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Ultra-deep Lysine Crotonylome Reveals the Crotonylation Enhancement on both Histones and Non-histone Proteins by SAHA Treatment Quan Wu, Wenting Li, Chi Wang, Pingsheng Fan, Lejie Cao, Zhiwei Wu, and Fengsong Wang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00380 • Publication Date (Web): 08 Sep 2017 Downloaded from http://pubs.acs.org on September 8, 2017

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Journal of Proteome Research

Ultra-deep Lysine Crotonylome Reveals the Crotonylation Enhancement on both Histones and Non-histone Proteins by SAHA Treatment Quan Wu1, Wenting Li2, Chi Wang1, Pingsheng Fan3, Lejie Cao4, Zhiwei Wu1, Fengsong Wang5, *

1

Central Laboratory of Medical Research Centre, Anhui Provincial Hospital, Anhui

Medical University, Hefei, 230001, China. 2

Department of Infectious Diseases, Anhui Provincial Hospital, Anhui Medical

University, Hefei, 230001, China. 3

Department of Oncology, Anhui Provincial Hospital, Anhui Medical University,

Hefei, 230001, China. 4

Department of Respiration, Anhui Provincial Hospital, Anhui Medical University,

Hefei, 230001, China. 5

School of Life science, Anhui Medical University, Hefei, 230032, China.

*

Correspondence:

[email protected], Ph: +86-551-62283574, fax: +86-551-62283292

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Abstract Lysine crotonylation is a newly discovered protein post-translational modification and was reported to share transferases and de-acylases with lysine acetylation. The acetyltransferase p300 was reported also contain crotonyltransferase activity and class I histone deacetylases were demonstrated to be the major histone decrotonylases. However, the decortonylases for non-histone proteins are unclear. Moreover, due to the lack of high-quality pan-antibodies, large-scale analysis of crotonylome still remains challenge. In this work, we comprehensively studied lysine crotonylome on both histones and non-histone proteins upon SAHA treatment and dramatically identified 10,163 lysine crotonylation sites in A549 cells. This is the first identification of 10,000s of lysine crotonylation sites and also the largest lysine crotonylome dataset up to now. Moreover, a parallel reaction monitoring-based experiment was performed for validation, which presented highly consistent results with the SILAC experiments. By intensive bioinformatic analysis, it was found that lysine crotonylation participate in a wide range of biological functions and processes. More importantly, it was revealed that both the crotonylation and acetylation level of most core histones sites and a number of non-histone proteins as well as some known substrates of class IIa and IIb HDACs were up-regulated after SAHA treatment. These results suggest that SAHA may have decrotonylation inhibitory activities on both histones and non-histone proteins by inhibiting HDACs. Keywords: Lysine crotonylation, crotonylome, SAHA, decortonylase

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Introduction Lysine crotonylation (Kcr) is a new protein post-translational modification (PTM), which was initially discovered by Zhao’s group in histones of human cell lines and mouse sperm1. Lysine crotonylation has drawn tremendous attentions by a number of researchers due to its underlying biological functions2-12. It is first reported that histone lysine crotonylation could act as a robust indicator of active promoters and could be an important signal in the control of male germ cell differentiation1. In a recent research, David Allis and colleagues demonstrated that the well-known acetyltransferase p300 also has crotonyltransferase activity, and p300-catalyzed histone Kcr directly stimulates transcription to a greater degree than histone acetylation (Kac)3. After the discovery of crotonyltransferase, the decrotonylation enzymes were also reported. Bao et al. found that Sirt1, Sirt2, and Sirt3 can catalyze the hydrolysis of lysine crotonylated histones and Sirt3 functions as a decrotonylase to regulate histone Kcr dynamics and gene transcription in living cells2. While in a recent study, Wong and co-authors presented evidence that class I histone deacetylases (HDACs) rather than sirtuin family deacetylases (SIRTs) are the major histone decrotonylases10. These studies greatly expanded our knowledge of the new PTM. More recently, several research groups presented deeper studies of lysine crotonylation and found several “readers” for Kcr. Li’s group reported that the AF9 YEATS domain displays selectively higher binding affinity for crotonyllysine over acetyllysine 5. Furthermore, Zhang and co-authors showed that the YEATS domain of AF9 preferentially binds crotonyl-lysine over acetyl-lysine in histone H38. These 3 / 22



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researches define the evolutionarily conserved YEATS domain as a family of crotonyllysine readers and specifically demonstrate that the YEATS domain of AF9 directly links histone crotonylation to active transcription. In other studies, Li’s group demonstrated that the histone acetylation-binding double PHD finger (DPF) domains of human MOZ (KAT6A) and DPF2 (BAF45d) accommodate a wide range of histone lysine acylations with the strongest preference for Kcr7, while Andrews et al. reported the Taf14 YEATS domain as an effective reader of histone lysine crotonylation4. All the above studies showed the important roles of Kcr in biological processes. However, large-scale analysis of Kcr in proteome level is still a challenge due to the lack of high-quality pan-antibodies for Kcr enrichment, especially for Kcr in non-histone proteins. Up to now, there is only a few researches focused on Kcr in non-histones. Liao’s group studied Kcr in non-histone proteins of Hela cells and identified 1185 crotonylated peptides in 453 proteins after treatment with sodium crotonate, while Zhang and co-authors reported a much larger dataset of Kcr with 2696 Kcr sites on 1024 proteins11, 12. They indicated that Kcr could have important roles in regulating multiple cellular processes. Suberoylanilide hydroxamic acid (SAHA) is a well-known HDACi which can suppress HDAC class I, IIa and IIb. It was already approved for the treatment of refractory cutaneous T-cell lymphomas (CTCL)13. Moreover, its activities for other tumors such as non-small cell lung cancer (NSCLC), breast cancer and so on were also confirmed14-18. In our previous work, the effect of SAHA on histone and non-histone lysine acetylome in A549 cells were both investigated19, 20, showing that

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SAHA treatment can enhance lysine acetylation level in both histones and non-histone proteins. According to the recent studies, acetylation and crotonylation may share catalyzed enzymes, both transferases and de-acylases2, 3, 10. Moreover, in the most recent study, it is demonstrated that class I histone deacetylases are the major histone decrotonylases10. In this work, we comprehensively studied lysine crotonylation in both histones and non-histone proteins upon SAHA treatment. We established an integrated system by using high-quality pan-antibody for Kcr enrichment and SILAC technique for relative quantification and dramatically identified 10,163 Kcr sites in A549 cells without treatment of sodium crotonate. To confirm the quantification results, a parallel reaction monitoring-based experiment was performed, which presented highly consistent results with the SILAC experiments. Further bioinformatic analysis was performed to reveal the biological functions of lysine crotonylation and to show the decrotonylation inhibitory activities of SAHA.

Experimental procedures SILAC and SAHA treatment for A549 cell line The A549 cells (non-small cell lung cancer, NSCLC) were cultured in DMEM SILAC medium (Invitrogen, Carlsbad, CA) with 10% FBS (Life Technologies, Grand Island, NY) at 37°C with 5% CO2 in humidified atmosphere. The SILAC (stable isotope labeling) was done as previously described20. The “light” labeled cells were treated with 3 µM final concentration of SAHA for 18 hours and the “heavy” labeled cells were treated with same volume of DMSO. After that, the cells were harvested and 5 / 22



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washed twice with ice-cold PBS. The cell pellets were snap frozen in liquid nitrogen and then stored in -80 °C freezer. Protein extraction was followed by previous method20. Protein digestion and peptide fractionation The proteins were reduced by adding dithothreitol (DTT) to the final concentration of 10 mM and then incubated at 37 °C for 60 min. After that, the alkylation reaction of proteins was performed by adding iodoacetamine (IAA) to the final concentration of 15 mM by incubation at room temperature in dark for 40 min. The reaction was then quenched by adding 30 mM cysteine at room temperature for another 30 min. Trypsin was then added at the trypsin to protein ration of 1:25 (w/w) at 37 °C for overnight digestion. The digest was then fractionated by high pH reverse-phase HPLC. The fractionation was performed on a Agilent 1260 HPLC system equipped with a 300Extend C18 column (5µm particles, 4.6 mm ID, 250 mm length). The procedures were as follows: peptide samples were first injected to the C18 column and then separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 8 over 60 min. The peptide samples were first fractionated into 60 fractions and then combined into 18 fractions for global proteome analysis and 6 fractions for crotonylation analysis. Lysine crotonylated peptides enrichment Before the enrichment experiments, the anti-lysine crotonylation (Kcr) antibody beads (PTM Biolabs Inc, Hangzhou) were washed twice with ice-cold PBS. To enrich Kcr

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peptides, 5 mg 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 then incubated with antibody beads (PTM Bio Inc, Hangzhou) at a ratio of 15 µL beads per milligram of protein at 4 °C overnight. The antibody beads were washed four times with NETN buffer and twice with ddH2O. The Kcr peptides were then eluted by adding elution buffer of 0.1% TFA. The eluted peptides were collected and vacuum-dried for further experiments. LC-MS/MS analysis The dried peptides were re-dissolved in solvent A (0.1% FA in 2% ACN, 98% H2O), directly loaded onto a reversed-phase analytical column (100 µm × 15 mm, 2 µm, 100 Å). The gradient was comprised of an increase from 6% to 22% solvent B (0.1% FA in 98% ACN) for 24 min, 22% to 36% for 10 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system. The electrospray voltage for electrospray was applied at 2.0 kV. The Orbitrap resolution was set at 70,000 for full MS and 17,500 for MS/MS, and the MS/MS NCE was set as 28. One MS scan was followed by the top 20 precursor ions for MS/MS above a threshold ion count of 1.5E4. The dynamic exclusion was set as 15 s. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for generation of MS/MS spectra. The m/z range was set as 350 to 1600 for full MS scan. Database mining The database mining was performed at MaxQuant (version 1.5.2.8) software. The MS/MS spectra were searched against SwissProt Human database (20,274 sequences).

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Trypsin/P was set as enzyme with up to 4 missing cleavages. Mass error was set to 5 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, crotonylation on lysine and acetylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for protein, peptide and modification site were specified at 0.01. The site identifications with localization probability less than 0.75 were removed. For protein and Kcr quantification, the SILAC methods on MaxQuant (version 1.5.2.8) was selected, and Arg0, Lys0 was selected for light label while Arg10, Lys6 was selected for heavy label. As three biological repeats were done in this work, the raw files of three repeats were set as three different “experiments” in the Maxquant software for quantification. T test was performed to calculate the P value of three repeats and only the quantification ratios with P value less than 0.05 was adopted. Bioinformatic analysis Gene Ontology (GO) annotation and enrichment analyses were done by using DAVID (the Database for Annotation, Visualization and Integrated Discovery). Encyclopedia of Genes and Genomes (KEGG) database was adopted for the enrichment of pathways by Functional Annotation Tool of DAVID against the background of Homo sapiens. InterPro database was researched using Functional Annotation Tool of DAVID against the background of Homo sapiens. To perform a protein–protein interaction network analysis, the STRING database was used and then functional protein interaction networks were visualized by using Cytoscape.

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PRM-based validation The tryptic peptides were re-dissolved in 0.1% formic acid (solvent A), and loaded onto a reversed-phase analytical column (15-cm length, 75 µm i.d.). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 38 min, 23% to 35% in 14 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1000 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 35,000. Peptides were then selected for MS/MS using NCE setting as 27 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-independent procedure that alternated between one MS scan followed by 20 MS/MS scans. Automatic gain control (AGC) was set at 3E6 for full MS and 1E5 for MS/MS. The maxumum IT was set at 20 ms for full MS and auto for MS/MS. The isolation window for MS/MS was set at 2.0 m/z. The resulting MS data were processed using Skyline (v.3.6). Peptide settings: enzyme was set as Trypsin, Max missed cleavage set as 2. The peptide length was set as 8-25, variable modification was set as Carbamidomethyl on Cys and oxidation on Met, and max variable modifications was set as 3. Transition settings: precursor charges were set as 2, 3, ion charges were set as 1, 2, ion types were set as b, y, p. The

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product ions were set as from ion 3 to last ion, the ion match tolerance was set as 0.02 Da.

Results Comprehensive analysis of the whole proteome and lysine crotonylome in A549 cells upon SAHA treatment In this study, we combined SILAC, HPLC fractionation, high-quality pan-antibody enrichment, high resolution mass spectrometry and intensive bioinformatics for comprehensive study of lysine crotonylome upon SAHA treatment in A549 cells. The whole workflow includes 6 steps: (1) stable isotope labeling of A549 cells by SILAC; (2) protein extraction and digestion; (3) HPLC fractionation of peptides into fractions; (4) affinity enrichment of lysine crotonylated peptides; (5) analysis of the enriched peptides using LC-MS/MS; (6) bioinformatic analysis of the data (Figure 1). Altogether, we identified 10,163 Kcr sites on 2,445 proteins from A549 cells in three biological replicates, and 8,475 sites from 2,021 proteins were quantifiable. This is the largest dataset for Kcr. Proteome-wide enrichment of Kcr is based on a high-quality pan-antibody (PTM Bio). All the data was listed in Supplementary Information Table S1. For normalization, global proteome data was also collected with the identification of 6,059 protein groups as shown in Table S2. Altogether, three biological replicates were performed in this work. The quantification reproducibility was also investigated as shown in Figure 2A and the venn diagrams for Kcr sites and proteins were presented in Figure 2B and 2C. We got 7765, 7387 and 7514 Kcr sites, corresponding to 2034, 1959 and 1999 proteins in 10 / 22


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the three experiments, respectively. According to obtained data, the identification of Kcr in three experiments were highly reproducible, with more than 70% of sites and proteins identified in at least two replicates. It is obviously that the three quantitative experiments were highly reproducible. Functional classification of the lysine crotonylome The subcellular localization of the identified lysine crotonylome was characterized (Figure 3A). It was found that the majority of Kcr proteins were distributed in the cytosol (35.7%), nuclear (27.4%) and mitochondria (13.5%). The subcellular localization of global proteome was also calculated for comparison (Figure 3A). According to the data, the protein localization of Kcr proteins and global proteome has no significant difference. Then we compared the molecular functions of Kcr proteins and global proteome. It was found that both Kcr proteins and global proteins present similar pattern in molecular functions (Figure 3B). The majority of proteins were participated in binding and catalytic activity. The percentage of proteins participated in other functions of Kcr and global proteins was also very close. These data as well as the localization data suggest that lysine crotonylation could have a broad localization distribution and biological functions. SAHA treatment increased lysine crotonylation level in A549 cells Previously we found that lysine acetylation level in both core histones and non-histone proteins were markedly increased with the dose-dependent SAHA treatment. As lysine crotonylation was reported to be catalyzed by acetyltransferase

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p300 and class I HDAC, it is speculated that the modification level of Kcr will also be enhanced by SAHA, a well-known HDACi which inhibit class I, IIa and IIb HDAC21. In this study, we systematically compared the Kcr changes before and after SAHA treatment. Firstly, we analyzed the Kcr changes in histones. Altogether, 35 Kcr sites on histones were identified, among which 24 sites were novel compared with previously reports (Figure 4). According to the quantification results, it is found that majority of the Kcr sites on core histones were up-regulated upon SAHA treatment, while most of the Kcr on histone H1 were down-regulated. In 22 Kcr sites on core histones, 18 sites were up-regulated, one site (H3K14) was down-regulated and three sites (H2BK20, H4K77 and H4K91) were not changed , while in the 13 Kcr sites in histone H1.4, 11 sites were slightly down-regulated (fold change 0.6-0.8) and two sites were not significantly changed (K16 and K96). All the data was presented in Table S1 and Figure 4. Then, the Kcr changes in non-histone proteins upon SAHA treatment was also analyzed. Among the dataset, 637 Kcr sites were up-regulated with quantification ratio over 1.5 (P value

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