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Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed Dongli He, Qiong Wang, Ming Li, Rebecca Njeri Damaris, Xingling Yi, Zhongyi Cheng, and Pingfang Yang J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 15 Jan 2016 Downloaded from http://pubs.acs.org on January 15, 2016

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Journal of Proteome Research

Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed

Dongli He1, Qiong Wang1,2, Ming Li1, Rebecca Njeri Damaris1,2, Xingling Yi3, Zhongyi Cheng3, Pingfang Yang1*

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Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Sino-African

Joint Research Center, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China, 2

University of Chinese Academy of Sciences, Beijing 100049, China

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Jingjie PTM Biolabs (Hangzhou) Co. Ltd, Hangzhou 310018, China

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ACS Paragon Plus Environment


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ABSTRACT: Regulation of rice seed germination has been shown to mainly occur at post-transcriptional levels, of which the changes on proteome status is a major one. Lysine acetylation and succinylation are two prevalent protein post-translational modifications (PTM) involved in multiple biological processes, especially for metabolism regulation. To investigate the potential mechanism controlling metabolism regulation in rice seed germination, we performed the lysine acetylation and succinylation analyses simultaneously. Using high accuracy nano-LC-MS/MS in combination with the enrichment of lysine acetylated or succinylated peptides from digested embryonic proteins of 24 h after imbibition (HAI) rice seed, a total of 699 acetylated sites from 389 proteins and 665 succinylated sites from 261 proteins were identified. Among these modified lysine sites, 133 sites on 78 proteins were commonly modified by two PTMs. The overlapped PTM sites were more likely to be in polar acidic/basic amino acid region and exposed on the protein surface. Both of the acetylated and succinylated proteins cover nearly all aspects of cellular functions. Ribosome complex and glycolysis/gluconeogenesis related proteins were significantly enriched in both acetylated and succinylated protein profiles through KEGG enrichment and protein-protein interaction network analyses. The acetyl-CoA and succinyl-CoA metabolism related enzymes were found to be extensively modified by both modifications, implying the functional interaction between the two PTMs. This study provides a rich resource to examine the modulation of the two PTMs on the metabolism pathway and other developmental processes in germinating rice seed. KEYWORDS: post translational modification, acetyllysine, succinyllysine, metabolism, seed germination, rice

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INTRODUCTION Because of their sessile feature, plants are constantly exposed to a changing environment. To ensure survival, plant cells have to adapt their endogenous status to these changes rapidly. Compared with transcription and translation, protein post-translational modifications (PTMs), which affect protein’s location, stability, and activity1-3, could help to trigger a response much faster. Because of this, studies on protein PTMs have become a major concern in scientific community. To date, there are more than 461 PTMs reported in the Uniprot database (http://www.uniprot.org/docs/ptmlist). Among all PTMs, modifications on ε-amino groups of lysine residues, identified over 50 years ago in histone are involved in gene expression regulation4. Subsequent studies indicated that a number of modifications, such as acetylation, succinylation, crotonylation, malonylationand and ubiquitination, could occur on lysine of both histone and non-histone proteins5-13. With the development of high-specific antibodies and high resolution mass spectrometry technique, more and more lysine modifications have been identified. In addition to protein phosphorylation, protein lysine acetylation is becoming another major PTM because it is an easily reversible modification and ideal candidate regulators for different signaling and regulatory pathways14. A database of Compendium of Protein Lysine Acetylation (CPLA) was compiled in 201115, accompanied with the increasing identified acetylated sites. Acetylation on histone in plant has been widely studied for its importance in epigenetic regulation16-18.Very recently, numerous acetylations that occur in non-histone proteins were also identified in different plants, including Arabidopsis19, 20, rice21, soybean22, grape23 and potato22. These studies showed that protein acetylation is involved in many metabolic pathways. Specifically, acetylation has been shown to be one of the most prevalent mitochondrial PTMs, as judged by the percentage of total acetylation sites found on the related proteins24, 25. 3



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Compared with acetylation, lysine succinylation is a newly discovered modification, which was initially identified in bacteria non-histone proteins26. Subsequent studies showed that it is widely present in different prokaryotes and eukaryotes27-30. Unfortunately, no study focusing on protein succinylation in plant has been reported until now. Similar to acetylation, succinylation is also prominent in mitochondria and bacteria. In liver tissue, more than 50% of the total detected succinylation sites occurred in mitochondrial proteins where acetylation had also been detected24. By the year of 2013, the statistics data showed over 2400 pairwise acetylation-succinylation crosstalks31. Based on these data, it is believed that both acetylation and succinylation were heavily involved in the regulation of central metabolisms, especially for tricarboxylic acid cycle. However, because the analyses on acetylation and succinylation were conducted independently, it is hard to establish the interaction between these two modifications. A systematic analysis of acetylation and succinylation on the same sample at the same time might provide more in-depth insights. Seed germination is a complicated physiological process during which structures of mitochondria and plastids are recovered and metabolisms are reactivated32. It is still unknown if protein acetylation and succinylation are involved in metabolism regulation during rice seed germination. To answer this question, we globally profiled the acetylated and succinylated proteins in rice embryos of the 24 HAI seeds. A number of acetylated and succinylated proteins were identified, among which many had overlapped sites. The results will provide more information about the metabolism regulation in germinating seeds. MATERIALS AND METHODS

Rice Seed Germination The dehulled rice (Oryza sativa L. japonica. cv. Nipponbare) seeds were washed with distilled water 4


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three times and then imbibed with distilled water in a dark growth chamber at 26 °C and 70% relative humidity. About 100 embryos (50 mg) of rice seeds were sliced manually and collected at intervals of 0 h, 12 h, 24 h, 36 h and 48 h after imbibitions (HAI), respectively. After being snap frozen with liquid nitrogen, the samples were stored at −80 °C until used for protein extraction.

Protein Extraction and Western Blot Proteins were extracted as previously described33. Briefly, embryos of rice seeds were ground in liquid nitrogen with a mortar and pestle, and then homogenized in buffer containing 250 mM sucrose, 10 mM EGTA, 10 mM Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 1 mM PMSF and 1 mM DTT. After centrifugation at 15000×g for 15 min at 4 °C, the supernatant was incubated in ice-cold acetone for more than 2 h at -20 °C, and then centrifuged at 15000×g for 15 min at 4 °C again. The obtained pellet was washed with cold acetone for three times, and then lyophilized and stored at −80 °C for further use. Protein pellet was dissolved in 100 mM NH4HCO3 (pH 8.0) and then the protein concentration was measured by the Bradford method according to the manufacturer’s instructions (Bio-Rad protein assay, USA). For western blot, protein was diluted with SDS loading buffer, and 20 ug protein of each sample was separated by 12% SDS-PAGE and electro-blotted onto PVDF. The blot was probed with Pan anti-acetyllysine antibody (PTM-102, PTM Biolab, China) or Pan anti-succinyllysine antibody (PTM-401, PTM Biolab, China) followed by horseradish peroxidase conjugated secondary antibody (Sigma, USA).

Protein Digestion, Acetylated and Succinylated Peptides Enrichment, and MS Analysis Protein (about 5 mg) from rice seed embryos at 24 HAI was reduced with 10 mM DTT for 1 h at

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56 °C and alkylated with 20 mM iodoacetamide for 45 min at room temperature in darkness. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratios for a second 4-hour-digestion. To enrich the acetyllysine （Kac）or succinyllysine (Ksu) peptides, tryptic peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and incubated with preconjugated pan-anti-acetyllysine or pan-anti-succinyllysine agarose beads (PTM Biolabs) at 4°C overnight with gentle rotation. The supernatant was discarded and the beads were washed four times with NETN buffer and twice with ddH2O. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. The eluted fractions were combined and desalted with self-packed C18 STAGE tips (Thermo, US) according to the manufacturer’s instructions. The enriched peptides were vacuum-dried and re-dissolved in HPLC solvent A (0.1% formic acid in 2% acetonitrile) for online nanoLC-MS/MS analysis using an EASY-nLC 1000 UPLC system (Thermo Scientific) connected to a Q-Exactive plusTM hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Briefly, peptides were loaded onto the reversed-phase analytical C18nanocapillary LC column (5 µm particle size, 100 Å pore diameter) and eluted with a linear gradient of 7%–24% solvent B (0.1% formic acid in 98% acetonitrile) for 24 min and 24%-36% solvent B for 8 min at a constant flow rate of 280 nl/min. The Q-Exactive plusTM was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. The resulting peptides were subjected to a nanospray ion source (NIS) followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM plus coupled to the online UPLC. The electrospray voltage applied was 2.0 kV. Full MS spectra for intact peptides from m/z 350 to 1800 were acquired in the Orbitrap with a resolution of 70,000, and the isolation window was 2.0 m/z. For MS survey scan, the top 20 6


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precursor ions above a threshold ion count of 2×104 with 5.0 s dynamic exclusion were picked for MS/MS fragmentation by higher energy C-trap dissociation (HCD) with normalized collision energy (NCE) of 28%, the repeat duration is 10s. Automatic gain control (AGC) was used to prevent overfilling of the ion trap, 5×104 ions were accumulated for generation of MS/MS spectra.

Database Searching The obtained MS/MS data were processed using MaxQuant with integrated Andromeda search engine (version 1.4.1.2). Tandem mass spectra were searched against UniProt database (Unipro release 2014_07, included 63195 protein sequences) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages, 5 modifications per peptide and 5 charges. Mass error was set to 10 parts per million (ppm) for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on cysteine was specified as fixed modification, whereas oxidation on methionine, acetylation or succinylation on lysine and acetylation on protein N-terminal were specified as variable modifications. Acetyllysine and succinyllysine add 42.0106 and 100.0160 mass shifts that are readily distinguishable from other acylations22. Peptide was identified with PEP score < 0.05 and andromeda peptide score > 40. False discovery rate (FDR) thresholds for protein, peptide and modification site were specified at 1%. Minimum peptide length was set at 7. All the other parameters in MaxQuant were set to default values. Briefly, the max peptide mass was 4600. The Min and Max peptide length for unspecific search were 8 and 25, respectively. Top MS/MS peaks per 100 Da was 12，MS/MS deisotoping was set as true.

Bioinformatic Analysis Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database. Kyoto

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Encyclopedia of Genes and Genomes (KEGG) database was used to annotate protein pathway, then the annotation result was mapped on the KEGG pathway database using KEGG online service tools KEGG mapper. Domain annotation was performed by using InterProScan on InterPro domain database via Web-based interfaces and services. Cello (version 2.5) was used for subcellular localization predication. GO enrichment analysis was performed using Cytoscape (version 3.1.1) plugin BiNGO. Soft motif-X was used for motif analysis, all protein sequences of rice database were used as background database parameter. The secondary structure types were determined for acylated lysine positions using NetSurfP (version 1.1). Functional interaction network analysis was performed using STRING software and visualized by Cytoscape. Molecular Dynamics Simulations were performed using GROMACS 4.5.5 referred to Yang et al34. The initial model of the PDHA1 module was prepared from the available crystal structure (PDB code 2ozl.1.A, 50.29% sequence identity) via structural modeling using CPHmodels 3.2 Server (http://www.cbs.dtu.dk/ services/CPHmodels).

Enzyme Activity Assays of the Pyruvate Dehydrogenase Complex E1 The E1 activity was measured with 2,6-dichlorophenolindophenol (2,6-DCPIP) methods referred to Nemeria et al35. Briefly, the reaction medium contained (in a 3 ml test volume) 50 mmKH2PO4 (pH 7.0), 1 mmMgCl2, 2.0 mm sodium pyruvate, 0.2 mm ThDP, and 0.1 mm 2,6-DCPIP. The reaction was initiated by adding the proteins and reacted at 30 °C, followed by monitoring the amount of DCPIP reduced over the reaction period of up to 2 h. One unit of activity is defined as the amount of 2,6-DCPIP reduced.

RESULTS Identification of Lysine Acetylated and Succinylated Proteins in Embryos of Germinating Rice

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Seeds The cellular Kac and Ksu degrees fluctuate with the in vivo level of acetyl-CoA and succinyl-CoA28. To acquire a global view of the intensity of protein Kac and Ksu during rice seed germination, we detected the dynamics of Kac and Ksu using western blot analysis with the proteins from embryos of rice seeds at 0, 12, 24, 36 and 48 HAI. As expected, the results showed that a number of proteins with a wide range of molecular masses were succinylated or acetylated, and both modifications achieved a higher level at 24 HAI (Figure 1A). For example, compared with 0 HAI and 12 HAI, the signals of 23 KDa, 35 KDa and 37 KDa bands were enhanced for anti-acetyllysine antibody detection at 24 HAI; meanwhile, the signals of 36 KDa, 42 KDa and 57 KDa bands were enhanced for anti-succinyllysine antibody detection. Therefore, the embryos at 24 HAI were chosen to profile the acetylated and succinylated proteins. Proteins extracted from the 24 HAI embryos were subjected to trypsin digestion, and then the acetylated or succinylated peptides were enriched using agarose beads conjugated acetyllysine or succinyllysine specific polyclone antibody28, 36, respectively. The enriched peptides were then analyzed using online nanoLC-MS/MS (Figure 1B). The obtained MS/MS data were processed with MaxQuant to identify the peptide sequence with a maximum false discovery rate of 1%. Before the protein identification, the mass errors of all detected peptides were checked. For both enriched acetylated and succinylated peptides, the distributions of mass errors were less than 4 ppm, showing that the mass accuracy of the MS data fits the requirement for further analyses (Figure 1C). The lengths of most identified peptides were in the range of 8 to 16 amino acids, which is consistent with the property of tryptic peptides (Figure 1C). Based on the criteria mentioned in M&M, a total of 699 acetylation sites from 389 proteins and 665 succinylation sites from 261 proteins were identified from rice embryos at 24 HAI. Among them, 142 9



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proteins were detected to be both acetylated and succinylated, specifically, 133 sites from 78 proteins were simultaneously acetylated and succinylated (Figure 1B). The detailed information is presented in Table S-1.

Analysis of PTMs Frequency and Sites Properties Most of the identified proteins were modified at only one or two sites, few at multiple sites (Figure 2A). The highest numbers of Kac and Ksu sites were 11 and 17, both of which occurred on the glycolysis related proteins, phosphoglycerate kinase (Q655T1) and fructose-bisphosphate aldolase (Q5N725), respectively. Ksu was found to occur more intensively on its target proteins than Kac in germinating rice seed. For acetylome, the mean modified sites per 100 amino acids were 0.66 with the highest being 5.36; while for succinylome, the average PTM frequency was 0.93 with the highest value being 6.67 (Figure 2B). Particularly, the LEA proteins were found more likely to be succinylated while less acetylated. Detailed information is presented in Table S-2. To identify the possible specific motifs flanking acetylated and succinylated lysine, all the identified acetylated and succinylated peptides were imported into motif-X software to extract the overrepresented motifs of amino acids (Figure S-1). Six acetylation motifs were defined on 400 unique sites accounting for 57.2% of the total Kacs. The sequence logos revealed that both tyrosine (Y) and histidine (H) were significantly overrepresented in the +1 position, and the phenylalanine (F) was highly presented around acetylated lysine, such as +1, -2 and +2 positions. As previously reported, the positively charged amino acids, such as arginine (R) and lysine (K) were excluded from –1 position of acetylated lysine36. For the succinylated peptides, five sequence motifs were defined on 279 (41.9%) unique sites. Enrichment of glutamine (Q) and glutamic acid (E) at position -1, and threonine (T) and valine (V) at position +2 of the succinylated lysine were presented. Just as Kac’s 10


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motifs, R and K were also excluded from the position –1 of the succinylated lysine. In T. gondii succinylome, Q also had been found highly presented at -1 of the succinylated lysine and K was excluded from the –1 position37. The 133 overlapped sequence windows were also extracted for motif analysis, four motifs were defined on 64 (48.1%) unique sites (Figure 2C). It can be seen the overlaps were preferentially to occur on the lysine with glutamic acid (E) at position -1 and histidine (H) at position +1. As well, we observed phenylalanine (F) and glycine (G) were most commonly found at -1 and -10, respectively. These observations suggested that the polar acidic/basic amino acid and non-polar hydrophobic regions were more easily to be attacked by both Kac and Ksu. To determine whether Kac or Ksu occurred frequently within particular structures in proteins, secondary structure surrounding Kac or Ksu sites were extracted using NetSurfP 1.1(Figure 2D). Comparing with the secondary structure probabilities of the modified and unmodified lysine residues, generally, both Kac and Ksu were moderately biased occurring in β-strand and α-helix regions, and a moderate bias against non-secondary coil regions, meanwhile, the overlapped modified sites were moderately bias to be exposed on the protein surface.

Conservation of Acetylated and Succinylated Proteins Acetylated and succinylated proteins have been validated in some bacteria, animals, and plants. To explore the evolutionary conservation of our identified lysine modification sites from prokaryote to eukaryote, we searched the consistent trypsin peptides of 699 acetylated and 665 succinylated peptides in this study against the previously reported acylomes: Saccharomyces cerevisiae, Homo sapiens, Mus musculus, Arabidopsis28, 38 (Table S-3). The consistency showed that 14 Kac and 12 Ksu peptides were conserved in at least two organisms, involving 5 cellular metabolism, 5 stimulus response, 2 protein degradation, and 5 translation related proteins. In addition, Ksu peptides for 3 11



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HSPs and two chaperon proteins were highly conserved, suggesting the Ksu widely participating in the response of stimulus. Five metabolism related proteins were conservatively acetylated across various organisms, including triosephosphate isomerase (TPI, Q0JQP8), adenylate kinase (Q0IP84), nucleoside-diphosphate kinase (Q5TKF4), dihydrolipoamide dehydrogenase (DLD, Q9ASP4), pyruvate dehydrogenase E1 component alpha subunit (PDHA, Q6YZV6) implying pivotal functions of acetylation in metabolism regulation.

Function Analysis of the Lysine Acetylated and Succinylated Proteins To understand the possible roles of Kac and Ksu in germinating rice seed, Kac and Ksu proteins were classified by Gene Ontology (GO) annotation based on three categories: cellular component, biological process, and molecular function. Both Kac and Ksu proteins covered nearly all aspects of cellular functions, and were distributed in different cellular compartments (Figure 3, Table S-1). In terms of biological process, metabolic process, cellular process, and response to stimuli were the three major groups for both Kac and Ksu proteins, which together accounted for about 66% and 64% of the total acetylated and succinylated proteins, respectively. In terms of molecular function, binding, and catalytic activity were the two major groups and accounted for over 70% for both Kac and Ksu proteins. These findings were consistent with previous results in E.coli and Hela cells28, indicating that both Kac and Ksu were the frequently occurring PTMs and might perform the essential regulatory roles in germinating rice seed. GO enrichment was further performed to elucidate the biological functions of Kac and Ksu proteins using Cytoscape plugin BinGO. As shown in Figure 4, for both Kac and Ksu proteins, response to stimuli, translation, glucose catabolic process, and glycolysis groups were significantly enriched in biological process category. Besides, generation of precursor metabolites and energy, 12


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nucleotide metabolic process related proteins, carbohydrate metabolic process, protein catabolic process, and GTP metabolic process were also enriched in acetylome. This result indicated that the Kac exerted more influence on multiple metabolic processes than Ksu. For cellular component category, the nucleolus, cytosol, chloroplast, and mitochondrion proteins were significantly enriched for both PTMs. The detailed enrichment information is presented in Table S-4. In this study, the proportion of Kac and Ksu proteins in mitochondria was not as high as those reported in yeast, hela cells, mouse liver and T. gondii28, 37, which might be ascribed to the different organism types and physiological status of the tissues.

Acetylation and Succinylation of Enzymes Involved in Metabolism Increasing evidence shows central metabolic pathways are regulated by Kac and Ksu on the key enzymes in various organisms34,

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. In this study, the KEGG pathway enrichment analysis was

performed to obtain further insights into the metabolism regulation of protein acetylation and succinylation（Figure 4）. Totally, 206 Kac and 136 Ksu proteins were identified to involve in metabolism pathways. The ribosome complex, glycolysis/gluconeogenesis, pyruvate metabolism, carbon fixation in photosynthetic organisms, and citrate cycle (TCA cycle) were enriched for both PTMs. In addition, pentose phosphate pathway and proteasome related proteins were also enriched in acetylome, and glyoxylate and dicarboxylate metabolism related proteins were enriched for succinylome. As shown in Figure 5, a large proportion of modified proteins participated in central metabolism of rice seed germination, including starch metabolism, sucrose biosynthesis, pentose phosphate pathway, glycolysis, TCA cycle, amino acid metabolism and oxidative phosphorylation (OPP) pathways related proteins. Particularly, six lysine positions of a PDHA (Q654V6) were modified, which were all located on 13



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the dehydrogenase region (E1_dh, 74-369 AA) and most exposed on the protein surface (Figure S-2A). Of these lysine positions, K90 was found to be possibly modified by both acetylation and succinylation, and conservative in yeast and mouse. To check the potential influence of the acylation modification on protein, we detected the enzyme activity of PDH-E1. The catalytic activity of PDH-E1 increased rapidly in the first 24 HAI, and then decreased (Fig. S-2B). Molecular dynamics (MD) simulations were used to determine how acetylation and succinylation of K90 and influences the structure of PDHA1 using GROMACS 4.5.5. Owning to locate in the stable alpha-helix region, the modification of K90 raised no significant structure change (Fig. S2C). However, as shown in the Fig. S2D, the root mean square fluctuations (RMSF) value of K90-su was different from that of the unmodified lysine. This difference might ascribe to the residue charge status change and result in perturbation of the conformational fluctuations.

Protein-Protein Interactions (PPIs) Networks of the Acetylated and Succinylated Proteins PPIs widely exist in all types of cells, and are critical for different biological processes. To obtain better understand of the biological function of protein acetylation and succinylationin in seed germination, we assembled the PPIs networks of the identified modified proteins based on the STRING database (Figure S-3 and S-4). Three highly connected subnetworks, including ribosome complex, glycolysis/gluconeogenesis and proteasome of Kac and Ksu proteins were enriched and visualized by Cytoscape software. In addition, the oxidative phosphorylation system was also enriched in the Ksu proteins. To be mentioned, none of the three association subnetworks for the Kac proteins was localized in mitochondrion, although mitochondrion is one of the main compartments for acetyl-CoA production. The most abundant subnetwork was ribosome complex for both Kac and Ksu proteins, which is consistent with previous report in prokaryote succinyl-proteome40, 41. PPIs 14


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network of the 78 overlapped modified proteins showed that the glycolysis, pyruvate metabolism, and ribosome complex proteins subnetworks were significantly enriched (Figure 6).

DISCUSSION

Regulation of rice seed germination has been shown to mainly occur at post-transcriptional level, of which changes on proteome status is the major one42-44. Kac and Ksu are two important PTMs on proteins, and involved in regulating multiple biological processes, especially for metabolism28, 36. In this study, both Kac and Ksu displayed dynamic patterns, and achieved higher intensity at 24 HAI during rice seed germination, indicating that acetylation and succinylation might function at this early stage, it is also a biomarker for the central metabolism recovery to catalyze acetyl-CoA or succinyl-CoA synthesis in vivo. Rice seed germination can be divided into three phases based on the water up-take. Normally, 24 HAI germinating seed is at the beginning of phase II, which is most important for metabolism reactivity. A total of 699 acetylation sites from 389 proteins and 665 succinylation sites from 261 proteins were successfully identified in the embryos at 24 HAI. Among these modified proteins, 206 acetylated and 136 succinylated proteins involve in a broad range of metabolic processes, which renders particular enrichment of metabolic process and facilitates in-depth investigation of the crosstalk between the two popular PTMs. As previously reported, protein succinylation extensively overlaps with acetylation28, 37. In this study, we simultaneously detected protein Kac and Ksu in the same sample, which will ascertain the comparability between the two PTMs. In total, 142 proteins were detected being both acetylated and succinylated, with 133 overlapped sites from 78 proteins, suggesting that the Kac and Ksu cooperate or compete with each other on the same protein. The overlapped PTM sites were more

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likely to be in polar acidic/basic amino acid region and exposed on the protein surface, which might affect the surface properties and change the activity. These overlapped proteins participated in multiple metabolism pathways including the glycolysis, pyruvate metabolism, TCA cycle, oxidative respiration and ribosome related functional proteins. Specifically, PPIs network analysis showed that the glycolysis and pyruvate metabolism related proteins were significantly enriched, which are important for carbon metabolism recovery and energy supply at early stage of seed germination. Almost all glycolytic enzymes involved in the conversion of glucose to pyruvate were either acetylated (15 proteins) or succinylated (11 proteins). Among them, six were targeted by both PTMs at the same sites, including two glyceraldehyde 3-phosphate dehydrogenases (GAPDH, Q0J8A4 and Q6K5G8), two phosphoglycerate kinases (PGK, Q6H6C7 and Q655T1), pyruvate decarboxylase (Q10MW3), and two fructose-bisphosphate aldolases (ALDO, Q5N725 and Q0DIB5). All the six proteins are acetylated or succinylated at multiple sites. Specifically, 17 Ksu and 9 Kac sites were detected on Q5N725, with 7 common sites, which may function on the activity of ALDO and regulate the synthesis of glyceraldehyde 3-phosphate (G3P), an important intermediate in several central metabolic pathways. Pyruvate dehydrogenase complex (PyDC) is a complex of three enzymes: pyruvate dehydrogenase (PDH), dihydrolipoyl transacetylase (DLAT), and dihydrolipoyl dehydrogenase (DLD) , which catalyzes the overall conversion of pyruvate to acetyl-CoA and CO2. In this study, 6 and 5 PyDC subunits were detected being modified by Kac and Ksu, respectively (Figure 5), with three being commonly modified at multi-sites: namely, PDH E1 component alpha subunit (PDHA, Q654V6), PDH E1 component beta subunit B (PDHB, Q6Z1G7) and DLD (Q9ASP4). The intensive succinylation of the acetyl-CoA metabolism related enzymes indicated complex interactions between 16


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two PTMs. Seven modified lysine sites of Q654V6 were located in the dehydrogenase region, and the overlapped site K90 was found to be conservative in plants and animals (Table S-3, Figure S-2). We observed that the enzyme activity of PDH-E1 increased rapidly in the first 24 HAI, but decreased in the following imbibition (Fig. S2), although the PDH-E1αprotein content accumulated during rice seed germination45, 46. In human cancer cells, K321 acetylation of the PDHA1 and K202 acetylation of PDP1 could inhibit the PyDC activities47. It is speculated that the reversible acylation might inhibit the PDHA catalytic activity and control its functional balance in the germinating rice seed. Under anaerobic condition, pyruvate is fermented to acetaldehyde by pyruvate decarboxylase (PDC) and further converted to ethanol by aldehyde dehydrogenase (ALDH). One PDC (Q10MW3) was detected being intensively modified by two PTMs (4 Kac and 6 Ksu sites, with 3 in common). A glutamate-pyruvate transaminase (GPT, Q338N8) catalyzing alanine synthesis was also detected as substrate of Kac and Ksu. The extensive modification on pyruvate metabolism related enzyme might play important roles in control of the pyruvate homeostasis between aerobic and anaerobic metabolism pathway at the early stage of rice seed germination. The Krebs cycle not only provides the energy for life activities but also contacts with sugar, fat and protein metabolism. Except for the fumarate hydratase, all enzymes involved in the TCA cycle were identified as targets of Kac (10 proteins) or Ksu (13 proteins), with four in common. From the PTMs frequency and KEGG enrichment, Ksu exerts more efforts on the TCA cycle modulation than Kac (Table S-2 and S-4). For example, oxoglutarate dehydrogenase complex (OGDC) catalyzes one rate-limiting step of TCA cycle, which contains 3 subunits: ketoglutarate dehydrogenase (KGD, Q6Z3X5), dihydrolipoyl succinyltransferase (DLST, Q7XVM2) and DLD. In addition to the shared enzyme DLD with PyDC, the other two components of OGDC are also sensitive to the Ksu, 7 and 8 17



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lysine sites on the OGDH and DLST were modified by Ksu, respectively. However, only one Kac site

was

found

on

the

OGDC.

OGDC

activity

can

be

inhibited

by

its

products, succinyl-CoA and NADH48. The high level of succinylation of OGDC might result from the product feedback. An additional enzyme, succinyl-CoA synthetase beta unit (LSC2, Q6K9N6) that catalyzes the reversible reaction of succinyl-CoA to succinate, was also detected to be highly succinylated, and 5 of the 6 Ksu sites located in the ATP-grasp fold region (36-262 AA). The succinyl-CoA metabolism related enzymes were intensively attacked by Ksu indicated the product/substrate feedback regulation might be an effective way of non-enzymatical Ksu, which will precisely regulate the succinyl-CoA production and further the overall Ksu level in cell. As previous reported in yeast cells, deletion of α-ketoglutarate dehydrogenase (Kgd1) and succinyl-CoA ligase alpha unit (LSC1) significantly affected mitochondrial succinylation28. Along with the reactivation of glycolysis and TCA cycle, the mitochondrial electron transport/ATP synthesis metabolism was recovered. During rice seed imbibition, the ATP concentration increased in vivo and peaked at 24 HAI43. In this study, 9 proteins of respiratory chain were identified to be acetylated or succinylated, but no modified proteins were found in complex III. Notably, proteins of complex V were intensively modified by Kac and Ksu, such as the ATP synthase complex subunits, ATPeF0D, ATPeF1A, ATPeF1B and ATPeF1G. Many of the ATP synthase subunits have been previously identified to carry multiple PTMs, such as phosphorylation, oxidation, glutathionylation, and nitrosylation49. It will be interesting to investigate the crosstalk among the different PTMs in regulation of complex V activities in the future. In the embryo of rice seed, the main reserve is protein (e.g. glutelin). Twenty seven Kac proteins and 10 Ksu proteins involved in protein degradation were detected, and two threonine-type 18


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endopeptidase (Q5VRG3 and Q0E466) of proteasome were modified at the same sites. The 20S core particle of the proteasome is easier to be modified by both Kac (7 proteins) and Ksu (3 proteins), specifically for α2, α3 and α7 subunits. Correspondingly, 4 and 6 amino acid degradation related enzymes, involved in degradation of threonine, glycine, tryptophan, and valine, were modified by Kac and Ksu, respectively. Fatty acids of germinating rice seed also degrade rapidly50. Some lipid degradation related enzymes were acetylated (6 proteins) and succinylted (2 proteins), and shared one misc protein (Q2R8Z5, ALDH) participating in the fatty acidω-oxidation. The abundance and enzymes activities of starch degradation related proteins had been shown to increase at the late stage (48-72 HAI) of rice seed germination51, 52. Accordingly, few enzymes involved in starch degradation were acetylated (Q0J528, alpha-amylase) or succinylated (Q8LQ33, alpha-glucan phosphorylase) at 24 HAI. These data suggested that Kac and Ksu on the catabolism related enzymes could play roles in control of the reserves degradation at early stage of rice seed germination. Studies has demonstrated that histones were conservative to be targeted by Kac53, 54. In this study, 9 Kac sites were detected on the core histones, including H2A (4 sites), H4 (4 sites) and H2B (1 site), and all were located at the N-terminal DNA binding domain11, 55. In contrast, no succinylated histone was identified in germinating rice seed, although lysine succinylation did widely occur on the histones in yeast, mouse, drosophila and human11, 36. In this study, protein synthesis and processing related proteins are the major groups for both acetylome and succinylome, with 82 and 49 related proteins being targeted by Kac and Ksu, respectively, and another 12 by both. Notably, the protein translation initiation factors were more liable to be targeted by Kac (9 proteins) than Ksu (1 protein). For fatty acid biosynthesis, 11 and 2 fatty acid synthesis related enzymes were modified by Kac and Ksu, respectively. Although both amylose and amylopectin were significantly increased around the 19



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vascular tissues in the embryo at 24 HAI of germinating rice seed33, 51, 56, no starch biosynthesis related enzyme was detected being modified by Kac or Ksu in this study, even though the these enzyme proteins had relative high abundance in the 24 HAI embryo33, 51. Signaling transduction play important role in the metabolism reactivation, in this study, we identified 19 Kac and 8 Ksu proteins involved in this process. Among them, calnexin (Q7XV86), calmodulin (Q6F332) and one 14-3-3 protein (Q2R2W2) were commonly modified by both Kac and Ksu. ROS also was regarded as signal molecular to help the cell counteract the oxidative damage in the stressed compartment57. Four redox related proteins were identified to be modified by both Kac and Ksu, including thioredoxin (Q53LQ0), ascorbate (Q6K680), 1-Cystein peroxiredoxin (P0C5C9) and superoxide dismutase [Mn] (Q43008). It is noteworthy that 1-Cystein peroxiredoxin was modified with high frequency, including 9 Kac and 13 Ksu sites on this small protein (220 AA), among which 7 sites were overlapped by the two PTMs. We speculate that the high frequency modification of Kac and Ksu might contribute to the signaling transduction and redox homeostasis in the germinating embryo through decrease/increase the related enzyme activity.

CONCLUSION

Lysine acetylation and succinylation are two prevalent protein post-translational modifications involved in metabolism regulation. To investigate the mechanism controlling the metabolism regulation in germinating rice seed, we performed the lysine acetylation and succinylation analyses in germinating rice seed at the same time. The main findings of this study are as follows: (a) both Kac and Ksu achieved a higher level at 24 HAI; (b) 389 acetylated proteins and 261 succinylated proteins were identified in rice embryos at 24 HAI, of them, 133 sites from 78 proteins were overlapped by

20


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two PTMs; (c) the overlapped PTM sites preferentially occurred in polar acidic/basic amino acid region and were exposed on the protein surface; (d) PPIs network and KEGG enrichment analyses indicated that the central metabolism pathways of rice seed germination were significantly modulated by Kac and Ksu. In M. tuberculosis, the enzymatic activity of acetyl-CoA synthase is affected by Ksu34, and bacterial enzyme CobB showed similar lysine desuccinylation and deacetylation activities58, which suggested that there exists an interaction between protein acetylation and succinylation. We also found that acetyl-CoA metabolism related enzymes (e.g. PDHA) were succinylated and succinyl-CoA metabolism related enzymes were acetylated (e.g. OGDH). In this study, we firstly displayed the detailed crosstalk information between the reversible Kac and Ksu in plants, which might provide rich resource to examine the modulation of the two PTMs on the metabolism pathway and other developmental processes in rice seed germination.

ASSOCIATED CONTENT

Supporting Information Figure S-1 Sequence Logo representation of significant motifs for acetyllysine (A) and succinyllysine (B) identified by Motif-X software. The motifs with significance of p < 0.000001 are shown.

Figure S-2 The lysine modification character of PDHA (Q654V6). (A) Identification of the modified sites of Q654V6. The mass spectrum displayed the overlapped acetylated and succinylated site K90. (B) Enzyme activity assays of the pyruvate Dehydrogenase Complex E1. (C) Structural modeling of PDHA. From left to right: Non-succinylated (WT), K90-ac and K90-su of PDHA. (D) Root mean

21



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square fluctuations (RMSFs) for the different PDHA systems.

Figure S-3 Protein-protein interaction network of acetylated proteins.

Figure S-4 Protein-protein interaction network of succinylated proteins.

Table S-1 Annotation of lysine acetylated (A) and succinyllated (B) peptides in germinating rice seed embryo.

Table S-2 PTM frequency statistical analysis. (A) The numbers of Kac, Ksu and overlapped sites in proteins. (B) The protein acetylation frequency. (C) The protein succinylation frequency. PTM frequency calculated with the number of the modified sites per 100 AA.

Table S-3 Conservation analysis for lysine acetylated (A) and succinylated (B) sites.

Table S-4 Functional enrichment of acetylated and succinylated proteins.

This material is available free of charge via the Internet at http://pubs.acs.org. The raw data of MS spectrometry and the results of protein and peptides identification in this paper had been uploaded to the proteomics repository PRIDE with accession number of PXD002739 (For acetylome, Username: [email protected], Password: 0YN3L32jv) and PXD002740 (For succinylome, Username: [email protected], Password: sfCFWIM1) using proteomeXchange software. AUTHOR INFORMATION Corresponding Author Pingfang Yang, *E-mail: [email protected]. Tel/Fax: 86-27-87510956.

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Author Contributions D.H performed all experiments and wrote the manuscript; Q.W and M.L analyzed data; Z.C and X.Y performed the MS sequencing and data processing; P.Y designed the experiments and revised the manuscript. All authors have given approval to the final version of the manuscript. NOTES The authors declare no competing financial interest. ACKNOWLEDGEMENTS We are thankful to Mingkun Yang and Feng Ge for the molecular dynamics simulations. This work was supported by the National Natural Science Foundation of China (NSFC, No. 31101213 and 31271805), 100 talents program of Chinese Academy of Sciences, and Sino-Africa Joint Research Project (SAJC201324).

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FIGURES LEGENDS Figure 1. (A) The dynamics of the succinyllysine and acetyllysine along with the germination process. Left: SDS-PAGE gel stained with blue as the loading control. Middle and right: proteins were blotted with a pan-anti-acetyllysine and pan-anti-succinyllysine antibody. Arrows indicated signals enhanced at 24 HAI compared with that at 0 HAI and 12 HAI. (B) Flowchart showing the establishment of the lysine succinylation and acetylation profile of rice seed embryo. (C) The mass error and peptide length distributions of the succinylation and acetylation profiles. Figure 2. Sequence properties of acetylated and succinylated peptides. (A) Distribution of acetylated or succinylated sites in proteins (B) PTM frequency of acetylation and succinylation. PTM frequency calculated with the number of the modified sites per 100 AA. (C) Sequence Logo representation of 31



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significant motifs for overlapped peptides identified by Motif-X software. The motifs with significance of p < 0.000001 are shown. (D) Distribution of modified lysine in the protein secondary structures. Probabilities for different secondary structures (α helix, beta-strand and coil) and relative surface accessibility (RSA) of succinylated lysines were compared with the secondary structure probabilities of lysines on all proteins identified in this study. Significance was calculated by T- test (P

Global Proteome Analyses of Lysine Acetylation ... - ACS Publications

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