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Systematic Analysis of the Lysine Acetylome in Candida albicans Xiaowei Zhou, Guanyu Qian, Xingling Yi, Xiaofang Li, and Weida Liu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00052 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 18, 2016

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Journal of Proteome Research

Systematic Analysis of the Lysine Acetylome in Candida albicans Xiaowei Zhou1,2, Guanyu Qian1, Xingling Yi3, Xiaofang Li1,2*, Weida Liu1,2* 1

Department of Medical Mycology, Institute of Dermatology, Chinese Academy of Medical Science and Peking

Union Medical College, Nanjing 210042, Jiangsu, People’s Republic of China; 2Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, Jiangsu, People’s Republic of China; 3Jingjie PTM Biolab (Hangzhou) Co., Ltd., Hangzhou 310018, zhejiang, People's Republic of China.

ABSTRACT: Candida albicans(C. albicans) is a worldwide cause of fungus infectious diseases. As a general post-translational modification(PTM), lysine acetylation of proteins play an important regulatory role in almost every cells. In our research, we used a high-resolution proteomic technique (LC-MS/MS) to present the comprehensive analysis of the acetylome in C. albicans. In general, We detected 477 acetylated proteins among all 9038 proteins(5.28%) in C. albicans, which had 1073 specific acetylated sites. The bioinformatics analysis of the acetylome showed significant role in the regulation of metabolism. To be more precise, proteins involved in carbon metabolism and biosynthesis were the underlying objectives of acetylation. Besides, through the study of acetylome, we found universal rule in acetylated motifs: the +4, +5 or +6 position which is an alkaline residue with a long side chain (K or R) and the +1 or +2 position which is a residue with a long side chain (Y, H, W or F). To the best of our knowledge, all screening acetylated histone sites of this study have not been previously reported. Moreover, protein-protein interaction network (PPI) study demonstrated that a variety of connections in glycolysis/gluconeogenesis, oxidative phosphorylation and ribosome were modulated by acetylation and phosphorylation, but the phosphorylated proteins in oxidative phosphorylation PPI network were not abundant, which indicated that acetylation may do more significant effect than phosphorylation on oxidative phosphorylation. This is the first study of acetylome in human pathogenic fungi, providing an important starting for the in-depth discovery of the function analysis of acetylated proteins in such fungal pathogens. KEYWORDS: Candida albicans, Lysine acetylation, acetylome, lysine acetylation motif, histone, interaction network INTRODUCTION C. albicans is a filamentous or yeast-like dipoid fungal pathogen, which can infect the skin or mucous membranes lead to vaginitis, candidiasis and systemic mycosis1. The results of the EPIC-II study, which involving in 13,796 analyzed patients in 1,265 ICU, showed 19% were fungi infections2. A retrospective study of these patients in EPIC-II revealed that C. albicans was the most common organism3. The incidence of systemic candidiasis is the fourth of the nosocomial infectious disease in USA. The mortality of candidemia is about 30~40% (30 days), and the cost of treatment reached up to $31,200 per person in 19984. There is an urgent need for more understanding of the pathogenic mechanism of C. albicans. In recent years, studies have tried to explore the pathogenesis and treatment of disease through the perspective of epigenetics. To date, epigenetics is a popular research spot in cancer,

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inflammatory, immunological, neurodegenerative disease, etc5-8, but has been little studied in fungal infection. Epigenetics is the research on DNA modifications, histone acetylation, histone lysine methylation or microRNA modifications but none alteration of the underlying nucleic acid sequence9-11. As a real-time change of PTM, protein lysine acetylation is important for the regulation of metabolism in microbiologists12, 13. In the last few years, LC-MS/MS were used in large-scale study of PTMs especially protein acetylation, have significant effect for research of protein function. Such as the recent study of Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis and Streptomyces roseosporus acetylated proteins were identified in cell physiology and biochemical reactions14-18. In C. albicans and S. cerevisiae, the pharmacological inhibition of lysine deacetylases (KDACs) affected the Hsp90-dependent resistance to the azoles, and Hsp90 was regulated by acetylation of lysine 27 and 270 in S. cerevisiae19. But there was no comprehensive or systematic study for the acetylome in C. albicans so far. In our research, we present the comprehensive analysis of the acetylome in C. albicans SC5314, which was obtained through a high-resolution proteomic technique. We detected 1073 specific acetylation sites on 477 acetylated proteins with remarkably dissimilar biological functions and cellular localizations in this strain(Table S-1). We comparatively analysed the evolutionarily conserved acetylated proteins between C. albicans and yeast(S. cerevisiae), mouse, human and E. coli. In addition, several conserved acetylated motifs similar to those extracted from eukaryotic cells were analyzed by a bioinformatics study of the amino acid surrounding the acetylated sites. We also identified acetylated sites distribution in histone, analysed crosstalk and PPI network between lysine acetylation and phosphorylation. These results provided the first comprehensive view of the acetylome of C. albicans. MATERIALS AND METHODS Sample Preparation Strains and Culture. Before crude protein extraction and western blotting analysis, SC5314 was shook at 28 °C at 220 rpm overnight in YPD medium and used as seed cultures. Seed culture of 10 ml was inoculated into 100 ml of YPD medium shook at 28 °C (220 rpm) for 4 hours at an OD600 of 0.8. Then we used highly sensitive immune-affinity purification and high-resolution LC-MS/MS to identify SC5314, which explored the first lysine acetylome in C. albicans. Crude Protein Extraction and Western Blotting Analysis. The cultured cells were centrifuged at 4℃(6000rpm) and washed by cold PBS in twice. Sample was first grinded by liquid nitrogen, then the cell powder was sonicated three times on ice in lysate(8 M urea, 65 mM DTT, 1% Triton-100 and 0.1% Cocktail). The chips was centrifugated at 4 °C (12,000g) for 40 min to wipe it off. Finally, the protein was precipitated in cold 15% TCA for 2h at -20°C. Then liquid was centrifugated at 4°C(12,000g) for 40min, and the supernatant was removed. The precipitate was washed in three times by cold acetone. After centrifugation, The precipitate was redissolved by buffer (8 M urea, 100 mM TEAB, pH 8.0) and the protein concentration was detected with 2-D Quant kit. Western blotting assays were performed using protein lysates from tachyzoites by 4-20% SDS-PAGE(Cat.No.456-1083, Bio-Rad, USA). After transferred to PVDF membrane (Cat.No.162-0177, Bio-Rad, USA), the PVDF was soaked in confining liquid(1% tween-20 and 3% nonfat milk powder in PBS). Acetylated lysines were detected using pan anti-acetyl antibodies

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(PTM Biolabs, China) and histone Kac site-specific antibody(anti-H2B, anti-H2BK12ac, anti-H2BK23ac from PTM Biolabs, China, anti-H4, anti-H4BK8ac from Abcam, UK) diluted in blocking buffer at 1:1,000. After incubated overnight at 4℃, PVDF were washed by PBST and incubated with (HRP)-conjugated Goat anti-rabbit antibody (Cat.No.31460, Pierce, USA)/ Goat anti-mouse antibody(Cat.No.CW0103S, cwbiotech, China) diluted at 1:5,000 and chemiluminescene(ECL) reagent (Cat.No. E411-04, Vazyme Biotech, China) was used for detection. Trypsin Digestion and Affinity Enrichment. The protein solution was diluted with 10 mM DTT at 56°C(1 h) and alkylated with 20 mM IAA(45 min) in darkness for digestion. Firstly, Protein was digested by trypsin at 1:50 ratio overnight then 1:100 ratio for 4h. Tryptic peptides was diluted in NETN buffer (100 mM NaCl, 50 mM Tris-HCl, 0.5% NP-40, 1 mM EDTA, pH 8.0) with mild shaking at 4°C overnight by pre-washed pan anti-acetyl antibody beads (PTM Biolabs, China). The beads were washed by NETN buffer for 4 times and ddH2O for twice. Enrich peptides were eluted from the beads by 0.1% TFA, then vacuum-dried for LC-MS/MS analysis. Analysis Proteomic by LC-MS/MS Analysis of LC-MS/MS. Peptides from strain SC5314 were melted into solvent A(0.1% FA in 2% ACN), then loaded up a reversed-phase pre-column(Acclaim PepMap 100, Thermo Scientific, USA) directly, and seperated by an EASY-nLC 1000 UPLC system at 280 nl/min in solvent B(0.1% FA in 98% ACN) respectively at gradient concentration of 6-35% (32min) and 35-80%(8min). The acquired peptides were detected by Q ExactiveTM hybrid quadrupole-Orbitrap mass spectrometer(Thermo Fisher Scientific, USA). The peptides were attached to NSI source then MS/MS in Q ExactiveTM Plus(Thermo Fisher Scientific, USA) bonded to the UPLC online. Orbitrap at 70,000 resolution was used to detect intact peptides, which were chosen for MS/MS with 28% NCE. Orbitrap at 17,500 resolution was used for ion fragments detection. A data-dependent procedure set as 10 MS/MS scans after 1 MS scan was applied for the top 10 precursor ions above a threshold ion count of 2E4 with 10.0s dynamic exclusion in the MS scan. The electrospray voltage was 2.0 kV. The charge state was set from +2 to +5. The overfilling of the ion trap was prevented with automatic gain control (AGC); 5E4 ions were accumulated to generate MS/MS spectra. For MS scans, the m/z scan ranged from 350 to 1800. Searching the Database. The MS/MS results were processed by MaxQuant(v1.4.1.2). C. albicans database(9043 proteins) linked to reverse decoy database was used for tandem mass spectra searching. Also, a database of known proteins provided with MaxQuant was employed in the search. Trypsin/P, a cleavage enzyme was set as 3 missing cleavages, 4 modifications tolerance and 5 charges per peptide. Mass error was 10 ppm(precursor ions) and 0.02 Da(fragment ions). Carbamido methylation of Cys was set as fixed modifications while lysine acetylation, oxidation of Met and acetylation of protein N-terminal were variable. False discovery rate of database were set at 1%. The least length of peptide was specified as 7. Other parameters of MaxQuant were set as default(Table S-2). The site localization probability was set as > 0.75. Bioinformatics analysis GO Annotation. The Gene Ontology(GO) is an important bioinformatics analysis of gene and gene product attributes classification among all species. GO annotation proteome was acquired in the UniProt-GOA database(http://www.ebi.ac.uk/GOA/). We converted the identified protein IDs as UniProt IDs then map to GO IDs. If some identified proteins have not been

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annotated in UniProt-GOA database, GO annotation carried out by the InterProScan soft. Then we used GO annotation based on three categories to classify proteins: biological process, cellular component and molecular function. Domain Annotation. Functional description of identified proteins domain were annotated by InterProScan(a sequence analysis application), and the InterPro domain database was used. InterPro(http://www.ebi.ac.uk/interpro/) is based on domains, protein families and functional sites, which is a database open to everybody. The important services of database are diagnostic models, which can use signatures to determine protein sequences’ function. InterPro is useful in study of whole genomes, characterizing protein sequences and meta-genomes. Annotation of KEGG Pathway. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways mainly involving in Environmental Information Processing, Drug development, Cellular Processes, Metabolism, Rat Diseases, Genetic Information Processing. Protein pathways were annotated by KEGG database. Firstly, We used KAAS to analyse and annotate proteins’ database. Then we used KEGG mapper in the KEGG pathway database to map the results. Subcellular Localization Annotation. Eukaryotic cells were classified into different compartments by subcellular localization. There, we used wolfpsort which was a predication soft of an updated version of PSORT/PSORT II to predict subcellular localization. Study of Motif. To analyse the sequences models involving in particular amino acids(6 upstream and downstream from the acetylated sites) in all peptides, soft motif-x was applied. And all protein sequences of database were set as background database parameter, while other parameters as default. Enrichment of GO analysis. In GO annotation, proteins were sorted out by biological process, cellular compartment and molecular function. We used Functional Annotation Tool of DAVID to identify enriched GO against the background of Uniprot_Candia_Albicans. To test the enrichment of the protein-containing IPI entries in all IPI proteins, a two-tailed Fisher’s exact test was used. Standard false discovery rate control methods were used to correct multiple hypothesis testing. Corrected p-value < 0.05 was important for GO. Enrichment of pathway analysis. KEGG database was classified to enriched pathways against the background of Uniprot_Candia_Albicans with Functional Annotation Tool of DAVID. To test the enrichment of the protein-containing IPI entries in all IPI proteins, a two-tailed Fisher’s exact test was used. Standard false discovery rate control methods were used to correct multiple hypothesis testing. Corrected p-value < 0.05 was important for the pathway. These pathways were identified as hierarchical categories based on the KEGG website. Enrichment of protein domain analysis. For each category proteins, InterPro database was researched by Functional Annotation Tool of DAVID against the background of Uniprot_Candia_Albicans. To test the enrichment of the protein-containing IPI entries in all IPI proteins, a two-tailed Fisher’s exact test was used. Standard false discovery rate control methods were used to correct multiple hypothesis testing. Corrected p-value < 0.05 was important for the domains. Study of Network. We used cytoscape software to study PPI Network. PPI was obtained from STRING database. STRING defined by a standard of a confidence score(≥0.7, high confidence). RESULTS AND DISCUSSIONS

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1073 sites of 477 acetylation proteins were identified from C. albicans To characterize the lysine acetylation proteins’ modification level of C. albicans, western blotting analysis was conducted using a pan anti-acetyl antibody. We detected many protein bands spanning a wide range, suggested a variety of acetylated proteins were present(Figure 1). We isolated proteins from exponentially growing SC5314, used pan anti-acetyl antibody to enrichment acetylated peptides which digested by trypsin for comprehensively identify the C. albicans acetyl proteome in vitro. LC-MS/MS was used to analyze enriched acetylated peptides from SC5314, and the results were searched by MaxQuant search algorithm in C. albicans databases. Total 447 proteins with 1073 sites were acetylated, these acetylated proteins comprised about 5.28% (477/9038) of C. albicans proteome，which was abundant in eukaryotes. Of the identified 447 acetylated proteins, about 55% included a single acetylated site (Figure 1B), 21% included two acetylated sites, and 12% included three acetylated sites. Notably, there were 40 proteins included 5 or more acetylated sites. Moreover, we found many identified acetylated proteins were the components of protein complexes, such as DNA biosynthesis and repair machines, RNA degradosome, ATPase and proteasome, which indicated the acetylation may have important role in glycolysis/ gluconeogenesis, oxidative phosphorylation, ribosome and PPI.

(A)

(B) Figure 1 Study of lysine acetylation in C. albicans.

(A) Analyze the overall acetylation of C. albicans. SDS-PAGE separated 20µg protein lysates from strain SC5314, then proteins were detected by pan anti-acetyl antibodies( from PTM Biolabs , PTM-401, 1:1000 dilution)； (B) Distributed of protein acetylation depending on the number of lysine acetylation points identified in C. albicans protein.

Comparative analysis of the evolutionarily conserved acetylated proteins between C. albicans and other species To confirm whether those identified acetylated 477 proteins and 1073 sites were conserved, we compared the C. albicans acetyl proteome to previously reported species acetyl proteomes. We found interesting parallels exist among acetyl proteomes of those species. Among the 447 acetylated proteins, 401 proteins had orthologs in the acetyl proteome of yeast(S. cerevisiae), 414 proteins had orthologs in the acetyl proteome of mouse 365 proteins had orthologs in the acetyl

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proteome of human, and 90 proteins had orthologs in the acetyl proteome of E. coli, respectively (Figure 2, Table S-3: Yeast, Table S-3A; Mouse, Table S-3B; Human, Table S-3C; E. coli, Table S-3D). Given that as high as 42.8% acetylated proteins in yeast(S. cerevisiae)20, 32.1% in mouse21, 29.5% in human21 and 27.5% in E. coli14 detected had orthologs with C. albicans, the C. albicans acetyl proteome profile exhibited greater similarity with yeast(S. cerevisiae) and less similarity with E. coli, which was in consistent with genetic relationship of four species.

Figure 2 Comparative analysis of C. albicans acetyl proteome with those of yeast(S. cerevisiae), mouse, human and E. coli. Venn diagram described the relationship of lysine acetylated proteins from yeast(S. cerevisiae), mouse, human, E. coli and C. albicans.

Functional Characterization of Lysine Acetylome of C. albicans To further explore the impact of acetylated proteins in cell physiological process and discover internal relations between acetylated proteins, we classified the functions of the acetylated proteins and then analyzed the significance of functional enrichment including GO (Figure 3 and Table S-4A，biological process, cellular component, molecular function), KOG(Figure 4A and Table S-4B) and subcellular location(Figure 4B and Table S-4C). According to GO annotation results, the acetylated proteins were distributed in all sorts of cell components, primarily in cytosol (41%), organelle (24%) and membrane (14%)(Figure 3C). The classification of acetylated proteins in biological process was further analyzed according to GO annotation. As shown in Figure 3B, there were 321 proteins involved in metabolic process (28%), 320 in cellular process(28%), 137 in single-organism process (12%), 90 in response to stimulus (8%), and 57 in biological regulation (5%) of all 477 lysine acetylated proteins. According to GO annotation, the molecular function of acetylated proteins were classified into several group, such as 262 proteins associated with catalytic activity (43%), 259 involved in binding (42%) and 45 related with structural molecule activity (7%), as shown in Figure 3D. According to KOG annotation information of identified protein, we calculated the number of differentially expressed proteins in each KOG categories. Results showed that 77 proteins associated with ribosomal structure, translation and biogenesis(16%), 10 proteins respectively related to “RNA processing and modification” or “chromatin structure and dynamicsin”(2%) in information storage and processing. In cellular processes and signaling, there were 52 proteins related to protein turnover, chaperones and posttranslational modification (11%), while 20 proteins related to vesicular transport, secretion, and intracellular trafficking. In metabolism, there are 61 proteins associated with conversion and energy production(13%), 31 proteins associated with

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metabolism and carbohydrate transport(6%), 33 proteins connected with metabolism and amino acid transport (7%), as shown in Figure 4A and Table S-4B. component organization or other biogenesis 3% growth 3% localization4% multi4% organism process 5% biological regulation 5%

350 321 320 277

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90 57 55 51 42 40 33

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metabolic process 28%

response to stimulus 8%

singleorganism process 12%

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cellular process 28%

B transporter activity 3%

extracellular region other 2% 3% membrane 14%

structural molecule activity 7%

other 5%

catalytic activity 43%

cell 41% macromolecula r complex 16%

binding 42%

organelle 24%

D

C

Figure 3 The classification of acetylated proteins according to GO annotation information. (A) Identified acetylated protein distribution in GO. (B) The classification of acetylated proteins in biological process. (C) Acetylated proteins distributed in the cell components. (D) Acetylated proteins classified in molecular function.

The subcellular location analysis of the acetylated proteins showed that 179 proteins were distributed in the cytoplasmic (37%), 123 located in the nucleus (26%), and 117 distributed in the mitochondria (25%), as shown in Figure 4B and Table S-4C. Functional Enrichment of Lysine Acetylome of C. albicans Furthermore, we built an enrichment to identify which type of proteins are prior targets for lysine acetylation from the acetylation data in three GO categories: cellular component, biological process and molecular function (Figure 5). We found protein-DNA complex, non-membrane-bounded organelle, and cell part were significantly enriched in the cellular component. And the biological processes associated with organic substance metabolic, cellular metabolic, biosynthetic, primary metabolic, single-organism metabolic, catabolic and interspecies interaction between organisms were found to be significantly enriched (Figure 5). This pattern suggested that those proteins related to metabolism were probably acetylated in C. albicans. In agreement with this opinion, the enrichment analysis ground on molecular function revealed that proteins related to oxidoreductase activity and isomerase activity had a higher tendency to be

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acetylated. We also found that organic cyclic compound binding, cofactor binding, small molecule binding, ion binding, heterocyclic compound binding, and ligase activity were significantly enriched (Figure 5). [S] Function unknown

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[R] General function prediction only POORLY CHARACTERIZED [Q] Secondary metabolites biosynthesis, transport and catabolism [P] Inorganic ion transport and metabolism

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[I] Lipid transport and metabolism

19

[H] Coenzyme transport and metabolism

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[F] Nucleotide transport and metabolism

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[E] Amino acid transport and metabolism

nucl 26%

33 31

[G] Carbohydrate transport and metabolism

cyto 38%

61

[C] Energy production and conversion METABOLISM [O] Posttranslational modification, protein turnover, chaperones [U] Intracellular trafficking, secretion, and vesicular transport [W] Extracellular structures

52 20 0

[Z] Cytoskeleton

mito 25%

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[N] Cell motility

0

[M] Cell wall/membrane/envelope biogenesis

extr 3%

cyto_nucl 4%

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[T] Signal transduction mechanisms [V] Defense mechanisms

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[Y] Nuclear structure [D] Cell cycle control, cell division, chromosome partitioning CELLULAR PROCESSES AND SIGNALING

1

plas 2%

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[B] Chromatin structure and dynamics

cysk 1% cyto_mito 1%

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[L] Replication, recombination and repair

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mito_nucl

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[K] Transcription [A] RNA processing and modification

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[J] Translation, ribosomal structure and biogenesis

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A

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Figure 4 Classify acetylated proteins by KOG and subcellular location annotation information. (A) KOG category distribution of identified acetylated proteins. (B) Identified acetylated protein distribution in Subcellular localization.

Proteins involved in the metabolism of three types of biological macromolecules were also found to be KEGG enriched: carbohydrate metabolism (including metabolic pathways, glycolysis/ gluconeogenesis, citrate cycle(TCA cycle，Figure 6B), pentose phosphate pathway, pyruvate metabolism, galactose metabolism, fructose and mannose metabolism), lipid metabolism (propanoate metabolism, glyoxylate and dicarboxylate metabolism, fructose and mannose metabolism), and amino acid metabolism(biosynthesis of amino acids) (Figure 6A). Moreover, special pathways such as biosynthesis of secondary metabolites，metabolic pathways，oxidative phosphorylation and methane metabolism were also enriched with acetylated proteins. In addition, structural constituent analysis of ribosome in GO enrichment and ribosome in KEGG pathway enrichment suggested that the cellular process of protein biosynthesis may be strictly regulated by acetylation modification and acetylated proteins involved in protein biosynthesis were thus particularly crucial in C. albicans. The TCA cycle general exists in cell metabolism, which play important role in energy transduction and synthetic intermediates. In this study, nineteen acetylated proteins were involved in TCA, among which 11 proteins were characterized, such as isocitrate dehydrogenase (IDP1, mitochondrial), succinate dehydrogenase(mitochondrial), aconitate hydratase (mitochondrial), likely pyruate carboxylase, pyruvate dehydrogenase E1 component subunit alpha, citrate synthase, malate dehydrogenase(cytoplasmic), dihydrolipoyl dehydrogenase, acetyltransferase component of pyruvate dehydrogenase complex, isocitrate dehydrogenase[NAD] subunit(mitochondrial), and malate dehydrogenase(Table S-1). We can see that most proteins were from mitochondria, which is important in energy metabolism22 for synthesizing more than 95% of ATP23, 24, and involved in many cellular activity23. Methylene blue(MB) that act on destruction of redox and membrane equilibrium, then trigger malfunction of mitochondrial to resist infection of C. albicans25. Our

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results indicated that lysine acetylation might be of great significances to maintaining the proper function of mitochondria in C. albicans.

Figure 5 GO-based enrichment analysis of identified proteins. cal00051 Fructose and mannose metabolism cal00071 Fatty acid degradation

9.87E-03 5.08E-03 4.87E-03

cal00052 Galactose metabolism cal00640 Propanoate metabolism

3.10E-03

cal00030 Pentose phosphate pathway cal00630 Glyoxylate and dicarboxylate metabolism cal01230 Biosynthesis of amino acids

1.22E-03 2.37E-05 1.29E-05

cal00680 Methane metabolism

6.24E-06 3.52E-06

cal00190 Oxidative phosphorylation

1.71E-07

cal01100 Metabolic pathways

9.97E-08

cal00620 Pyruvate metabolism

2.66E-09

cal00020 Citrate cycle (TCA cycle)

5.69E-12

cal00010 Glycolysis / Gluconeogenesis cal01110 Biosynthesis of secondary metabolites cal03010 Ribosome

1.50E-12 6.82E-18 1.00E-30

cal01200 Carbon metabolism

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Fold enrichment

A

B

Figure 6 The pathway obtained from KEGG pathway enrichment analysis. (A) KEGG pathway-based enrichment analysis of identified proteins. (B)The proteins in yellow were identified in TCA of this project. Cyclophilin-like domain

1.47E-03 1.31E-03

ATP-grasp fold, subdomain 2 Aldolase-type TIM barrel

1.10E-03

Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain

6.77E-04

Translation elongation factor EFTu/EF1A, C-terminal

6.77E-04

Transketolase-like, pyrimidine-binding domain

6.13E-04

Histone core

4.13E-04

Isopropylmalate dehydrogenase-like domain

4.13E-04

Heat shock protein 70kD, peptide-binding domain

2.47E-05

Biotin/lipoyl attachment

2.44E-05

Thiamin diphosphate-binding fold

1.68E-05

Heat shock protein 70kD, C-terminal domain

8.88E-06

NAD(P)-binding domain

7.34E-06

Single hybrid motif

2.39E-06 0

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Fold enrichment

Figure 7 Protein domain enrichment analysis of identified proteins.

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According to domain enrichment analysis information, we can see many acetylated proteins existed in the domain, such as single hybrid motif, NAD(P)-binding domain, heat shock protein 70kD, thiamin diphosphate-binding fold, biotin/lipoyl attachment, isopropylmalate dehydrogenase-like domain, histone core, transketolase-like(pyrimidine-binding domain), cyclophilin-type peptidyl-prolyl cistrans isomerase domain, aldolase-type TIM barrel, translation elongation factor EFTu/EF1A (C-terminal), ATP-grasp fold(subdomain 2), and cyclophilin-like domain(Figure 7). Among all domains, acetylated proteins were abundant in transketolase-like, pyrimidine-binding domain. Transketolase (TK) sequences widely exists in the eukaryotic and prokaryotic sources26, 27 showed that the enzyme has been highly conservative, and transketolase was an enzyme of the pentose phosphate pathway in all organisms. Analysis of Acetylated Lysine Motifs There were many studies on both eukaryotic and prokaryotic cells, which have been found preferences for amino acid residues at special locations enclosing the acetylated lysine28-31. Thus, to further explore the regular pattern of the acetylated lysines in C. albicans, we investigated all identified acetylated lysines’ motif sequences using the Motif-X program, which was a software tool used to sum up forms from any random of sequences32. Among all acetyl-lysine peptides, 1073 acetylated sites were found to include the amino acid sequence from the −6 to +6 positions surrounding the acetylated lysine, and these were matched to a total of seven definitively conserved motifs (Figure 8), namely, Kac****K, Kac***K, KacW, Kac***R, KacY, KacH, and KacF (Figure 8A). The analysis of all motifs suggested that two particular types of residues were discovery of the nearby sites of the acetylated lysine: a positively charged residue such as arginine (R), histidine (H) or lysine (K), or a residue with aromatic groups, for instance phenylalanine (F), tryptophan (W) and tyrosine(Y) (Figure 8A). According to the position of the residues and other properties of the residues around the acetylated lysine, these motifs could be classified into two categories: the +4 or +5 position is an alkaline residue with a long side chain (K or R) and the +1 or +2 position is a residue with a long side chain (Y, H, W or F) (Figure 8B).

A

B

10


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70.0% M e a n p ro b a b ility o f s e c o n d a ry s tru tu re

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


66.3% 65.5%

60.0% 50.0% 40.0% 30.0%

All Lysine

27.0% 27.5%

Acetyl

20.0% 6.7% 7.0%

10.0% 0.0% Alpha-Helix

Beta-strand

Coil

Type of secondary struture

C

D

Figure 8 Bioinformational study of lysine acetylated sites. (A)Motif analysis of all identified Kac acetylated sites. The analysis of enriched acetylated sites in particular positions(6 amino acids close to two sides of the acetylated site) through Motif-X software. Seven enriched acetylated site motifs were labeled in statistically significantly. (B)Heat map showed the periodicity of the diverse types of amino acids which nearby the acetylated lysine. (C) Analysis of motifs involving identified peptides with acetylated sites. (D)Statistics of secondary structures involving identified lysine acetylated sites. The contrast of a variety of secondary structures (α helix, β strand and coil) involving identified acetylated lysine and all lysine secondary structure.

The first motif was remarkably conservative, and the acetylated peptides with this motif accounted for approximately 24.2% of all identified acetylated peptides( Figure 8C, Table S-5). Moreover, the frequency of K and R in positions −2 to +2 in the motif was low, as determined by an inspection of the heat map(Figure 8B). These results indicated that the lysine residue of a polypeptide with a K or R amino acid at the +4, +5 or +6 position ,which was a preferred substrate of lysine acetyltransferase. Intriguingly, some acetylated lysine motifs were also observed in other species, such as Kac****K,Kac*** K, KacY, and KacH in human cells28, 31, 33, Kac*** K in rat34, KacY in grape35, Kac****K, KacY, and KacH in Vibrio parahemolyticus36, Kac***R, KacH,KacY, Kac****K , Kac***K and Kac****R in Mycobacterium tuberculosis16, which suggested that lysine acetyltransferases were broadly distributed in prokaryotes and eukaryotes. To detect the protein secondary structures which involving lysine acetylation, we further studied all lysine proteins’ secondary structures (Figure 8D). Among acetylated lysine proteins, approximately 27.5% sites were located in α-helix, 7% in β-sheet and 65.5% in coil. This was in consistent with results from acetyl proteome of Streptomyces reseosporus18 and Mycobacterium tuberculosis16 for an alike distribution mode from secondary structures. But it was different from HeLa cells28 for approximately 58.8% sites located in α-helix, 9.4% in β-sheet and 31.8% in coil of acetylated lysine proteins. Analysis of histone lysine acetylated modifications As we all know that sorts of modifications on histone tails has important implications for transcription initiation. For example, H3K56ac is specific for it occurs on the spherical structure of histone, and this histone acetylated structure is directly referring to DNA pre-initiation complex and transcriptional activation37-40. Wurtele et al. found that H3K56ac acetylation levels can cause alterations by genetic or pharmacological reasons then decreased C. albicans virulence in mouse,

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which told us reduction H3K56ac levels may be associated with pathogenicity of C. albicans41. In small cell lung cancer, KAT6B inhibited tumor by affected a newly discovered H3K23ac transferase activity42. H4K12ac was localized at promoters43 and may be important for gene expression44. H4K16ac was globally reduced in cancer45 and it was important for higher order chromatin conformation46. In this study, we analysed all acetylated histones in C. albicans to find the universal law. We discovered H2AK6, H2AK11, H2AK13, H2BK7, H2BK8, H2BK12, H2BK17, H2BK18, H2BK22, H2BK23, H2BK47, H2BK112, H3K10, H3K15, H3K19, H3K24, H3K28, H3K37, H3K57, H4K8, H4K11, H4K15, H4K19, H4K62, and H4K94 were acetylated (Table 1). As far as we know,those screening acetylated sites have not been previously reported in C. albicans. Table 1 Acetylation sites and acetylated histones of C. albicans identified in this Study Protein accession

Position

Amino acid

Protein names

Gene names

H2A Q5AEE1

6

K

Histone H2A.Z

H2AZ

Q5AEE1

11

K

Histone H2A.Z

H2AZ

Q5AEE1

13

K

Histone H2A.Z

H2AZ

H2B Q59VP1

7

K

Histone H2B.1

H2B1

Q59VP1

8

K

Histone H2B.1

H2B1

Q59VP1

12

K

Histone H2B.1

H2B1

Q59VP1

17

K

Histone H2B.1

H2B1

Q59VP1

18

K

Histone H2B.1

H2B1

Q59VP1

22

K

Histone H2B.1

H2B1

Q59VP1

23

K

Histone H2B.1

H2B1

Q59VP1

47

K

Histone H2B.1

H2B1

Q59VP1

112

K

Histone H2B.1

H2B1

H3 Q59VN2

10

K

Histone H3.3

H33

Q59VN2

15

K

Histone H3.3

H33

Q59VN2

19

K

Histone H3.3

H33

Q59VN2

24

K

Histone H3.3

H33

Q59VN2

28

K

Histone H3.3

H33

Q59VN2

37

K

Histone H3.3

H33

Q59VN2

57

K

Histone H3.3

H33

H4 Q59VN4

8

K

Histone H4

Q59VN4

Q59VN4

11

K

Histone H4

Q59VN4

Q59VN4

15

K

Histone H4

Q59VN4

Q59VN4

19

K

Histone H4

Q59VN4

Q59VN4

62

K

Histone H4

Q59VN4

Q59VN4

94

K

Histone H4

Q59VN4

12


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Figure 9 Western Blotting analysis of histone Kac modification. Primary antibody: anti-acetyl-histone H2BK12 (Lys12) mouse mAb（PTM-153; 1:1000 dilution); anti-acetyl-histone H2BK23(Lys23) rabbit pAb (PTM-171; 1:1000 dilution); anti-histone H2B mouse mAb (PTM-1006; 1:1000 dilution); anti-histone H4K8(Lys8) rabbit mAb (ab15823; 1:1000 dilution); anti-histone H4 rabbit mAb (ab61254; 1:1000 dilution). Secondary antibodies: (HRP)-Goat anti-mouse IgG(H+L)(Pierce-31460, 1:2000 dilution); (HRP)-Goat anti-Rabbit IgG(H+L)(CW0103S, 1:2000 dilution).

In order to verify the analysis results of mass spectrometry, we used sequence-specific antibodies which were commercially available to examine histone modification sites through western blotting analysis. We found that H2B, H2BK12ac, H2BK23ac were detected at 15kDa(Figure 9A) and H4, H4K8ac were detected at 12kDa(Figure 9B). H2BK12ac along with apoptosis was discovered as a target for auto antibodies in SLE47. H4k8ac has been particular studied in many illnesses, which was proved that involving in cognition. The latest evidence suggested that H4K8ac regulated learning capacity and memory48-49. Besides, E. coli induced mice mammary tissue inflammation led to H3K14 and H4K8 hyperacetylation in the immune gene’s promoter, which was a precondition of expressing inflammatory genes then starting a significant immune response50. Flow cytometry detected remarkable up-regulation of H4K5ac, H4K8ac, H4K12ac, and H4K16ac in systemic lupus erythematosus monocytes51. The significance of those histone modifications in fungal infection needs further research. Enrichment analysis of crosstalk between lysine acetylation and phosphorylation on GO categories , KEGG and protein domains Among the many PTMs, phosphorylation has been studied most extensively because of its critical role in the modulation of cellular processes52. Lysine phosphoproteome has been recently identified in C. albicans53. In our study , proteins have been found crosstalk between lysine acetylation and phosphorylation, which may play a major role in metabolic pathways. A comparison of the two modified proteome data was performed classified as enrichment GO categories(biological process, cellular compartment, molecular function), KEGG and protein domains(Figure S-1).

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In the biological process category (Figure S-1A), We can see that 11 biological processes were modified by acetylation(15%), 18 biological processes modified by phosphorylation(25%), 45 biological processes were modified by acetylation and phosphorylation(60%). Among those crosstalk biological processes, 20%(9/45) processes related to glycolysis/gluconeogenesis(carbon metabolism), such as carbohydrate catabolic, single-organism carbohydrate metabolic, single-organism carbohydrate catabolic, one-carbon metabolic, carbohydrate derivative catabolic, monosaccharide metabolic, carbohydrate metabolic, glycosyl compound catabolic, and glycosyl compound metabolic. Furthermore, the study of cellular compartment(Figure S-1B) indicated that 15 cellular compartments were modified by acetylation(30%), 18 biological processes modified by phosphorylation(36%), 17 biological processes modified by both acetylation and phosphorylation(34%). Among those 18 crosstalk cellular compartments, 22%(4/18) compartments were related to cell wall, such as fungal-type cell wall, hyphal cell wall, cell wall, and yeast-form cell wall. And about 33%(6/18) compartments related to ribosome, such as cytosolic ribosome, small ribosomal subunit, ribosomal subunit, ribonucleoprotein complex, cytosolic small ribosomal subunit, and ribosome. The enrichment study of molecular functions (Figure S-1C) revealed that 17 molecular functions were modified by acetylation(35%), 10 molecular functions modified by phosphorylation(20%), 22 molecular functions modified by both acetylation and phosphorylation(45%). Among those 22 crosstalk molecular functions, 18%(4/22) proteins related to oxidoreductase activity. 23%(5/22) proteins related to translation, such as RNA binding, structural constituent of ribosome, translation factor activity(nucleic acid binding), translation elongation factor activity, and translation initiation factor activity. To study cellular pathways which involving lysine acetylation and phosphorylation co-modification, we conducted a pathway enrichment analysis the KEGG. Our data showed that 11 pathways were modified by acetylation(31%), 7 pathways such as MAPK signaling pathway, phosphatidylinositol signaling system etc. were modified by phosphorylation(19%), 18 pathways were modified by both acetylation and phosphorylation(50%). Among those 18 crosstalk pathways(Figure S-1D), 39%(7/18) pathways related to glycometabolism, such as Galactose metabolism, Fructose and mannose metabolism, Pentose phosphate pathway, Amino sugar and nucleotide sugar metabolism, Glycolysis/Gluconeogenesis, Starch and sucrose metabolism, and Citrate cycle (TCA cycle). An important functional indication of proteins is particular domain structure. Therefore, we studied the domain modification characteristics of acetylated proteins. We found 3 domains were modified by acetylation(7%), 28 modified by phosphorylation(67%), 11 modified by both acetylation and phosphorylation(26%). Histone core was lysine acetylation modified, Peptide-binding domain(Heat shock protein 70kD) and C-terminal domain(Heat shock protein 70kD) were found modified by both acetylation and phosphorylation(Figure S-1E). Cells may express Heat shock proteins(HSP) to feedback the stressful54-57. In both mammals and yeast, function of Hsp90 was regulated by phosphorylation of several serine, threonine, and tyrosine residues58, 59. Phosphorylation of S. cerevisiae Hsp90 on tyrosine 24 by the Swe1 kinase and on threonine 22 by protein kinase CK2 was important for Hsp90’s ability to interact with and stabilize several cochaperones and client proteins60, 61. Hsp90 played an important function in pushing the quick reaction of resistance through different mechanisms in C. albicans and

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S.cerevisiae62. Hsp90 via calcineurin regulated crucial cellular function, got feedback to the cell wall stress then governed echinocandins resistance in C. albicans63. In C. albicans and S.cerevisiae, the lysine deacetylases(KDACs) pharmacological inhibition affected the Hsp90-dependent resistance to the azoles19. Hsp90 interacted with 10% of the S. cerevisiae proteome64 and the C. albicans Hsp90 genetic interaction network reaffirmed Hsp90’s high degree of connectivity65. Posttranslational control of Hsp90 function was likely to be fundamental for its capacity to modulate client proteins in a dynamic and environmentally contingent manner. In our study, Hsp90 of C. albicans on lysine 199, 477 and 591 positions were acetylated(Table S-1). Given that Hsp90 is regulated by acetylation of lysine 27 and 270 in S. cerevisiae19, whether those acetylated positions we detected in Hsp90 of C. albicans can influence fungal drug resistance needs further research. PPI network study of crossover between lysine acetylation and phosphorylation in C. albicans In C. albicans, we found 477 proteins were modified by acetylation, 2896 proteins were modified by phosphorylation53, and 267 proteins were modified by both acetylation and phosphorylation. We used PPI to analyze acetylated and phosphorylated proteins through Cytoscape software. The C. albicans PPI network used all acetylated proteins as nodes, direct physical linked with 1861 proteins which analyzed from the STRING database of physical and functional interactions. We formed a thorough acetylated proteins network. Consequently, Figure 10 offered an understanding of the relationship of interactions among acetylated proteins in C. albicans. The node degree was an useful measurement to assess the relationship of proteins from network(Table S-6). Six proteins which revealed the maximum degree in network were modified by both acetylation and phosphorylation including likely cytosolic ribosomal protein S3(Q59N00), likely cytosolic ribosomal protein L5(Q5AGZ7), likely cytosolic ribosomal protein S2(Q5A900), 40S ribosomal protein S6(Q5AMI6), likely cytosolic ribosomal protein L11(Q59Z66) and likely cytosolic ribosomal protein L17(Q59TE0). The former studies of acetylome of eukaryotic cells66 and E. coli14 showed that multiple functions of acetylated lysine existed in various pathways, but whether those functions existed in C. albicans needed more study. In our research, we studied functions of acetylated lysine and analyzed interaction of each other. We further illustrated the occurrence of lysine acetylation and phosphorylation in four cluster (Figure S-2) and three key metabolic pathways: glycolysis/ gluconeogenesis, oxidative phosphorylation, and ribosome (Figure 10). Many acetylated proteins were involving in multifarious cellular process and PPI. Such as 42 acetylated proteins of cluster1 (ribosome) were linked to complex PPI network (Figure 10 and Figure S-2A). About 86 %(36/42) acetylated proteins modified by phosphorylation in cluster1 were related to significant biological process. There was strongest interaction of 60S ribosomal protein L20 (Q5AML4, both acetylation and phosphorylation) and likely cytosolic ribosomal protein S25(Q59NZ2, acetylation). 60S ribosomal protein L20 was identified to be acetylated at lysine 23, 38,47,92,141 and 146, also found two phosphorylation sites(see Table S-1 and Table S-6). And 60S ribosomal protein L20 was discovered to combine the 35S pre-rRNA67 and has significant functions in series of processes including macromolecule biosynthetic process, cellular metabolic process, cellular macromolecule metabolic process, organic substance biosynthetic process, gene expression, cellular biosynthetic process, cellular protein metabolic process, protein

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metabolic process, organic substance metabolic process, biosynthetic process, metabolic process, primary metabolic process, cellular macromolecule biosynthetic process, macromolecule metabolic process, translation, and cellular process.

Figure 10 lysine acetylation and phosphorylation of proteins involved in the glycolysis/gluconeogenesis, oxidative phosphorylation, and ribosome. Acetylation proteins identified in C. albicans are shown in red ovals, sites that were found to be both acetylated and phosphorylation are shown as blue and red circles.

Thirteen acetylated proteins were involved in cluster2 (oxidative phosphorylation, Figure 10 and Figure S-2B) and 12 acetylated proteins in cluster3 (oxidative phosphorylation, Figure 10 and Figure S-2C). Among those acetylated proteins, 30%(4/13) proteins were modified by both acetylation and phosphorylation in cluster2, and 33%(4/12) modified by both acetylation and phosphorylation in cluster3. At the same time, 100%(9/9) proteins involved in cluster4 (glycolysis/gluconeogenesis, Figure 10 and Figure S-2D) were modified by both acetylation and phosphorylation. Compared with other clusters, we can see that the phosphorylated proteins in acetylated oxidative phosphorylation PPI network was not abundant. CONSLUSIONS This is the first study of acetylome in human pathogenic fungi, we analyzed the acetylated sites of C. albicans proteome and got the first systematic study of the acetylome from this human pathogen. We identified 1073 lysine acetylated sites in 477 acetylated proteins, and these proteins accounted for 5.28% of the total proteins in C. albicans. In addition, through extensive characterization of the acetylome, we found that the acetylated proteins were mostly associated with cellular functions and were distributed in different cellular compartments. The lysine modification of proteins linked with protein biosynthesis or carbon metabolism may play particularly important physiological roles in C. albicans. Moreover, the study of the amino acid of motifs told us that the acetylated lysine was enclosed by an alkaline residue(K or R)

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at the +4, +5 or +6 position or by a residue(Y, H, W or F) at the +1 or +2 position. These motifs were also found in human cells and other bacterial cells. We found many lysine acetylation on histone tails which might important for transcription initiation. To the best of our knowledge, all screening acetylated histone sites of this study have not been previously reported in C. albicans. The PPI networks resulting from the acetylome clearly indicated that multitudinous regulatory roles were modulated by acetylation and/or phosphorylation in C. albicans. Compared with 86%(36/42) acetylated proteins of cluster1(ribosome) and 100%(9/9) acetylated proteins of cluster4 (glycolysis/gluconeogenesis) modified by phosphorylation, only 30%(4/13) acetylated proteins were modified by phosphorylation in cluster2(oxidative phosphorylation), and 33%(4/12) in cluster3(oxidative phosphorylation). Compared with other clusters, the phosphorylated proteins in acetylated oxidative phosphorylation PPI network was not abundant, which indicated that acetylation may do more active role than phosphorylation in oxidative phosphorylation PPI network. Our research proved that the LC-MS/MS is useful for a massive study of protein acetylation. These data represented an important development of our latest knowledge of acetylome and suggested that abundant of lysine acetylation were associated with many cellular processes in C. albicans. As the first study of acetylome in human pathogenic fungi, the study provided an available resources for the in-depth study on the functional mechanism of lysine acetylation in such fungal pathogens. ASSOCIATED CONTENT Supporting Information Additional experimental details. Table S-1, lysine acetylation protein annotation; Table S-2, the default parameters for acetylomic raw data searching; Table S-3, the original data of Venn diagram(Yeast, Table S-3A; Mouse, Table S-3B; Human, Table S-3C; E. coli, Table S-3D); Table S-4, Functional Characterization of Lysine Acetylome: the distribution of identified Kac proteins in GO terms of level 2(Table S-4A), Identified Kac protein distribution in KOG categories (Table S-4B), The subcellular location of identified Kac proteins(Table S-4C); Table S-5, motif analysis; Table S-6, PPI network; Figure S-1, Enrichment analysis of crosstalk between lysine acetylation and phosphorylation on biological process category(Figure S-1A), cellular compartment(Figure S-1B), molecular functions(Figure S-1C), KEGG(Figure S-1D) and protein domains(Figure S-1E); Figure S-2: PPI network analysis of crosstalk between lysine acetylation and phosphorylation in four cluster: cluster1(ribosome, Figure S-2A), cluster2(oxidative phosphorylation, Figure S-2B), cluster3(oxidative phosphorylation, Figure S-2C), cluster4 (glycolysis/gluconeogenesis, Figure S-2D).

AUTHOR INFORMATION Common Corresponding Author *Xiaofang Li: E-mail: [email protected]; Tel: +086 25-85478983; Fax number: +086 25-85414477; *Weida Liu: E-mail: [email protected]. ACKNOWLEDGMENTS Our research was funded by National Natural Science Foundation of China (Grant number 81573059) and Jiangsu Provincial Special Program of Medical Science (Grant number BL2012003).

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