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Glutaredoxin-1 silencing induces cell senescence via p53/p21/p16 signaling axis Fan Yang, Meiqi Yi, Yan Liu, Qingtao Wang, Yadong Hu, and Haiteng Deng J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00761 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Proteome Research

Glutaredoxin-1 silencing induces cell senescence via p53/p21/p16 signaling axis Fan Yang 1, Meiqi Yi1, Yan Liu1, Qingtao Wang2, Yadong Hu1, 3, Haiteng Deng1* 1. MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China, 100084 2. Beijing Chaoyang Hospital Affiliated to Capital Medical University, Beijing, China 3. Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China

* To whom correspondence should be addressed: Haiteng Deng, School of Life Sciences, Tsinghua University, Beijing, 100084 China, Tel: 8610-62790498; Fax: 8610-62797154;

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HIGHLIGHTS

Grx1 knockdown increases the reduced to oxidized glutathione ratio.

Grx1 knockdown increases reactive oxygen species production

Grx1 knockdown in 293T and U87 cells activates the p53/p21/p16 axis.

Grx1 knockdown causes cell senescence in 293T and U87 cells.

ABSTRACT Glutaredoxin-1 (Grx1) catalyzes deglutathionylation with glutathione as a cofactor. Accumulating evidence indicates important roles for Grx1 and S-glutathionylation in the aging process; however, further exploration of Grx1-regulated cellular processes is important to understand the functions of Grx1 in aging. In the present study, we constructed stable Grx1 knockdown or overexpression human cell lines. Grx1 silencing significantly decreased the cellular ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (GSH/GSSG ratio), resulting in excessive reactive oxygen species (ROS) accumulation, whereas Grx1 overexpression decreased cellular ROS levels. Grx1 silencing also increased glutathionylation of DJ-1 and HSP60, contributing to decreased mitochondrial spare respiration capacity and ATP production. We applied quantitative proteomics to identify differentially expressed proteins between Grx1 knockdown and control cells and showed that Grx1 silencing inactivated DNA replication and damage repair pathways. p53 signaling was activated by Grx1 silencing, which inhibited the CDK4-mediated G1-S transition, resulting in G1 phase cell cycle arrest and cell senescence, a known hallmark of aging. Taken together, our results indicate that Grx1 regulates DNA replication and damage repair processes and is a potential therapeutic target for aging-related diseases.

KEYWORDS: Glutaredoxin-1; S-glutathionylation; cell senescence; proteomics; redox signaling

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INTRODUCTION Reactive oxygen species (ROS) are highly electrophilic molecules generated by partial reduction of oxygen and consist of radical and non-radical oxygen species, such as hydroxyl radicals (HO•), hydrogen peroxide (H2O2), and superoxide anions (O2-). These small molecules can cause severve damage to macromolecules in vivo, such as proteins, lipids and nucleic acids, which may lead to aging and aging associated diseases.1 Glutathione (GSH) is one of the most abundant molecules in cells with a concentration up to 10 mM in mammalian cells. GSH is an important antioxidant and plays an essential role in maintaining redox homeostasis.2 S-glutathionylation protects proteins from excessive and irreversible oxidation,3 and plays an important role in regulating redox signaling in multiple biological processes.4-7 However, glutathionylation also affects protein activities and deglutathionylation is needed for protein homeostasis. Human glutaredoxins (Grxs), specific glutathionyl-mixed-disulfide oxidoreductases, catalyze protein deglutathionylation.8-9 The Grx family consists of five members, among which Grx1 is widely distributed in the nucleus, cytoplasm and the mitochondrial inner membrane space. It has been proposed that Grx1 acts as the main deglutathionylation enzyme and plays a key role in redox signaling and redox homeostasis.10 Recent studies have shown that both Grx1 expression and activity decrease with age.11 However, the molecular mechanisms of Grx1-mediated aging-related physiological functions remain elusive.12 To explore the effects of Grx1 on cellular processes, we established stable cell lines in which Grx1 was knocked down or overexpressed. Using these cell lines we showed that Grx1 silencing significantly inhibited cell growth. Quantitative proteomics and metabolomics analysis revealed that Grx1 knockdown decreased the cellular level of GSH and increased ROS production, resulting in activation of p53 and associated signaling pathways, which promoted cells to undergo senescence. These results provide a comprehensive view of Grx1-mediated cellular processes and establish that Grx1 is essential for maintaining cellular proteostasis and redox 3



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homeostasis.

EXPERIMENTAL METHODS Cell Culture Human embryonic kidney 293T, human glioblastoma cell line U87 and human liver HepG2 cell lines were obtained from cell bank of Chinese Academy of Sciences (Shanghai, China). 293T and U87 cells were grown in Dulbecco’s modified Eagle medium (Wisent, Montreal, QC) supplemented with 10% fetal bovine serum (Wisent, Montreal, QC) and 1% streptomycin/penicillin (Wisent, Montreal, QC) at 37°C in a humidified incubator with 5% CO2. Establishment of Stable Grx1 Knockdown and Overexpression Cell Lines The human Grx1 cDNA was synthesized from total RNA of the HepG2 cell line. The Grx1 coding region with C-terminal flag tag sequence was cloned into the pLVX-IRES-ZsGreen1 vector. Blank pLVX-IRES-ZsGreen1 vector was used as control. The shRNA sequence used for targeting Grx1 was based on preliminary research13-14 and further confirmed by NCBI BLAST. A scrambled shRNA with no homologous sequence reported in human genome was applied as control. The shRNA sequences were shown in Table S1. The shRNAs were cloned into plasmid pLL3.7. Production of lentiviral particles of recombinant Grx1 were carried out based on the protocol by Tiscornia et al.15 We constructed pLVX-Grx1-IRES-ZsGreen1 or pLVX-IRES-ZsGreen1 stable transfected cell lines, respectively, with packing vectors into 293T cells when they reached 80-90% confluence. After 48 hours’ culture, the cell culture supernatant was then collected and concentrated with PEG6000. The precipitated lentiviral particles were resuspended in PBS. The recombinant pLL3.7 vector carrying Grx1 shRNA or control shRNA was processed as the overexpress vector. The lentivarial particles were then used to infect 293T and U87 cells in the presence of 6 µg/mL of polybrene for 6h. Cells were cultured in fresh medium for 96 h after infection and sorted by flow cytometer with GFP positive to generate stable cell lines. 4


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Cell Proliferation Assay with CCK-8 Cells were seeded in 96-well plates (2,000 cells per well). Cell proliferation rate was determined with the Cell Counting Kit-8 (CCK-8) according to the manufacturer’s instructions (Dojindo Laboratoies, Japan). Optical density (OD) was measured in 2 hours after CCK-8 addition at 450 nm with a microplate reader when cells grew for 0, 12, 24, 36, 48, 60, 72h. Cell Viability Assay of Grx1 Knockdown Cells Treated with Hydrogen Peroxide Effects of hydrogen peroxide on control and Grx1 knockdown cells were analyzed with the CCK-8 kit. Cells were treated with hydrogen peroxide (0, 250, 500 and 750 µM) for 12h. The CCK-8 reagent was added to treated cells and the cells were incubated for 2 hours at 37°C. Absorbance at 450nm was measured after incubation. Mitochondrial Respiration Analysis Oxygen consumption rates of 293T and U87 cells were measured using the Seahorse XF Cell Mito Stress Test and the Seahorse XFe96 Analyzer (Seahorse Bioscience,

Agilent

Technologies,

Waldbronn,

Germany).

Drugs

including

oligomycin, carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and antimycin A/rotenone were added to the reaction system in time series. The following parameters were calculated: basal respiration, maximal respiration and ATP production. After the experiment, the cells were lysed and the proteins of each sample were quantitated by BCA method. The experiment data was analyzed and visualized using wave software (version 2.3.0, Seahorse Bioscience, Agilent Technologies, Waldbronn, Germany) and normalized by the protein content. Detection of Cellular Reactive Oxygen Species (ROS) The cellular ROS in Grx1 knockdown cells was detected by CellROX® Deep Red Reagents (Invitrogen, Grand Island, NY) following manufacturer’s instructions. Briefly, cell medium was added 5µM CellROX® Deep Red probe and then cells were incubated at 37°C for 30 minutes. Then, the cells were washed twice with PBS and analyzed on a BD FACS Aria Flow Cytometer (Becton Dickinson, NJ). 5



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Sample Preparation and Quantitative Proteomic Analysis by 2D LC-MS/MS Proteomic analysis was carried out as described in our previous research.16 Briefly, proteins were extracted from Grx1 cells with 8 M urea, and 200 µg of protein was reduced and alkylated. Then the proteins were digested with trypsin (Promega, Fitchburg, WI) at 37°C overnight. Tryptic peptides were desalted and labeled with the tandem mass tag (TMT, Thermo Waltham, MA) according to manufacturer’s protocol. Labeled peptides from different samples were mixed together, desalted and separated by two dimensional HPLC system. At the first step, reversed phase separation was performed on an HPLC system at pH10.0. 12 fractions were collected, and then resuspended in 0.1% formic acid for next seperation. For LC-MS/MS analysis, the TMT-labeled peptides were separated by a 120 min gradient elution at a flow rate 0.250 mL/min using HPLC at a lower pH. A Q-Extractive mass spectrometer was applied with the data-dependent acquisition mode. Each full-scan mass spectrum in the Orbitrap (300-1800 m/z, 70, 000 resolution) was followed by 20 data-dependent MS/MS scans. The MS/MS spectra from each LC-MS/MS run were searched against the Uniprot human database using the SEQUEST searching engine of Proteome Discoverer software (version 2.1). Detailed search criteria were included in Supporting Information. The false discovery rate was estimated and the cutoff score of 1% was accepted based on the decoy database. Relative protein quantification was performed using PD software according to manufacturer’s instructions on the intensity of six TMT reporter ions per peptide. Proteomic analysis was carried out in biological triplicates. Metabolomics Analysis The metabolomics analysis was performed as previous described.17 Briefly, the cells were washed and extracted using 80% pre-chilled methanol. The metabolites were analyzed by LC-MS/MS. To quantify the change level of metabolites of control and Grx1 knockdown cells, we applied two mass spectrometry methods. For untargeted metabolomics profiling, the Q-Extractive Mass Spectrometer was used. The metabolites were identified based on the retention time on the LC analysis and 6


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the accurate mass with less than 5 ppm mass accuracy. TraceFinder was used for peaks identification and quantification. For targeted quantitative analysis, the Triple Quadrupole Mass Spectrometer with positive/negative ion switching was used for target molecules quantitation with selective reaction monitoring (SRM). The method was constructed with parameters acquired with standard metabolites. Identification of Changes in Protein Redox States by a Combination of Double – labeling and LC-MS/MS We devised an in-solution double-labeling method to identify the thiol-redox state change of proteins in the control or Grx1 knockdown cells referring to Gilbson’s method.18 The experimental procedures were diagrammed in Figure S1. To trap different state of thiols, we performed a two-step in-solution labelling method in this experiment. Protein solution was alkylated with 5 mM

13

C-iodoacetic acid for 30

minutes in the dark and then reduced by 15 mM DTT. At last, 30 mM 12C-iodoacetic acid was added to the solution and reacted for another 30 minutes in dark. Samples were digested by trypsin in solution as described and analyzed using LC-MS/MS. Only the peptides detected in both control and Grx1 knockdown groups were selected for subsequent analysis.

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C-carboxymethyl modified cysteines indicated reductive

state while 12C-carboxymethyl labeled cysteines indicated oxidative state. Cellular Senescence Analysis Cells were stained using Senescence Cell Histochemical Staining Kit (Sigma, St Louis, MO) for detection of SA-β-Gal expression. The staining of cells was performed as recommended by the manufacturer. 293T or U87 cells were stained with an X-gal containing staining buffer at 37°C overnight in a CO2-free atmosphere. Positive staining was evaluated compared to the control cells. Cell Cycle Analysis Cells were harvested, washed by ice-cold PBS and fixed with 70% ethanol for two hours on ice. After that, the fixed cells were resuspended in PBS, treated with RNase and stained with PI. The cell cycle analysis was performed on BD FACS Aria Flow Cytometer (Becton Dickinson, NJ). The percentage of cells in each cell cycle 7



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phase was assessed on Modfit software. Western Blotting Cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH7.5, 1% sodium pyrophosphate, Protease Inhibitor Cocktail). Equal amounts of proteins were separated on the 15% SDS-PAGE gel and then transferred onto a PVDF membrane. Western blot analysis followed a standard procedure. Anti-Grx1 antibody, anti-CDK4 antibody and anti-Nrf2 antibody were from Abcam (Cambridge, MA). Anti-p53 antibody was obtained from Sigma (St Louis, MO). Anti-p21 antibody and anti-p16 antibody were purchased from Proteintech (Chicago, IL). Anti-SOD2 antibody was from Cell Signaling Technology (Danvers, MA). Bioinformatics Analysis The metabolism analysis was processed on MetaboAnalyst 3.0 website.19 The differentially expressed protein-involved signaling pathways were analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID)20 and Ingenuity Pathway Analysis (IPA, QIAGEN). The upstream regulators of differentially expressed protein were predicted based on the Ingenuity Knowledge Base. Statistical Analysis Statistical analysis was carried out with GraphPad Prism 6.0 software by two sided unpaired t test. Multiple comparisons were performed using ANOVA. P values of

p16

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