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Quantitative dynamics of proteome, acetylome and succinylome during stem cells differentiation into hepatocyte-like cells Zekun Liu, Qing-bin Zhang, Chen Bu, Dawei Wang, Kai Yu, Zhixue Gan, Jianfeng Chang, Zhongyi Cheng, and Zexian Liu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00238 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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Journal of Proteome Research

Quantitative dynamics of proteome, acetylome and succinylome during stem cells differentiation into hepatocyte-like cells Zekun Liu1,#, Qing-bin Zhang2,#, Chen Bu3,#, Dawei Wang4,#, Kai Yu1, Zhixue Gan5, Jianfeng Chang5, Zhongyi Cheng3,*, Zexian Liu1,*

1

Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China,

Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China 2

Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Stomatology Hospital of

Guangzhou Medical University, Guangzhou 510140, China 3

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

4

Department of Thoracic Surgery, China Meitan General Hospital, Beijing 100028, China

5

Research Center for Translational Medicine at East Hospital, School of Life Sciences and

Technology, Tongji University, Shanghai 200092, China

#

*

These authors contributed equally to this work.

To whom correspondence should be addressed.

Dr. Zhongyi Cheng: Tel/Fax: +86 571 2883 3588; Email: [email protected] Dr. Zexian Liu: Tel: +86 20 8734 2025; Fax: +86 20 8734 2522; Email: [email protected]

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ACS Paragon Plus Environment

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Abstract Stem cell differentiation is a complex biological process controlled by a series of functional protein clusters and signaling transductions especially metabolism related pathways. Although previous studies have quantified the proteome and phosphoproteome for stem cell differentiation, the investigation of acylation-mediated regulation is still absent. In this study, we quantitatively profiled the proteome, acetylome and succinylome in pluripotent human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). In total, 3,843 proteins, 185 acetylation sites in 103 proteins and 602 succinylation sites in 391 proteins were quantified. The quantitative proteome showed that, in differentiated HLCs, the TGF-β, JAK-STAT and RAS signaling pathways were activated while ECM related processes such as sulfates and leucine degradation were depressed. Interestingly, it was observed that the acetylation and succinylation were more intensive in hESCs, while protein processing in endoplasmic reticulum and the carbon metabolic pathways were especially highly succinylated. Since the metabolism patterns in pluripotent hESCs and the differentiated HLCs were different, we proposed that the dynamic acylations especially succinylation might regulate the Warburg-like effect and TCA cycle during differentiation. Taken together, we systematically profiled the protein and acylation levels of regulation in pluripotent hESCs and differentiated HLCs, and the results indicated the important roles of acylation in pluripotency maintaining and differentiation.

Keywords stem cell; differentiation; proteome; acetylation; succinylation; metabolism

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Introduction The 2012 Nobel prize for Physiology or Medicine was awarded to John Gurdon and Shinya Yamanaka for their pioneering discovery of reprogramming mature cells to pluripotent stem cells 1. Besides the reprogramming studies, the differentiation/programming of pluripotent stem cells into functional specialized cells which might be useful for potential therapeutic applications also attracted great attentions

2, 3

. Thus, it is interesting and important to reveal the molecular characteristics of

pluripotent stem cells/differentiated cells and dissect the molecular mechanisms of cell (re)programming. Recent studies have identified a number of critical regulators such as LIN28, OCT4, SOX2, and NANOG for early development such as LIF/STAT3 signaling

7, 8

4-6

. Pathways responsible for cell behaviors controlling

and gene transcription related pathways such as TGF-β/SMAD

signaling were also identified to participate in differentiation processes

9-11

. However, since there

were various destinations for cell differentiation, the molecular mechanisms were extremely complicated and needed further dissection. Besides the investigations on transcription factors based gene transcription regulations, various studies were contributed to characterize the stem cells and reveal their differentiation related orchestrators. For example, Rigblot et. al. systematically quantified the proteome and phosphoproteome dynamics during human embryonic stem cell differentiation, while the results showed that post-translational modifications (PTMs) including phosphorylation played critical roles in differentiation processes

12

. Recently, the specific metabolism modes in stem cells and the

metabolism programming during differentiation attracted great attentions

13, 14

. For instance,

Moussaieff et. al. discovered that embryonic stem cells maintained high level of cytosolic acetyl-CoA synthesis through glycolysis to block histone deacetylation and differentiation and preserve pluripotency

15

. These studies proposed the Warburg-like effect in stem cells that they might utilize

anaerobic respiration for energy source instead of the aerobic respiration in mitochondrion

13-17

.

Furthermore, anaerobic respiration related signaling pathways such as HIF-α signaling was found to be highly activated during stem cells differentiation

18

. Thus, it should be helpful to study the

regulatory mechanism of metabolism programming during stem cell differentiation. Moreover, recent studies identified that metabolic coenzymes including acetyl-CoA and succinyl-CoA mediated protein acetylation and succinylation were critical in regulation of metabolism

19, 20

. Taken together,

systematical analyses of protein acetylation and succinylation might provide helpful insights of 3



molecular mechanisms of pluripotency maintaining and differentiation for stem cell. In this study, we quantitatively compared the proteome, acetylome and succinylome between human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). Divergence of protein expression profiles and differences of quantitative acetylation and succinylation were observed between these two cell lines. The signaling pathways differently regulated at protein and acylation level in these two cell lines were analyzed, and the results showed that acetylation and succinylation might be highly involved in the regulation of metabolism in pluripotent hESCs. Taken together, the first quantitative study of proteome, acetylome and succinylome provide helpful insights of molecular dynamics in pluripotent hESCs and differentiated HLCs.

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Materials and methods hESCs maintenance The hESCs (H9) were cultured under feeder-dependent condition on a 6-well plate at 37 ℃, 5% CO2, 20% O2. Before differentiation, hESCs were switched to feeder-free by seeding on Matrigel (BD Biosciences, San Jose, CA, USA) coated plate in mTeSR1 medium (STEMCELL Technologies, Vancouver, BC, Canada) at 37 ℃, 5% CO2, 20% O2. hESCs in vitro differentiation into HLCs When reaching 80% confluence, hESCs were switched to differentiation medium consisting RPMI 1640 medium (Invitrogen, San Diego, CA, USA), 2% B27 Serum-Free Supplement (Invitrogen, San Diego, CA, USA), 1% Non-Essential Amino Acids (Invitrogen, San Diego, CA, USA), 1% penicillin/streptomycin (MP BIOMEDICALS, Santa Ana, CA, USA) and supplemented with 100 ng/mL Activin A (338-AC-050/CF, R&D, Minneapolis, MN, USA) for 5 days. In days 5-10, cells were cultured in differentiation medium supplemented with 20 ng/mL FGF4 (235-F4-025/CF, R&D, Minneapolis, MN, USA), 10ng/mL BMP4 (314-BP-010/CF, R&D, Minneapolis, MN, USA). In days 10-14, cells were maintained in differentiation medium supplemented with 20 ng/mL FGF4, 10 ng/mL BMP4 and 20 ng/mL HGF (294-HG-025/CF, R&D, Minneapolis, MN, USA). In days 14-18, cells were cultured in differentiation medium supplemented with 20 ng/mL HGF. In days 18-22, cells were matured in differentiation medium supplemented with 20 ng/mL HGF, 10 ng/mL oncostatin M (295-OM-050, R&D, Minneapolis, MN, USA) and 100 nM dexamethasone (D2915, Sigma-Aldrich, St. Louis, MO, USA) for hepatocyte maturation. Protein extraction and digestion Cell samples were lysed in 2 × NETN buffer (200 mM NaCl, 100 mM Tris-Cl, 2mM EDTA, 1.0 % NP-40, 1.0 % Cocktail, pH 7.2) with 0.5% Triton X-100 for 30 min on ice. Lysed sample were sent for a centrifugation at 20,000 g for 10 min at 4 ℃, the supernatant was collected and the protein concentration was determined. After that, 15% final concentration (v/v) was added into the sample solution for precipitation. -20 ℃ acetone was used for protein washing twice. The protein pellets were dissolved in 100 mM NH4HCO3 (pH 8.0) for trypsin digestion. A twice digestion method was applied in our study. For the first digestion, trypsin (Promega, Madison, WS, USA) was added into protein solution at a ratio of 1:50 (trypsin: protein, w/w) at 37 ℃ for 16 hours. After that, DTT was added into the solution to a final concentration of 5 mM at 37 ℃ for 5



1h. And then, IAA was added for alkylation at a final concentration of 15 mM for 30 min in dark at room temperature. The alkylation reaction was quenched by adding cycteine to a final concentration of 30 mM at room temperature for another 30 min. At last, the second digestion was performed by adding trypsin again at a ratio of 1:100 (trypsin: protein, w/w) at 37 ℃ for 4 hours to complete the digestion cycle. TMT labeling After digested by trypsin, a desalting treatment was applied using Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried. Peptides was reconstituted in 0.5 M TEAB and processed according to 6-plex TMT kit manufacturer’s protocol. One unit of TMT reagent (defined as the amount of reagent required to able 1mg of protein) were thawed and reconstituted in ACN. After that, the peptide samples were then incubated for 2h at room temperature and pooled, desalted and dried by vacuum centrifugation for the following HPLC fractionation. HPLC fractionation The fractionation of peptide sample was performed by high pH reverse –phase HPLC using Agilent 300Extend C18 column (5 µm particles, 4.6 mm ID, 250 mm length). First, peptides were separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate (pH 10) over 80 min into 80 fractions. Then, the peptides were combined into 18 fractions for proteomic study and 8 fractions for acylomic study and dried by vacuum centrifugation. Affinity enrichment For acetylated and succinylated peptide enrichment, the peptide samples were first dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and incubated with pre-washed antibody beads (PTM Biolabs, Hangzhou, Zhejiang, China) at 4 ℃ overnight with gentle shaking. After that, the beads were washed with NETN buffer for four times and with ddH2O twice. Then, 0.1% TFA was used to elute the bounded peptides. The eluting fractions were combined and then vacuum-dried. At last, the enriched samples were desalted with C18 ZipTips (Millipore, Burlington, MA, USA) for LC-MS/MS analysis. LC-MS/MS analysis Peptide samples were dissolved in buffer A (0.1% FA in 2% ACN) and directly loaded onto a reversed phase pre-column (Acclaim PepMap 100, Thermo Fisher Scientific, Waltham, MA, USA). The separation of peptides was conducted at the following reversed-phase analytical column 6


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(Acclaim PepMap RSLC, Thermo Fisher Scientific, Waltham, MA, USA) with a linear gradient of 6–22% buffer B (0.1% FA in 98% ACN) for 26 min, 22–35% buffer B for 8 min, 35–80% buffer B for 3 min and stay at 80% buffer B for the last 3 min at a constant flow rate of 300 nL/min with an EASY-nLC 1000 UPLC system.

The separated peptides were analyzed

by Q

ExactiveTM Plus

hybrid

quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were sent to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific, Waltham, MA, USA) coupled online to the UPLC. For mass spectrometer identification parameter, the detail information was set as follows. Intact peptides and ion fragment peptides were detected in the Orbitrap at the resolution of 70,000 and 17,500 respectively. The peptides were seletcted for MS/MS using NCE at 28% and 32%. The electrospray voltage was set to 2.0 kV. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions. The threshold of ion count in MS survey scan is set to 1E4 under a 30s dynamic exclusion. Automatic gain control (AGC) was applied and 5E4 ions was set as the threshold for MS/MS spectra accumulation.For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z. Database searching For database searching, MaxQuant software (Ver. 1.4.1.2) was applied for the database searching and quantification. MS spectra were searched against SwissProt_Human database (20,203 sequences). Cleavage enzyme was chosen as Trypsin/P. Missed cleavages were set to 4. Mass error of precursor ions and fragment ions were set to 10 ppm and 0.02 Da respectively. FDR was set to less than 0.01. Peptide score was set to equal to or more than 40. Carbamidomethyl on Cys, TMT-6plex (N-term) and TMT-6plex (K) were specified as fixed modification and oxidation on Met, acetylation on protein N-terminal was specified as variable modifications. For acetylation or succinylation data searching, acetylation or succinylation on lysine was further added into the searching parameter and the site localization probability was set as > 0.75. The mass spectrometry data have been deposited to the iProX (http://www.iprox.org) via the the data set identifier IPX0001098000. Bioinformatics analysis UniProt-GOA database (http://www.ebi.ac.uk/GOA/) provides the Gene Ontology (GO) annotation data, and the GO clusters were manually performed. To identify enriched GO clusters 7



against background Homo sapiens GO clusters, Functional Annotation Tool of DAVID Bioinformatics Resources 6.7 was introduced and the enrichment results were obtained. Protein complex information was searched against CORUM – the Comprehensive Resource of Mammalian protein complexes (http://mips.helmholtz-muenchen.de/genre/proj/corum/).

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Result Quantitative profiling

of

proteome,

acetylome

and succinylome

during

stem cell

differentiation In this study, to quantify the protein expression, acetylation and succinylation dynamics during stem cell differentiation, the Tandem Mass Tag (TMT) labeling and high-throughput mass spectrometry based quantitative proteomic profiling was performed in human embryonic stem cells (hESCs) with p53 knockout to maintain pluripotency and the differentiated hepatocyte-like cell line (HLCs) (Figure 1). The proteins were extracted from the cell lysis of the two types of cells, followed by trypsin digestion. Then the peptides generated from digestion were labeled with two different TMT reagents and mixed at 1:1 for further quantification. High-pH reverse-phase liquid chromatography (HPLC) was employed to separation the peptides into 18 and 9 fractions for proteome and acylome profiling, respectively. For acetylation and succinylation profiling, the pan anti-acetyl antibody and pan anti-succinyl antibody were employed to enrich the acetylated and succinylated peptides, which were then submitted into mass spectrometer (MS) for LC-MS/MS analysis. For proteome profiling, the peptide fractions were submitted into MS directly (Figure 1). In total, 4,991 proteins, 298 acetylation sites in 185 acetylated proteins, and 918 succinylation sites in 554 succinylated proteins were identified in the study (Table S1), and 31 sites in 30 proteins were both acetylated and succinylated. Furthermore, 3843 proteins, 185 acetylation sites in 103 proteins, and 602 succinylation sites in 391 proteins were quantified among the proteome, acetylome, and succinylome profiling, respectively. The differential expression of proteins was presented as volcano plot in Figure S1A, and most quantified acetylated or succinylated proteins were also quantified at protein level (Figure S1B). It was observed that both acetylation and succinylation were quantified on 14 lysine residues in 14 proteins (Figure S1C). Accidentally, acetylation and succinylation quantifications were significantly positively correlated other than competitively exclusive (Figure S1D). The ratio distributions for proteome, acetylome and succinylome were shown in Figure 2A. It seemed that a little more proteins were up-regulated in HLCs, and the protein expression levels in these two types of cells were balanced by and large. These results indicated that the protein expression was shifted during stem cell differentiation. Interestingly, there were much more down-regulated acetylation and succinylation sites in HLCs, which indicated that acetylation and succinylation levels should be higher in hESCs. Furthermore, with ratios of 1.5 and 0.6667 as the 9



cutoffs for up and down differential expression, 824 and 588 proteins were up- and down-regulated at protein level in hepatocyte-like cells, respectively. Among the quantitative acylome, 16 and 122 acetylation sites were up- and down-regulated, while 18 and 396 succinylation sites were up- and down-regulated in HLCs (Figure 2B). Taken together, the proteins were intensively and dynamically regulated at protein expression, acetylation and succinylation levels during stem cell differentiation. Alteration of protein abundance during stem cell differentiation Since it was observed that the abundances of more than 1/3 quantified proteins were altered, the protein functional modules should be dynamically regulated during stem cell differentiation. In this study, the differentially expressed proteins were classified into four groups of Q1 (HLCs/hESCs < 0.5), Q2 (0.5 < HLCs/hESCs

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