Systematic Analysis of the Lysine Succinylome in Candida albicans

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Systematic Analysis of the Lysine Succinylome in Candida albicans Hailin Zheng, Yun He, Xiaowei Zhou, Guanyu Qian, Guixia Lv, Yongnian Shen, Jiyun Liu, dongmei Li, Xiaofang Li, and Weida Liu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00578 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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Systematic Analysis of the Lysine Succinylome in Candida albicans 、



Hailin zheng1 2, Yun He1, Xiaowei Zhou1 2, Guanyu Qian1, Guixia Lv1, Yongnian Shen1, Jiyun Liu3, Dongmei Li1,4, Xiaofang Li1

、2*

, Weida Liu1

、2*

1Department 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;4Georgetown University Medical Center, Department of Microbiology & Immunology, Washington, DC, 20057,USA.

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ABSTRACT

Candida albicans is the most common human fungal pathogen for both immunocompetent and immunocompromised individuals. Lysine succinylation is a frequently occurring posttranslational modification (PTM) that is found in many organisms, however, the roles of succinylation is still under investigation. Here, we initiated a first screening of lysine succinylation in C. albicans. We identified 1550 succinylation sites from 389 proteins in C. albicans; demonstrating succinylation is conservative in this organism. However, the lysine succinylation sites showed some difference in C. albicans, the overlapping rates between C. albicans and other species were ranging from 55% for S. cerevisiae, 40% for human, 35% for mouse and to only 16% for E. coli. The further bioinformatics analysis indicated that the succinylated proteins were involved in a wide range of cellular functions with diverse subcellular localizations. Furthermore, we discovered that lysine succinylation could coexist with phosphorylation and/or acetylation in C. albicans. The KEGG enrichment pathway analysis of these succinylated proteins suggested that succinylation may play an indispensable role in the regulation of the TCA cycle. The bioinformatic data obtained from this study therefore enables the depth-resolved physiological roles of lysine succinylation in C. albicans.

KEYWORDS: Lysine succinylation, succinylation motif, crosstalk, function, Candida albicans

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INTRODUCTION

Systemic fungal infections have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation)1 and have become a major health concern for the patients (e.g., on an intensive care unit) who may suffer hospital-related infections 2-4. C. albicans is one of the most commonly encountered human pathogen that relies upon reversible morphogenetic transitions between budding, pseudohyphal and hyphal forms for their virulence

5, 6

. C. albicans also uses complex networks of

transcriptional activators and repressors to modulate the switch in its role as a commensal inhabitant of the gastrointestinal tract to becoming a pathogen in the bloodstream. 6, 7. Since the mammalian host and invading fungi are both eukaryotic, it is difficult to apply antifungal agents that only target the pathogen, which is evident from the facts that none of the existing antifungal drugs is free from side effects in humans 8.

In order to avoid or at least minimize these side effects, the comparative

genomics approach has been widely applied recent years to search for an appropriate drug target in C. albicans. Some progress have been made in screening possible drugs that target a variety of cellular activities including enzymes, transporters, receptors and transcription factors 8. Most of these are proteins, regardless of the specific function each may perform. Protein posttranslational modifications (PTMs) are covalent processing events that often change the properties of a protein. PTMs not only change the protein structure, it also affects the protein functions such as trafficking and protein-protein interactions 3 ACS Paragon Plus Environment

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through a proteolytic cleavage or by the addition of a modifying group to one or more amino acid residues of a given protein

9, 10

. The common types of PTMs including

phosphorylation, acetylation, methylation, glycosylation, ubiquitination, sumoylation, and neddylation9-16 are able to modulate a wide variety of cellular processes in eukaryotes and prokaryotes, to regulate cell growth and division as well as to adapt environmental changes and guide the developmental processes. The posttranslational modification either makes chemical changes on a candidate protein, such as the glycosylation of a cell wall mannoprotein, or it alters the function of a specific protein, such as changing the state of enzyme activity, localization, stability, and interactions with other proteins. Protein phosphorylation is one of the best known PTMs, which is a ubiquitous and reversible modification that is crucial for the regulation of cellular events. Signaling pathways often culminate in the phosphorylation of specific transcription factors that direct gene expression patterns which modulate in turn the corresponding adaptive or developmental process. For example, the heat shock transcription factor Hsf1, which controls thermal adaptation in C. albicans and other eukaryotic cells, is phosphorylated in response to heat shock and this phosphorylation is also essential for the virulence of C. albicans

10

. In addition, protein

phosphorylation via cAMP-protein kinase A contributes to fungal virulence through regulation of S. cerevisiae hyphal morphogenesis and stress resistance in C. albicans. Up to date, lysine acetylation is the best studied PTM and can be served as a good model for investigation of other PTMs. Accumulating evidence indicates that lysine acetylation is a conserved PTM that is involved in almost any aspect of cellular 4 ACS Paragon Plus Environment

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process and also links Ac-CoA metabolism to the cellular signaling. It is well known that the genome organization, gene regulation, DNA replication, and DNA repair are all essential for cell growth and pathogenicity. These processes are frequently controlled by histone acetylation

13, 17

. Histone (de)acetylation, on the other hand

contributes to morphogenetic regulation and phenotypic switching in C. albicans. Both these two PTM steps have been linked to virulence in this organism 9, 18. Protein lysine succinylation, in contrast to other PTM types mentioned above, has been recently recognized as one of conserved protein posttranslational modifications that play the important roles in regulation of cellular bioprocesses.

19, 20

. It is widely

distributed among prokaryotes and eukaryotes, such as E. coli, S. cerevisiae, mouse and human cells. Lysine succinylation is a highly dynamic process, in which a succinyl group (-CO-CH2-CH2-CO-) is transferred to a lysine residue of a protein molecule21.

As the result, two negative charges (from a ﹢1 to a 1 charge) are

added to a lysine residue during lysine succinylation at physiological pH. This addition of a modifying group and the two extra charges in turn modulate the structure and functions of a substrate protein21. For example, lysine succinylation has been noted to regulate the functions of histones and affect chromatin structure and gene expression in previous studies22, 23. Lysine succinylation was also reported to be involved in the lipid oxidation under oxidative stress and in regulation of the activity of metabolic enzymes. To date, most studies on lysine succinylation point out that this type of PTM is a dynamic, abundant, and evolutionarily conserved event among diversified species

19, 24, 25

. In eukaryotic cells, posttranslational modifications of 5

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proteins drive a wide variety of cellular processes and regulate cell growth and division as well as adaptive and developmental processes26.

More recently, the

relevance of posttranslational modifications on virulence of other pathogens highlights the necessary to study the roles of lysine succinylation during the fungal infection

9, 27

In C. albicans, it remains unknown if lysine succinylation relates to

Candida virulence or there is a possibility to develop the antifungals on basis of this PTM event 19. In present study, we performed a large scale analysis of lysine succinylated proteins in C. albicans as the first step in trying to understand the function of succinylation in this organism. The lysine succinylomewas obtained from C. albicans SC5314 using an integrated proteomic method. A total of 1550 unique lysine succinylation sites in 389 succinylated proteins were identified, which carry on remarkably diverse biological functions and have different cellular localizations in this fungus. In addition, several hundreds of proteins were found to have succinylation simultaneously with other species such as bacteria (E. coli), S. cerevisiae, mouse liver tissue, and human (HeLa) cells, demonstrating a conservative succinylation in diverse organisms. Moreover, we found that a majority of succinylated proteins of C. albicans are also able to be phosphorylated and/or acetylated at the same time. The KEGG pathway analysis indicates that a very wide range of regulatory roles are modulated by succinylation. These results provide the first comprehensive view of the succinylome of C. albicans.

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MATERIALS AND METHODS Strains and Culture. The C. albicans strain SC5314 was grown at 28 °C on a rotary shaker (220 rpm) overnight in YPD medium and used as seed cultures. 10 mL of seed culture was inoculated into flasks containing 100 mL of fresh YPD medium, and the latter were then cultured with shaking (220 rpm) at 28 °C for 4 hours until OD600 reached to 0.8. Protein Extraction and Digestion. The cultured cells were harvested by centrifugation at 6,000 rpm and 4 °C for 10 min then washed twice with cold PBS. The pellets were first grinded by liquid nitrogen, then the cell powder was transferred to 5 mL centrifuge tube and sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer (8 M urea, 1% Triton-100, 65 mM DTT and 1% Protease Inhibitor Cocktail). The remaining debris was removed by centrifugation at 20,000 g at 4 °C for 10 min. The protein was finally precipitated with cold 15% TCA for 2 h at -20 °C. After centrifugation at 4 °C for 10 min, the supernatant was discarded. The remaining precipitate was washed with cold acetone for three times. The protein was re-suspended in buffer (8 M urea, 100 mM NH4CO3, pH 8.0) and the protein concentration was determined with 2-D Quant kit according to the manufacturer’s instructions. For protein digestion, the protein solution was pretreated with 10 mM DTT for 1 h at 37 °C and then alkylated with 20 mM IAA for 45 min at room temperature in darkness. After these pretreatment, the protein sample was diluted by a mixture of 100 mM NH4CO3 into urea solution less than 2 M. Finally, trypsin was 7 ACS Paragon Plus Environment

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added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and at 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. Western Blotting Western blotting assays were performed using protein lysates from whole cell that were separated by 12 % SDS-PAGE. After transferred to nitrocellulose membrane (Millipore Corp), the membranes were incubated in blocking buffer (0.05 % Tween 20 and 5 % nonfat milk powder in PBS). Succinylated lysines were detected using rabbit-derived polyclonal anti-succinyl lysine antibody (PTM Biolabs) that had been diluted in blocking buffer at 1:1000 overnight at 4℃. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Sigma) at 1:2,000 and chemiluminescence substrate for detection (Sigma). Enrichment of Lysine Succinylated Peptides To enrich Ksu peptides, tryptic peptides in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with pre-washed antibody beads (PTM Biolabs) at 4°C overnight with gentle shaking. The beads were washed four times with NETN buffer and twice with ddH2O. The bound peptides were eluted from the beads with 0.1% TFA (.The resulting peptides were cleaned with C18 ZipTips (Millipore) according to the manufacturer’s instructions, followed by LC-MS/MS analysis. In this research, 5 parallel enrichments were performed and analyzed, respectively. LC-MS/MS Analysis by Q Exactive Peptides were dissolved in 0.1% FA (formic acid), directly loaded onto a 8 ACS Paragon Plus Environment

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reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) with an 6% to 23% solvent B (0.1% FA in 98% ACN) gradient increase for 24 min, an 23% to 35% increase for 8 min, following an 80% raising in 4 min and sustaining at this gradient for extra 4 min.

The flow rate

was constantly maintained at 280 nl/min on an EASY-nLC 1000 UPLC system, the resulting peptides were analyzed by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptides were subjected to NSI source followed by a tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) that is 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 NCE setting as 30; 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 1.5E4 in the MS survey scan with 30.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Database Search The resulting MS/MS data were processed using MaxQuant with integrated Andromeda search engine (v.1.4.1.2) and tandem mass spectra were then searched 9 ACS Paragon Plus Environment

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against Uniprot_Candia_Albicans database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 3 missing cleavages, 4 modifications per peptide and 5 charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, succinylation on Lys and acetylation on protein N-terminal were specified as variable modifications. The thresholds of false discovery rate (FDR) for protein, peptide and modification site were specified at 1%. Minimum peptide length was set as 7. All the other parameters in MaxQuant were set to default values. The probability of site localization was set as > 0.75. Go Annotation Enrichment Analysis Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/) and proteins were then classified into three categories according to GO annotation: biological process, cellular component and molecular function. For Domain Annotation, the functional description of identified protein domains were referred from InterProScan (a sequence analysis application) based on protein sequence alignment method and the InterPro domain database. We also use InterPro (http://www.ebi.ac.uk/interpro/), a freely available to the public database that integrates diverse information of protein families, domains and functional sites, to interpret our proteomic data. KEGG Pathway Annotation The proteomic data were further analyzed with KEGG (Kyoto Encyclopedia of Genes and Genomes) for pathways annotation that includes Metabolism, Genetic 10 ACS Paragon Plus Environment

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Information Processing, Environmental Information Processing, Cellular Processes, Rat Diseases and Drug development. Subcellular Localization For protein subcellular localization, we used subcellular localization predication software- an updated version of PSORT/PSORT II of Wolfpsort against eukaryotic sequences database. Motif Analysis Soft motif-x was used to analyze the model of sequences that contains amino acids in specific positions of modifier-21-mers (10 amino acids upstream and downstream of the site) for all proteins. And all the protein sequences in the database were used as background database parameter, with other parameters following the default values. Functional Enrichment A two-tailed Fisher’s exact test was employed to test the enrichment of the protein-containing IPI entries against all IPI proteins. Correction for multiple hypothesis testing was carried out using standard false discovery rate control methods. Any pathway with a corrected p-value < 0.05 was considered significant.

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RESULTS AND DISCUSSION Proteome-Wide Analysis of Lysine Succinylation Sites and Proteins in C. albicans The overall succinylated proteins of C. albicans were first evaluated by Western blot, in which succinyl-lysine antibody was used against whole cell lysate. As shown in Figure S1, a great number of proteins that had been succinylated with a wide range of molecular masses were detected in Western blot. To further determine the protein succinylome in C.albicans, a proteomic method based on affinity purification and LC−MS/MS was then applied

11

. In this research, 5 parallel enrichments were

performed and analyzed, respectively. The overlap of the 5 repeats were shown in Figure S2. The distribution of mass error in general is near zero and most of them are less than 3 ppm which satisfies the mass accuracy of the MS.

Lengths of most

peptides range from 8 to 20 as the typical tryptic peptides, which further confirms that the sample preparation met the requirement. Figure S3 shows three MS/MS spectra of succinylated peptides of protein ACO (P82611) with their succinylation sites at K43. K44, and K53, respectively. With this LC−MS/MS analysis, 1550 succinylation sites from 389 proteins were identified as shown in Table S1, which accounts for 2.6 % of the total proteins (14633) of C. albicans 8. In order to characterize the nature of the succinylation in C. albicans, the sequence motifs of succinylated lysines in all of the succinylated sites were analyzed using the Motif-X program, a software tool that has been designed to extract overrepresented patterns in any giving set of sequences. Of all of the succinyl-lysine peptides, 735 12 ACS Paragon Plus Environment

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peptides were found to include the desired amino acid sequence from the −6 to the +6 positions surrounding the succinylated lysine. The succinylated sites in these peptides range from 1 to 25, and all match to a total of seven defined motifs, namely, A*****KSucc, KSucc*A, A**KSucc, KSuccG, KSucc*G, A*KSucc, and V*KSucc* in different abundances (Figure 1A & 1B), in which KSucc indicates the succinylated lysine and * indicates a random amino acid residue. Among these motifs, the first motif in Figure 1A is strikingly conserved, covering approximately 38 % of all the succinylated peptides we have identified. A comparison of these motifs indicates that three residues - alanine (A), glycine (G) and valine (V) are commonly found around succinylated lysine. Alanine at positions -6 to +6 and glycine/valine at positions -2 to +2 have higher frequencies when the amino acid compositions surrounding the succinylation sites were inspected (Figure 1c). Furthermore, we found it intriguing that some succinylated lysine motifs of C. albicans are conserved among species. For example, KSucc*A in S. cerevisiae, A**KSucc in mouse, KSuccG and A*KSucc in humans cells 22, KSucc*G in the Protozoan Toxoplasma gondii, and the alanine residue at positions of -4 to +3 around Ksucc sites in E. coli9, 28 can also be found respectively in succinylated lysine proteins from each species. These results are consistent with the fact that lysine succinyltransferases are broadly distributed in eukaryotes and prokaryotes by genetic BLAST analysis

29

.

Unexpectedly, we have also discovered that V*KSucc* is the only motif that has never been reported as a succinylated lysine motif in any organism. Whether this unique motif of C. albicans is meaningful for its functions is a motivation for a further study. 13 ACS Paragon Plus Environment

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In order to determine if succinylation was biased to occur within particular secondary structural domains of proteins, the secondary structure of each protein was assigned using the UniProt database, and whenever found, succinylated and non-modified lysines are then compared. We find it interesting that no significant difference in usage has been found between lysine and succinylated lysine in each type of secondary structure (Figure S4). The ratios of all lysine and succinylated lysine in helical regions and coiled regions of C. albicans are almost equal, suggesting that succinylation does not significantly change protein secondary structure. Instead, succinylation may predominantly affect the charges of modified proteins by addition of a modifying group. Lysine succinylation in eukaryotes and prokaryotes Lysine succinylation has been assigned as one of important regulatory PTMs that may affect a variant of metabolic pathways. In E. coli, the initial step of methionine biosynthesis is activation of homoserine via succinylation reaction that is catalyzed by homoserine trans-succinylase (HTS) encoded by the metA gene20. Lysine succinylation is crucial for maintaining structure and function of histones in S. cerevisiae

22

. In mouse liver tissue, all the succinylated proteins are metabolic

enzymes, suggesting this type of PTM may important in metabolic regulation

13, 30

.

Evidence gathered recently also supports the notion that lysine succinylation dynamically regulates enzymes that are specifically related to carbon metabolism in both bacteria and human cells 29. To improve our understanding of succinylation in C. albicans, we compared 14 ACS Paragon Plus Environment

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succinylated proteins that have identified in this study with succinylated proteins that have been reported in other prokaryote and eukaryotes such as E. coli, budding yeast (S. cerevisiae), mouse (liver tissue), and human (cervical cancer (HeLa) cells)12. The succinylated data listed in Table 1 are quite compatible since same strategy had been used in each species for the respective succinyl-proteomes s 12, that is enrichment of the succinylated peptides by immune affinity purification before proteome performed by LC−MS/MS. The results shown in Table 1 revealed significant differences of succinylation among species in either number of succinylated sites or number of succinylated proteins. We found a total of 1550 succinylation sites and 389 succinylated proteins in C. albicans. The average number of succinylation sites carried by each protein in C. albicans is 3.98 when compared to 2.6 of E. coli (2572 succinylation sites over 989 succinylated proteins), 2.83 of S. cerevisiae (1345 succinylation sites over 476 succinylated proteins), 3.03 of mouse (2140 succinylation sites over 705 succinylated proteins) and 2.89 of human HeLa cells (2004 succinylation sites over 695 succinylated proteins)12,

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. Apparently, C. albicans

possesses the highest level of succinylation per protein although the total number of succinylated proteins is much less than other species. On the other hand, prokaryote E. coli has more succinylated proteins than all eukaryotes in Table 1 with lowest number of modified sites per protein. The distribution of succinylated proteins differs among species as well. Some overlaps can be found between C. albicans and other species when focused on the same positions of succinylation. As shown in Figure 2 and Table S2, the overlap rates are as follows: 16 % between C. albicans and E. coli, 55% 15 ACS Paragon Plus Environment

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between C. albicans and S. cerevisiae, and 35% or 40%, respectively, between C. albicans and mouse or human. It is not surprising to have the lowest overlap rate between C. albicans and E. coli since prokaryotes and eukaryotes are very different in their cellular structures. Among eukaryotes, succinylated proteins of C. albicans are more close to the ones of S. cerevisiae, almost certainly due to the close genetic relationship between these two species. Therefore, the higher overlap rate between C. albicans and mouse liver or HeLa cells than prokaryote can be explained by the genetic relatedness between them and is likely due to the similar subcellular distribution of succinylated proteins 12. These results indicate that succinylation occur frequently in both eukaryotes and prokaryotes with certain species specificity. Characterization of Lysine Succinylome of C. albicans All succinylated proteins that we identified from C. albicans were annotated via the GO functional classification. The annotation revealed that succinylated proteins are involved in diverse biological processes and molecular functions, suggesting that succinylation may be one of the important PTMs in C. albicans. With regards to biological process, molecular function and subcellular location, these succinylated proteins are mainly involved in metabolic process (29%) for binding (41%) or catalytic activity (41%) and most of them are cellular (41%) or organelle (24%) localized as shown in Figure 3. In terms of biological process,the second largest group is composed of proteins associated with cellular processes, and the number of these proteins in this group is 28% of all of the identified proteins (Figure 3A). The higher distributions in metabolic process and catalytic activity in Figure 3A & 3B 16 ACS Paragon Plus Environment

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demonstrate that succinylation is necessary for metabolism in C. albicans. The subcellular location results (Figure 3C) of 41% in the cytoplasm, 24% in the organelle and 16% in micromolecular complex (6%) are consistent with results from the bacteria and eukaryotic cells as previously reported

12, 24

, indicating that

succinylation of these candidate proteins may participate in similar biological processes or carry similar molecular functions when localized at each specific location of the cell. With GO classification and KEGG pathway enrichment analyses, the succinylation data from C.albicans shows that a wide range of metabolic processes are related to succinylated proteins such as structural constituents of ribosomes protein-DNA complex, interaction between organism (Figure 4A). In KEGG enrichment analysis (Figure 4B), multiple metabolic pathways are highly enriched in succinylome, which are associated with a reduction of organic peroxides (via alkyl hydroperoxide reductase), NADPH production, glycolysis carbon metabolism (via transketolase-like proteins), stress adaptation (heat-shock proteins) and other degradation pathways. The broad distribution of succinylated proteins in C. albicans suggests that lysine succinylation has an impact on most fundamental cellular processes of C. albicans. Overlap between Lysine Succinylation and Phosphorylation or Acetylation To date, 469 types of PTM have been entered in the UniProt database, of which 326 in eukaryotes, 250 in bacteria, and 80 in archeae respectively

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. These PTMs

including phosphorylation, acetylation, malonylation, crotonylation, propionylation, butyrylation and succinylation, are all crucial for the activation of protein functions in 17 ACS Paragon Plus Environment

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many prokaryotes and eukaryotes. Succinylation is unique among all the acyl lysine modification due to the presence of the negatively charged carboxylate group13. The carboxylate group can interact with arginine residues, which is similar to the phosphate group found in protein phosphorylation. The regulatory roles of phosphorylation in multiple cellular events have been extensively studied in diverse organisms, which is an ubiquitous and reversible modification of the proteins. Compared to phosphorylated proteins previously found in C. albicans16, we find that 63 % of succinylated proteins are also able to be simultaneously phosphorylated in C. albicans. The enrichment analysis of phosphorylome suggests that the phosphorylated proteins in this organism are related to positive regulations in response to the stimuli, negative regulation of molecular function or catalytic activity and the mitotic cell cycle. On the other hand, organic acid catabolic process, small molecule catabolic process, single-organism catabolic process, organic acid biosynthetic process and small molecule metabolic process are more significantly enriched in the succinylome. However, ribose phosphate metabolic process, ribosomal small subunit biogenesis, responses to stress and toxic substance are found to be significantly enriched in the proteins that have both phosphorylated and succinylated sites (Figure 5A and Table S1). As with lysine acetylation25, lysine succinylation was also found in proteins involved in lipid oxidation processes under oxidative stress or in regulating of the activities of metabolic enzymes. Acetylation is another PTM type that is well known for its regulatory roles in chromatin structure and in transcription by either modifying 18 ACS Paragon Plus Environment

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histones or transcription factors18. This type of PTM is also associated with almost every aspect of cellular bioprocess, such as genome organization, gene regulation, DNA replication and DNA repair, all of which are essential for cell growth and pathogenicity of organisms. In C. albicans, histone (de)acetylation has been associated with morphogenetic regulation and phenotypic switching, both of which bioprocesses are related to virulence. When compared the succinylated proteins identified in this study with the acetylome that was obtained very recently in our lab31, we found that 422 succinylation sites on succinylated 278 proteins of C. albicans are also acetylated simultaneously. To better depict the relationship between succinylation and acetylation, we divided these proteins into four types (Figure 5B and Table S1) as follows: Type 1, acetyl and succinyl modifications occur at the same position; Type 2, both acetyl and succinyl events occur but not at the same position; Type 3, exhibits acetyl modification only and Type 4, exhibits succinyl modification only. Notably, the proteins which participate in energy derivation from oxidation of organic compounds and in cellular respiration are enriched in type 1; proteins having function in RNA localization, establishment of RNA localization and oxoacid metabolic process are enriched in type 2; small molecule catabolic process, single-organism catabolic process, and macromolecular complex disassembly are enriched in type 3; carbohydrate metabolic process, positive regulation of response to stimulus, response to host, regulation of defense response, interaction with host are enriched in type 4. In type 1 and type 2, succinylation and acetylation coexist at several fundamental physiological processes although each PTM type has quite unique functions. The 19 ACS Paragon Plus Environment

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overlaps between succinylation and the other two important PTMs indicated that cellular regulation in C. albicans is a highly dynamic process. Succinylation of Metabolic Proteins within Tricarboxylic Acid Cycle in C. albicans GO functional classification of the identified succinylated proteins in terms of biological process indicates that 29 % of these proteins are related to variant metabolic processes. The succinylated proteins are also commonly found in a number of metabolic pathways with KEGG pathway enrichment analysis, particular in tricarboxylic acid cycle (TCA). TCA is the essential metabolic pathway for carbohydrate, lipid and protein decomposition in all organisms and couples with mitochondrial oxidative phosphorylation to meet most energy requirements for living lives. Interestingly, our result showed that every enzyme in the TCA cycle is succinylated, as shown in Figure 6. For example, we found 19 succinylated sites on aconitate hydratase (ACO), a coenzyme is required to convert citric acid into isocitrate. The greatest number of succinylated sites in ACO may be partially due to the fact that ACO is the second largest protein (containing 777 amino acid acids) among TCA enzymes. Three categories of rate-limiting enzymes were also found to be succinylated enriched. First, citrate synthase (CS) has 9 succinylated sites. Second, isocitrate dehydrogenase (IDH1) and isocitrate dehydrogenase (NAD+) (IDH3) are succinylated at 2 and 7 sites, respectively. Third, 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase) (DLST) has 4 succinylated sites and 2-oxoglutarate dehydrogenase complex E1 component (OGDH) has 2 sites. These 20 ACS Paragon Plus Environment

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results suggest that this type of modification may play an important role in regulating TCA metabolic processes. Perhaps the occurrence of succinylation at each position may change the activity of each enzyme, thereby providing the necessary fine-tuning for the regulation of TCA for cellular activity. The further characterization of these succinylated modifications by studying the mutants with abrogated succinylation remain before us for future investigation. CONCLUSIONS In this study, we provided the first in-depth analysis of the lysine succinylome for the human fungal pathogen C. albicans, using a series of highly sensitive proteomic methods. A total of 1550 succinylation sites were identified from 389 proteins of C. albicans, which account for 2.6 % of the total proteins in this fungus. Through the analysis of the amino acid sequence motifs, we found that three distinct types of residues were common for the succinylated lysine: alanine (A), glycine (G) and valine (V). And the V*KSucc* motif was first reported in organisms especially. Moreover, the average number of succinylation sites carried by each protein in C. albicans was the highest among all the organisms reported12, 25, 28, 29, 32-38. In addition, through extensive characterization of the succinylome, we found that the succinylated proteins are mostly associated with cellular functions and are distributed in different cellular compartments. Major metabolic pathways are targeted by succinylation. These pathways include the TCA cycle, glycolysis, fatty acid oxidation, and mycolic acid biosynthesis, all of which are crucial components of C. albicans energy metabolic networks.

This study is the first succinylome in human pathogenic fungi which may 21 ACS Paragon Plus Environment

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serve as an important starting point for further characterization of the pathophysiological role of lysine succinylation in C.albicans and other fungal pathogens.

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ASSOCIATED CONTENT Supporting Information The following files are available free of charge at ACS website http://pubs.acs.org: Figure S1. Western blot analysis of whole cell lysate using succinyl-lysine antibody demonstrates the presence of succinylated proteins. Figure S2. Venn diagram show the overlap among 5 parallel enrichments of total protein solution. Figure S3. Representative MS/MS spectra of succinylpeptides from protein ACO (P82611). Figure S4. Assignment of secondary structure types in C.albicans. Table S1. Protein and functional annotation. Table S2. Comparison with other species . Abbreviations list PTM, posttranslational modification; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. AUTHOR INFORMATION Corresponding Author *Xiaofang Li. E-mail: [email protected]; Tel: +086 025 85478983; Fax: +086-025-85414477 *Weida Liu. E-mail: [email protected]; Tel: +086 025 85478983; Fax: +086-025-85414477

Notes The authors declare no competing financial interest. 23 ACS Paragon Plus Environment

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Author Contributions All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (Grant No. 81573059), funded by National Key Basic Research Program China (973 Program ) (Grant No. 2013CB531605) and Jiangsu Provincial Special Program of Medical Science (Grant No. BL2012003).

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19. Xie, L.; Liu, W.; Li, Q.; Chen, S.; Xu, M.; Huang, Q.; Zeng, J.; Zhou, M.; Xie, J., First Succinyl-Proteome Profiling of Extensively Drug-Resistant Mycobacterium tuberculosis Revealed Involvement of Succinylation in Cellular Physiology. J Proteome Res 2015, 14, (1), 107-119. 20. Rosen, R.; Becher, D.; Buttner, K.; Biran, D.; Hecker, M.; Ron, E. Z., Probing the active site of homoserine trans-succinylase. FEBS Lett 2004, 577, (3), 386-392. 21. Chao Peng‡, Z. L., Zhongyu Xie‡, Zhongyi Cheng‡,, The First Identification of Lysine Malonylation Substrates and Its Regulatory Enzyme.PDF. Molecular & Cellular Proteomics 2011, 10, (10), 1-12. 22. Zhongyu Xie‡§, J. D., Lunzhi Dai‡§,, Lysine succinylation and lysine malonylation in histones. Molecular & Cellular Proteomics 2012, 11, (5), 100-107. 23. Seet, B. T.; Dikic, I.; Zhou, M.-M.; Pawson, T., Reading protein modifications with interaction domains. Nat Rev Mol Cell Biol 2006, 7, (7), 473-483. 24. Xiaolong Li, Xin Hu, Yujing Wan, Guizhen Xie, Systematic Identification of the Lysine succinylation in the Protozoan Parasite Toxoplasma gondii. Journal of Proteome Research 2014, 13, (12), 6087-6095. 25. Pan, J.; Chen, R.; Li, C.; Li, W.; Ye, Z., Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus. J Proteome Res 2015, 14, (10), 4309-4318. 26. Pagel, O.; Loroch, S.; Sickmann, A.; Zahedi, R. P., Current strategies and findings in clinically relevant post-translational modification-specific proteomics. Expert Rev Proteomics 2015, 12, (3), 235-253. 27. Verma-Gaur, J.; Qu, Y.; Harrison, P. F.; Lo, T. L.; Quenault, T.; Dagley, M. J.; Bellousoff, M.; Powell, D. R.; Beilharz, T. H.; Traven, A., Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans. PLoS Genet 2015, 11, (10), e1005590. 28. Colak, G., Identification of Lysine Succinylation Substrates and the Succinylation Regulatory Enzyme CobB in Escherichia coli*. Molecular & Cellular Proteomics 2013, 12, (10), 3509-3520. 29. Mingkun Yang, Y. W., Ying Chen, Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis H37Rv. Mol Cell Proteomics 2015, 14, (4), 796-811. 30. Du, J.; Zhou, Y.; Su, X.; Yu, J. J.; Khan, S.; Jiang, H.; Kim, J.; Woo, J.; Kim, J. H.; Choi, B. H.; He, B.; Chen, W.; Zhang, S.; Cerione, R. A.; Auwerx, J.; Hao, Q.; Lin, H., Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science 2011, 334, (6057), 806-809. 31. Zhou, X.; Qian, G.; Yi, X.; Li, X.; Liu, W., Systematic Analysis of the Lysine Acetylome in Candida albicans. J Proteome Res 2016. 32. Cheng, Y.; Hou, T.; Ping, J.; Chen, G.; Chen, J., Quantitative succinylome analysis in the liver of non-alcoholic fatty liver disease rat model. Proteome Sci 2016, 14, 3. 33. He, D.; Wang, Q.; Li, M.; Damaris, R. N.; Yi, X.; Cheng, Z.; Yang, P., Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed. J Proteome Res 2016, 15, (3), 879-890. 34. Jin, W.; Wu, F., Proteome-Wide Identification of Lysine Succinylation in the Proteins of Tomato (Solanum lycopersicum). PLoS ONE 2016, 11, (2), e0147586. 35. Mizuno, Y.; Nagano-Shoji, M.; Kubo, S.; Kawamura, Y.; Yoshida, A.; Kawasaki, H.; Nishiyama, M.; Yoshida, M.; Kosono, S., Altered acetylation and succinylation profiles in Corynebacterium

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glutamicum in response to conditions inducing glutamate overproduction. Microbiologyopen 2016, 5, (1), 152-173. 36. Pan, J.; Ye, Z.; Cheng, Z.; Peng, X.; Wen, L.; Zhao, F., Systematic analysis of the lysine acetylome in Vibrio parahemolyticus. J Proteome Res 2014, 13, (7), 3294-3302. 37. Kosono, S.; Tamura, M.; Suzuki, S.; Kawamura, Y.; Yoshida, A.; Nishiyama, M.; Yoshida, M., Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source. PLoS ONE 2015, 10, (6), 1-24. 38. Li, X.; Hu, X.; Wan, Y.; Xie, G.; Li, X.; Chen, D.; Cheng, Z.; Yi, X.; Liang, S.; Tan, F., Systematic Identification of the Lysine Succinylation in the Protozoan Parasite Toxoplasma gondii. J Proteome Res 2014, 13, (12), 6087-6095.

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FIGURES ANS LEGENDS Figure1

Figure1. Properties of the succinylated peptides. (A) Succinylation motifs and conservation of succinylation sites. The height of each letter corresponds to the frequency of that amino acid residue in that position. The central K refers to the acetylated lysine. Succinylated lysine motif analysis using Motif-X software. (B) Number of identified peptides containing succinylated lysine in each motif. (C) Heat map of the amino acid compositions of the succinylation sites showing the frequency of the different types of amino acids around the succinylated lysine.

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Figure2

Figure2. The venn diagrams show the succinylated protein overlapping between C. albicans and E.coli, S.cerevisiaeor, Mouse or Human.

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Figure3

Figure3. Gene Ontology functional classification of the identified succinylated proteins in terms of (A) biological process, (B) molecular function, and (C) subcellular location.

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Figure4

Figure4. Enrichment analysis of the succinylated proteins in C. albicans. (A) Enrichment analysis of the succinylated proteins based on the classification of GO annotation in terms of biological process, cellular component and molecular function (green bars) (p < 0.05). (B) KEGG pathway enrichment analysis (p < 0.05).

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Figure5

Figure5. Properties of the modified proteins. (A) Heat map of the modified compositions of the succinylation and phosphorylation separately, and both modified by succinylation and phosphorylation. Colors indicate the frequency of the different types of modification around the succinylated lysine. (B) Heat map of the modified compositions of the succinylation and acetylation. Type 1: protein of acetyl- and succinyl- modifications occur in same position; Type 2: protein of acetyl and succinyl both occur; Type 3: protein of only acetyl identified; Type 4: protein of only succinyl identified. The colors indicate the frequency of the different types of modification 32 ACS Paragon Plus Environment

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around the succinylated protein. Figure 6

Figure 6. Succinylation of enzymes involved in the Tricarboxylic Acid Cycle. Succinylated enzymes identified in C.albicans are shown in green ovals; sites that were found to be succinylated are shown as blue dots. The enzymes shown are pyruvate dehydrogenase E1 component alpha subunit (PDHA), dihydrolipoamide acetyltransferase (DLAT), citrate synthase (CS), aconitate hydratase (ACO), isocitrate dehydrogenase (IDH1), isocitrate dehydrogenase (NAD+) (IDH3), 2-oxoglutarate dehydrogenase complex E1 component (OGDH), dihydrolipoamide dehydrogenase (DLD),

2-oxoglutarate

dehydrogenase

E2

component

(dihydrolipoamide

succinyltransferase) (DLST), succiny-coA synthetase alpha subunit (LCS1), succinyl-CoA synthetase beta subunit (LCS2), succinate dehydrogenase flavoprotein submit (SDHB), succinate dehydrogenase (ubiquinone) iron-sulfur subunit (SDHA), fumarate hydratase (FH), malate dehydrogenase (MDH2). 33 ACS Paragon Plus Environment

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Table1. Succinylation sites and succinylated proteins identified in this study compares with previous studies. Table1 succinylation sites and succinylated proteins identified in this study compares with previous studies species

sites

proteins

reference

C. albicans E.coli S.cerevisiae M.musculus(liver) H.sapiens

1550 2572 1345 2140 2004

389 989 476 705 692

This study Brian et al., 2013 Brian et al., 2013 Brian et al., 2013 Brian et al., 2013

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For TOC only

Abstract Graphic

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