3 μm trichostatin A, 50 mm nicotinamide, 2 mm EDTA and 1% protease inhibitor mixture set VI [Calbiochem, Darmstadt, Germany]). The whole cell lysate was then centrifuged (20,000 × g at 4 °C for 10 min) to remove the remaining debris, after which the protein was precipitated with cold 15% trichloroacetic acid at −20 °C for 2 h. Following another round of centrifugation at 4 °C for 10 min, the supernatant was discarded, leaving the precipitate which was washed with cold acetone three times. The precipitated protein was redissolved in buffer (8 m urea, 100 mm NH4CO3, pH 8.0) and the protein concentration was determined using the BCA Protein Assay Kit (TIANGEN, Beijing, China). The protein in solution was reduced with 10 mm DTT for 1 h at 37 °C and alkylated with 20 mm iodoacetamide for 45 min at room temperature in the dark. Then, the protein sample was diluted by 100 mm NH4CO3 to urea concentration less than 2 m. Finally, sequencing grade Trypsin (Promega, Madison, WI) was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1: 100 trypsin-to-protein mass ratio for a second 4 h-digestion. The digested peptides were fractionated by high pH reverse-phase HPLC using an Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated into 80 fractions with a gradient of 2 to 60% acetonitrile in 10 mm NH4CO3 at pH 10 for over 80 min. Then, the peptides were combined into 8 fractions and then collected and dried by vacuum centrifuging. The resulting peptides were enriched by agarose-conjugated the anti-acetyllysine antibody (PTM Biolabs, Chicago, IL). Briefly, tryptic peptides dissolved in NETN buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40) were incubated with anti-acetyllysine antibody conjugated protein A agarose beads at 4 °C overnight with gentle rotation. The beads were washed four times with NETN buffer and then with ddH2O two times. The bound peptides were eluted from the beads by 0.1% TFA, combined and vacuum-dried. The resulting peptides were cleaned with C18 ZipTips from Millipore (Billerica, MA) according to the manufacturer's instructions. The enriched peptides were dissolved in 0.1% FA, directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100; Thermo Fisher Scientific, Waltham, MA), and separated using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Fisher Scientific). The gradient was comprised of an increase from 5 to 20% solvent B (0.1% FA in 98% ACN) for 20 min, 20 to 35% for 8 min and climbing to 80% in 2 min then holding at 80% for the last 5 min. The resulting peptides were eluted at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system and analyzed by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptides were subjected to a nanosprayionization source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using a NCE setting of 30 and ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1E4 in the MS survey scan with 10.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for the generation of the MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. MS2 fixed first mass was set as 100. The MS/MS data was processed using MaxQuant with an integrated Andromeda search engine (v.1.3.0.5). Tandem mass spectra were searched against the P. tricornutum (10,402 sequences) database concatenated with reverse decoy database. Trypsin/P was specified as a cleavage enzyme allowing up to 4 missing cleavages. Mass error of precursor ions was set to 20 ppm for the first search, 5 ppm for the second search and 0.02 Da for fragment ions. Carbamidomethylation (Cys) was set as fixed modification and oxidation (Met), and deamidation (Asn/Gln), acetylation (Lys) and protein N-terminal were specified as variable modifications. Minimum peptide length was set at 6. Search results were filtered out by eliminating all putative hits with an Andromeda score < 40. The estimated false discovery rate (FDR) thresholds for proteins, peptides and modification sites were specified at a maximum 1%. The site localization probability was set as ≥ 0.75. All the other parameters in MaxQuant were set to default values. All the raw data were deposited in a publicly accessible database, PeptideAtlas (www.peptideatlas.org) (26.Desiere F. Deutsch E.W. King N.L. Nesvizhskii A.I. Mallick P. Eng J. Chen S. Eddes J. Loevenich S.N. Aebersold R. The PeptideAtlas project.Nucleic Acids Res. 2006; 34: D655-D658Crossref PubMed Scopus (590) Google Scholar). All the MS/MS spectra were manually inspected to improve the reliability of the results, and the spectra were kept only if a series of at least three successive b- or y-ions were present (27.Macek B. Gnad F. Soufi B. Kumar C. Olsen J.V. Mijakovic I. Mann M. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation.Mol. Cell. Proteomics. 2008; 7: 299-307Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 28.Macek B. Mijakovic I. Olsen J.V. Gnad F. Kumar C. Jensen P.R. Mann M. The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis.Mol. Cell. Proteomics. 2007; 6: 697-707Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Finally, those MS/MS spectra with high-quality were used for further analyses in this study. Annotation of acetylproteins based on the Gene Ontology (GO) terms including biological process and molecular function was performed using Blast2GO software (29.Conesa A. Götz S. Blast2GO: A comprehensive suite for functional analysis in plant genomics.Int. J. Plant Genomics. 2008; 2008: 619832Crossref PubMed Scopus (1572) Google Scholar, 30.Conesa A. Götz S. Garcia-Gomez J.M. Terol J. Talon M. Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.Bioinformatics. 2005; 21: 3674-3676Crossref PubMed Scopus (8558) Google Scholar, 31.Götz S. Arnold R. Sebastián-León P. Martín-Rodríguez S. Tischler P. Jehl M.A. Dopazo J. Rattei T. Conesa A. B2G-FAR, a species-centered GO annotation repository.Bioinformatics. 2011; 27: 919-924Crossref PubMed Scopus (117) Google Scholar). Protein subcellular localization was predicted by ngLOC (32.King B.R. Vural S. Pandey S. Barteau A. Guda C. ngLOC: software and web server for predicting protein subcellular localization in prokaryotes and eukaryotes.BMC Res. Notes. 2012; 5: 351Crossref PubMed Scopus (37) Google Scholar). Because nuclear-encoded plastid-localized proteins in Phaeodactylum generally possess bipartite N-terminal pre-sequences that contain a conserved sequence motif (‘ASAFAP’ motif), we additionally used a customized prediction tool (ASAFind) to predict plastid localization of identified acetylated proteins as previously described (33.Gruber A. Rocap G. Kroth P.G. Armbrust E.V. Mock T. Plastid proteome prediction for diatoms and other algae with secondary plastids of the red lineage.Plant J. 2015; 81: 519-528Crossref PubMed Scopus (106) Google Scholar). Functional enrichment analysis of the identified acetylated proteins was performed by BinGO 2.44 plugin (34.Maere S. Heymans K. Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks.Bioinformatics. 2005; 21: 3448-3449Crossref PubMed Scopus (3091) Google Scholar) in the Cytoscape platform (35.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: 2498-2504Crossref PubMed Scopus (25321) Google Scholar). Lys-acetylated motifs were analyzed by soft motif-x (36.Chou M.F. Schwartz D. Biological sequence motif discovery using motif-x.Curr. Protoc. Bioinform. 2011; (Chapter 13, Unit 13.15–24)Crossref Scopus (298) Google Scholar, 37.Schwartz D. Gygi S.P. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets.Nat. Biotechnol. 2005; 23: 1391-1398Crossref PubMed Scopus (716) Google Scholar). All the database protein sequences of Phaeodactylum were used as a background database parameter and other parameters as default. To analyze Lys acetylation sites, the ratios of 6 amino acids surrounding the acetylation sites upstream and downstream were calculated, and the position-specific heat map was generated by plotting the log10 of the ratio (38.Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12130-12135Crossref PubMed Scopus (1236) Google Scholar, 39.Treeck M. Sanders J.L. Elias J.E. Boothroyd J.C. The phosphoproteomes of Plasmodium falciparum Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries.Cell Host Microbe. 2011; 10: 410-419Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 40.Yang M.K. Qiao Z.X. Zhang W.Y. Xiong Q. Zhang J. Li T. Ge F. Zhao J.D. Global phosphoproteomic analysis reveals diverse functions of serine/threonine/tyrosine phosphorylation in the model cyanobacterium Synechococcus sp. strain PCC 7002.J. Proteome Res. 2013; 12: 1909-1923Crossref PubMed Scopus (61) Google Scholar). Predictions of secondary structures were performed using NetSurfP (41.Petersen B. Petersen T.N. Andersen P. Nielsen M. Lundegaard C. A generic method for assignment of reliability scores applied to solvent accessibility predictions.BMC Struct. Biol. 2009; 9: 51Crossref PubMed Scopus (493) Google Scholar). The mean secondary structure probabilities of modified lysine residues were compared with those of control residues for all identified acetylated proteins, and p values were calculated as previously described (42.Wagner S.A. Beli P. Weinert B.T. Nielsen M.L. Cox J. Mann M. Choudhary C. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles.Mol. Cell. Proteomics. 2011; 10 (M111 013284)Abstract Full Text Full Text PDF Google Scholar). Further, orthologs of acetylproteins that were identified were searched using BLASTP to evaluate the conservation across species (43.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70353) Google Scholar). KEGG mapper was used to analyze the KEGG pathway to evaluate the metabolic pathway involving acetylproteins (44.Kanehisa M. Goto S. KEGG: kyoto encyclopedia of genes and genomes.Nucleic Acids Res. 2000; 28: 27-30Crossref PubMed Scopus (17971) Google Scholar, 45.Kanehisa M. Goto S. Sato Y. Kawashima M. Furumichi M. Tanabe M. Data, information, knowledge and principle: back to metabolism in KEGG.Nucleic Acids Res. 2014; 42: D199-D205Crossref PubMed Scopus (2298) Google Scholar). The structural models of FABZ (bd1143), FABFa (37367), FABI (10068), FABG (13073) and FABD (37652) were prepared from the previously determined crystal stru" @default.
• W2766171218 created "2017-11-10" @default.
• W2766171218 creator A5000653817 @default.
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• W2766171218 date "2018-03-01" @default.
• W2766171218 modified "2023-10-02" @default.
• W2766171218 title "Acetylome Profiling Reveals Extensive Lysine Acetylation of the Fatty Acid Metabolism Pathway in the Diatom Phaeodactylum tricornutum" @default.
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• W2766171218 doi "https://doi.org/10.1074/mcp.ra117.000339" @default.
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