Overexpression of CD38 Decreases Cellular NAD Levels and Alters

Dec 2, 2013 - Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells and mediates multiple cellular signaling pathways. In th...
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Overexpression of CD38 Decreases Cellular NAD Levels and Alters the Expression of Proteins Involved in Energy Metabolism and Antioxidant Defense Yadong Hu,† Helin Wang,† Qingtao Wang,‡ and Haiteng Deng*,† †

MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China Beijing Chaoyang Hospital Affiliated to Capital Medical University, Beijing 100020, China

‡

S Supporting Information *

ABSTRACT: Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells and mediates multiple cellular signaling pathways. In the present study, a 35% decrease of cellular NAD level is achieved by stable expression of the N-terminal truncated CD38, a NAD hydrolase. CD38expressing (CD38(+)) cells have the lower growth rate and are more susceptive to oxidative stress than the wild type cells and empty vector-transfected (CD38(−)) cells. Quantitative proteomic analysis shows that 178 proteins are down-regulated in CD38(+) cells, which involve in diverse cellular processes including glycolysis, RNA processing and protein synthesis, antioxidant, and DNA repair. Down regulation of six selected proteins is confirmed by Western blotting. However, down-regulation of mRNA expressions of genes associated with glycolysis, antioxidant, and DNA repair is less significant than the corresponding change in protein expression, suggesting the low NAD level impairs the protein translational machinery in CD38(+) cells. Down-regulation of antioxidant protein and DNA-repair protein expression contributes to the susceptibility of CD38(+) cells to oxidative stress. Taken together, these results demonstrate that CD38(+) cells are a useful model to study effects of the cellular NAD levels on cellular processes and establish a new linker between cellular NAD levels and oxidative stress. KEYWORDS: CD38, proteomics, chaperones, oxidative stress, glycolytic enzymes

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INTRODUCTION Nicotinamide adenine dinucleotide (NAD) is one of the most fundamental components in life, which plays an important role in all major biological processes.1−4 In cells, NAD is partitioned into NAD, reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) pools. NAD and NADP are cofactors for a variety of enzymes. Generally, NAD(H) functions in dehydrogenase (DH)-catalyzed reactions involved in biodegradation reactions, whereas NADP(H) functions in DHcatalyzed reactions involved in biosynthesis. Ratios of NAD/ NADH and NADP/NADPH regulate cellular redox states and mitochondria functions.5,6 NADPH is the key component of the cellular defense system against oxidative stress. Homeostasis of NAD involves three major processes: biosynthetic process, catabolic process, and nonredox NAD-mediated enzymatic process. In all vertebrates, tryptophan is the de novo precursor of NAD synthesis that is involved in seven major intermediates. Besides its role as a cofactor of oxidoreductases, NAD is a substrate for various ADP-ribosyltransferases, including poly(ADP-ribose) polymerase (PARPs), mono(ADP-ribosyl)-transferases (ARTs), NAD(+)-dependent deacetylases (sirtuins), © XXXX American Chemical Society

tRNA 2′-phosphotransferases, and ADP-ribosyl cyclases (CD38 and CD157). CD38 is a type II transmembrane glycoprotein expressed on cell membrane and is used to convert NAD to cyclic ADP-ribose to initiate calcium efflux.7,8 Sirtuins or Sir2related enzymes conserved from bacteria to humans use NAD as a substrate to deacetylate acetyl-lysine residues of various proteins and to generate 2-O-acetyl-ADP-ribose.9−14 In yeast, Sir2 plays an important role for transcriptional silencing, in suppressing rDNA recombination, and in controlling life span.9,10 In mammalians, NAD-dependent deacetylation of chromatin and other substrates is the basis for the broad range cellular functions carried out by sirtuins.15−33 In addition, NAD is the substrate for ADP-ribosylation of nuclear proteins that plays a key role in cellular stress responses.34,35 The induction of poly-ADP-ribosylpolymerase (PARP) by genotoxicity can deplete NAD and raise nicotinamide levels. These findings indicate that NAD is a key mediator for cellular metabolisms, stress responses, genomic silencing and aging. Despite extensive studies on regulations and functions of NAD and related compounds, systematic and quantitative understanding of the Received: August 21, 2013

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dx.doi.org/10.1021/pr4010597 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

2−43 amino acids at the N-terminal of CD38 was deleted, and a Flag-tag sequence was added onto the C-terminus to create a recombinant human CD38 DNA. The recombinant human CD38 DNA was cloned into pLVX-IRES-ZsGreen1 to create the pLVX-CD38-IRES-ZsGreen1 vector. Production of lentiviral particles of recombinant CD38 was carried out based on the protocol published by Tiscornia et al.39 Briefly, we cotransfected pLVX-CD38-IRES-ZsGreen1 or pLVX-IRES-ZsGreen1, respectively, with packing vectors into 293T cells when they reached 80−90% confluence. The cell culture supernatant was then collected after 48 h and was concentrated with PEG6000. The precipitated lentiviral particles were resuspended in PBS.

global impact of cellular NAD levels on protein expression, cell proliferation and stress responses has not yet been achieved. Cellular NAD levels are subject to fluctuation in cells through nonredox enzymatic reactions. CD38 functions as a NAD glycohydrolase as well as an ADP-ribosyl cyclase. Recent studies have demonstrated that CD38 hydrolyze a hundred molecules of NAD to generate only one molecule of cADPR, suggesting that the major function of CD38 is a NADase to regulate cellular NAD levels.36,37 Indeed, the tissue NAD levels in CD38 deficient mice are 10- to 20-fold higher than those in wild type mice,36 indicating that CD38 mediates continuous NAD degradation and thereby contributes to NAD homeostasis. Herein, we established a stable CD38-expressing cell line and carried out a comprehensive analysis to investigate how cellular NAD levels affect protein expression, cell growth rates, and cellular responses to oxidative stress.

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Isolation of a Monoclonal Stable Cell Line Expressing Recombinant CD38

293T cells were cultured in a six-well plate, and lentiviral particles with 5 μg·mL−1 Polybrene were added into 293T cells when they reached 30−40% confluence. After 72 h, a large population of cells expressed GFP and emitted green fluorescence. Cells were then harvested and resuspended in PBS with 1.5% FBS and 1% penicillin/streptomycin. The suspended cells were injected into a flow cytometer for fluorescence-activated cell sorting. A single GFP-positive cell was seeded into one single well in a 96-well plate. The clone with intense and uniform GFP expression was selected and used in the present study.

MATERIALS AND METHODS

Chemicals and Reagents

Dulbecco’s modified Eagle’s medium (DMEM), phosphatebuffered saline (PBS), penicillin/streptomycin, normal and dialyzed fetal bovine serum (FBS and D-FBS) were purchased from Wisent (Montreal, Canada). SILAC DMEM medium, isotope labeling L-13C6-lysine·HCl, standard L-lysine·HCl, standard L-arginine·HCl, and mass spectrum grade acetonitrile were purchased from Thermo (Waltham, MA). Dithiothreitol (DTT) was purchased from Merck (Whitehouse Station, NJ). Sequencing grade trypsin was purchased from Promega (Fitchburg, WI). Iodoacetamide (IAA), Polybrene, NAD/ NADH quantification kit, anti-PKM2 antibody, anti-Flag tag antibody, and anti-HMGB1 antibody were purchased from Sigma (St Louis, MO). Anti-GAPDH antibody, anti-ENO1 antibody, and anti-β-actin antibody were purchased from Abmart (Shanghai, China). Anti-HSP60 antibody was purchased from Enzo (New York, NY). Anti-LDHA antibody, anti-mouse secondary antibody and anti-rabbit secondary antibody were purchased from Cell Signaling Technology (Boston, MA). The Total RNA Isolation System and Reverse Transcription kit were purchased from TIANGEN (Beijing, China). Cell counting kit-8 was purchased from Dojindo (Kumamoto, Janpan). H2O2 was purchased from Aladdin (Shanghai, China). BCA protein assay kit was purchased from Solarbio (Beijing, China).

NAD and NADH Content Assay

NAD and NADH contents were measured using a NAD/ NADH quantification kit according to the manufacturer’s instruction (http://www.sigmaaldrich.com/content/dam/ sigma-aldrich/docs/Sigma/Bulletin/1/mak037bul.pdf). Briefly, cells were washed with cold PBS and then extracted with a NADH/NAD extraction buffer for three freeze/thaw cycles. The mixture was centrifuged, and the supernatant was collected. For detection of total NAD (NAD + NADH), the supernatant was reacted with the NAD cycling enzyme mix to convert NAD to NADH. For detection of NADH only, the supernatant was heated to 60 °C to decompose NAD. Then, samples and NADH standards reacted with a NADH developer reagent, and the absorbance at 450 nm (A450) was measured to quantify NAD and NADH concentrations. Cell Proliferation Assay with CCK-8

Cells were seeded in 96-well plates with 2000 cells/well. Cell proliferation rate was determined with the Cell Counting Kit-8 (CCK-8) according to the manufacturer’s protocol (Dojindo Laboratories, Japan). Briefly, CCK-8 reagents were added into wells after cells grew for 0, 8, 16, 24, 32, 40, 48, 72, 96, and 120 h. Absorbance at 450 nm was measured 3 h after CCK-8 addition.

Cell Culture and SILAC Labeling

Human embryonic kidney 293T cell line was obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. For SILAC labeling, cells were washed with PBS prior to culture in SILAC culture medium, which was made by mixing SILAC DMEM medium with 10% D-FBS, 1% penicillin/streptomycin, 146 mg·L−1 isotope labeling L-13C6-lysine·HCl and 84 mg·L−1 standard Larginine·HCl. 293T cells were grown for 8−10 passages in SILAC medium and tested for full incorporation.

Susceptibility of 293T Cells and Empty-Vector- and CD38-Transfected Cells to Hydrogen Peroxide and Cisplatin

Effects of hydrogen peroxide and cisplatin on cell proliferation in 293T cells and empty-vector- and CD38-transfected cells were analyzed with the CCK-8 kit. Cells (4000 each) were seeded into wells in 96-well cell culture microplates and incubated for 48 h prior to hydrogen peroxide or cisplatin treatment. Cells were then treated with hydrogen peroxide (200, 400, and 600 μM) or cisplatin at different concentrations (10 and 50 μM) in triplicate for 12 h. The CCK-8 reagent was added to treated cells and incubated at 37 °C for 3 h. Optical

Production of Recombination CD38 Lentiviral Particles

Lentiviral expression vector pLVX-IRES-ZsGreen1 with reporter gene of GFP and the package vectors were obtained by courtesy of Dr. Jun Xu (Tongji University, Shanghai, China). The human CD38 cDNA was synthesized from the total RNA of the U266 cell line. The DNA sequence encoding B

dx.doi.org/10.1021/pr4010597 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

Figure 1. Graphical representation of mean values and standard deviation of expression of CD38 and cellular NAD levels in wild type cells, CD38(−) cells, and CD38(+) cells. (a) qPCR analysis of mRNA levels of CD38 in wild type, CD38(−) cells, and CD38(+) cells. (b) Western blot analysis of CD38 protein expression in three different cell lines. (c) Cellular NAD levels in wild type, CD38(−) cells, and CD38(+) cells. (d) Cellular NADH levels in three different cell lines. *p < 0.001; **p < 0.01; n = 3.

and there was a single full-scan mass spectrum in the orbitrap (400−1800 m/z, 30 000 resolution) followed by 20 datadependent MS/MS scans in the ion trap at 35% normalized collision energy (CID). The MS/MS spectra from each LC-MS/MS run were searched against the selected database (IPI human v3.84) using an in-house Proteome Discoverer Searching Algorithm (version 1.3). The search criteria were as follows: full tryptic specificity was required; one missed cleavage was allowed; carbamidomethylation was set as the fixed modification; the oxidation (M) and SILAC 13C6-lysine (+6.020 Da at lysine) were set as the variable modification; precursor ion mass tolerances were set at 10 ppm for all MS acquired in an orbitrap mass analyzer; and the fragment ion mass tolerance was set at 0.8 Da for all MS2 spectra acquired in the linear ion trap. An identified peptide with a confidence value of high was considered as a positive identification. Database searching against the corresponding decoy database was also performed to evaluate the false discovery rate (FDR) of peptide identification. Protein quantitation was also carried out with Proteome Discoverer Searching Algorithm (version 1.3). Briefly, ratios of relative protein expressions for each lysine-containing peptide were calculated using the peak area of Lys6 divided by the peak area of Lys0. The protein ratio is then calculated by averaging all peptide ratios for that protein. Quantitative precision was expressed as protein ratio variability.

density (OD) was measured at 450 nm with a microplate reader (Bio-Rad, Hercules, CA). Cell viability was represented as the percentage of viable cells compared to untreated cells. The experiment was repeated three times. Protein Separation by 1D SDS-PAGE and Proteomics Analysis

The 293T cells infected by the empty pLVX-IRES-ZsGreen1 lentiviral particles were cultured in normal DMEM medium designated as CD38(−) cells, and the monoclonal stable cell line of 293T infected by pLVX-CD38-IRES-ZsGreen1 lentiviral vector was designated as CD38(+) cells and cultured in the SILAC culture medium. Equal amount of proteins from CD38(−) cells and CD38(+) cells was mixed together and separated by 1D SDS-PAGE. The gel bands were excised from the gel into twelve slices, reduced with 25 mM DTT and alkylated with 55 mM IAA. In gel digestion was then carried out with sequencing grade trypsin in 40 mM ammonium bicarbonate at 37 °C overnight. The peptides were extracted twice with 0.1% formic acid in 50% acetonitrile aqueous solution for 30 min. Extracts were then centrifuged in a speedvac to reduce the volume. For LC-MS/MS analysis, the digestion product was separated by a 65 min gradient elution at a flow rate 0.250 μL/min with an EASY-nLCII integrated nano-HPLC system (Proxeon, Denmark) which was directly interfaced with a Thermo LTQ-Orbitrap mass spectrometer. The analytical column was a homemade fused silica capillary column (75 μm ID, 150 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 Å, 5 μm, Varian, Lexington, MA). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The LTQOrbitrap mass spectrometer was operated in the datadependent acquisition mode using Xcalibur 2.0.7 software

Western Blot Analysis

CD38(−) and CD38(+) cells were harvested and lysed on ice with RIPA lysis buffer. The supernatants were collected after centrifugation at 14 000g for 10 min at 4 °C. Protein concentrations were measured with the BCA protein assay kit. Proteins were separated in 12% 1D SDS-PAGE gel and transferred onto a PVDF transfer membrane with electroC

dx.doi.org/10.1021/pr4010597 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

blotting. After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibody, washed with TBST buffer for three times, and then incubated with anti-mouse or anti-rabbit secondary antibody labeled with HRP at room temperature for 1 h. The membrane was further washed with TBST buffer three times and developed with ECL reagents (Engreen, China). βActin was detected with anti-β-actin antibody as an internal control. Quantitative Real-Time PCR (qPCR)

CD38(−) and CD38(+) cells were cultured in 60 mm culture dishes. Total RNA was extracted with the Total RNA Isolation System. cDNA was synthesized from 3 μg total RNA with the Reverse Transcription kit. All qPCR was performed with the Roche LightCycler 480II Detection System with SYBR green incorporation according to the manufacturer’s instructions and β-actin was detected as an internal control. The primers were acquired from Primer Bank (http://pga.mgh.harvard.edu/ primerbank/). The primers are listed in Supporting Information Table 1. Statistical Method

Statistical analysis was carried out using GraphPad Prism 5.0 software. Significant differences in the data were determined by Student’s t test. P values of 1.5 or