Redox Sensitivities of Global Cellular Cysteine Residues under

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Redox sensitivities of global cellular cysteine residues under reductive and oxidative stress Kazutaka Araki, Hidewo Kusano, Naoyuki Sasaki, Riko Tanaka, Tomohisa Hatta, Kazuhiko Fukui, and Tohru Natsume J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00087 • Publication Date (Web): 28 Jun 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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Journal of Proteome Research

Redox sensitivities of global cellular cysteine residues under reductive and oxidative stress Kazutaka Araki1, *, Hidewo Kusano1, Naoyuki Sasaki2, Riko Tanaka1, Tomohisa Hatta2, Kazuhiko Fukui1, Tohru Natsume1, 2 1

Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo 135-0064, Japan

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Robotic Biology Institute, Inc., Tokyo 135-0064, Japan

* Corresponding author : Kazutaka Araki : Tel: +81-3-3599-8140, Fax: +81-3-3599-8134, Email: [email protected] Hidewo Kusano : Tel: +81-3-3599-8850, Fax: +81-3-3599-8134, E-mail: [email protected] Naoyuki Sasaki : Tel: +81-3-3599-8127, Fax: +81-3-3599-8134, E-mail: [email protected] Riko Tanaka : Tel: +81-3-3599-8850, Fax: +81-3-3599-8134, E-mail: [email protected] Tomohisa Hatta : Tel: +81-3-3599-8140, Fax: +81-3-3599-8134, E-mail: [email protected] Kazuhiko Fukui : Tel: +81-3-3599-8667, Fax: +81-3-5530-2064, E-mail: [email protected] Tohru Natsume : Tel: +81-3-3599-8100, Fax: +81-3-5530-2064, E-mail: [email protected]

RUNNING TITLE: Redox sensitivities of global cellular cysteine residues

KEYWORDS: Oxidation, Cysteine, Quantification, Redox, Proteomics

ABBREVIATIONS: ROS, reactive oxygen species; H2O2, hydrogen peroxide; GFP, green fluorescent protein; roGFP, reduction oxidation-sensitive green fluorescent protein; HyPer, hydrogen peroxide sensor; PRDX, peroxiredoxin; Grx, glutaredoxin; NEM, N-ethymaleimide; DTT, dithiotreitol; GSH, reduced glutathione; GSSG, oxidized glutathione; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TXN, thioredoxin

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ABSTRACT

The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteinetargeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. It also caused some unique oxidative shifts, including for peroxiredoxin 2 (PRDX2), uroporphyrinogen decarboxylase (UROD) and thioredoxin (TXN), proteins reportedly affected by cellular reactive oxygen species production. Modest H2O2 treatment (50 µM for 5 min) did not cause global oxidations but, instead, had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins, like PRDX2, peroxiredoxin 1 (PRDX1), TXN and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Overall, our quantitative data illustrated both H2O2- and reduction-mediated cellular responses, whereby, while redox homeostasis is maintained, highly reactive thiols can potentiate the specific, rapid cellular signaling to counteract acute redox stress.


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INTRODUCTION Cellular redox homeostasis is recognized as playing diverse roles in protection against oxidative stress and in regulation of multiple physiological processes. Living cells are always exposed to intracellular and extracellular challenges that alter redox processes, generally known as oxidative stress. Proper maintenance of physiological redox states is crucial to maintaining the appropriate cellular machinery and function1, 2. Thus, cells have acquired broad adaptive mechanisms, including activation of redox regulatory and defense machineries (short-term), and activating gene expression (long-term)3-5. Collapses in physiological redox states have been recognized as being pathogenic in numerous diseases, including diabetes, cancer and neurodegenerative disorders6, 7. Cysteine is intrinsically vulnerable to oxidative stress because it has a highly reactive nucleophilic thiol and can be reversibly or irreversibly modified. Thiol modifications are key regulatory events in redox processes. The state of such modifications can give insights into cellular redox status, potentially altering protein activity, location, function or interaction partners7, 8. Reversible modifications include disulfide (S–S) formation, S-nitrosylation (SNO), S-sulfenylation (SOH), S-glutathionylation (SSG) and S-sulfhydration (SSH) (Figure 1)1, 9-11. Irreversible modifications include formation of S-sulfinic acid (SO2H) and S-sulfonic acid (SO3H). Detection and quantification of these cysteine modifications is technically demanding because of the varied and transient nature of these reversible redox states and the relatively low cysteine content of the entire proteome (~2 %)12. Introduction of the ICAT (isotope-coded affinity tags) method has enabled enrichment and mapping of cysteine-containing peptides13, 14. Recently, another advanced labeling strategy, called cysteine thiol-reactive iodoacetyl tandem mass tag (iodoTMT), was introduced, which enables 6-plex experiments (iodoTMT6plex)


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(Figure 1)15. So far, this strategy has been applied to mapping and quantification of Snitrosylation and S-sulfenylation16, 17. While it is crucial to identify and quantify redox-sensitive cysteine residues to explore their physiological and pathological functions, the lack of quantitative data on their sensitivities to redox stresses is still a limitation. We introduced a feasible multiplexing protocol to simultaneously measure cysteine redox states. With human cultured cells as a model system, we evaluated oxidation levels of cysteines under exogenous perturbations, employing DTT and H2O2. We believe that ours are the first reported quantitative data on reductive stress, with bolus DTT treatment (1 mM for 5 min) causing cysteine thiols to globally become converted to a reduced state, irrespective of their cellular localizations. We also discovered some interesting proteins whose levels of oxidation became, instead, increased with DTT treatment, including TXN and PRDX2. Regarding H2O2 treatment, we applied modest treatment conditions (50 µM H2O2 for 5 min) because data using only higher dose bolus H2O2 treatment (1 mM) were previously available for mammalian cells. Collectively, our findings illustrate relevant and unique cellular responses to redox stress and highlight the importance of detailed quantitative analysis to evaluate volatile cellular redox states18.

EXPERIMENTAL PROCEDURES Materials The iodoTMT6plex reagents, anti-TMT antibody, immobilized anti-TMT antibody resin and bicinchoninic acid (BCA) protein assay kit were from Thermo Fisher Scientific (Rockford, IL,


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USA). Fetal bovine serum (FBS) was from Atlanta Biologicals, Inc. (Lawrenceville, GA, USA). MonoSpin C18 columns were from GL sciences Sciences Inc. (Tokyo, Japan). Amersham ECL Prime Western Blotting Detection Reagent was from GE Healthcare (Buckinghamshire, UK). Trypsin (modified, sequencing grade) was from Promega (Madison, WI, USA). Dulbecco’s modified Eagle’s medium (DMEM) and L-glutamine were from Wako, Inc. (Kyoto, Japan). The following antibodies were used for western blotting: anti-thioredoxin (sc-13130; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-PDIA6/P5 (ab37756; Abcam, Cambridge, UK), anti-PDIA4/ERp72 (SPS720; Enzo Life Sciences, Farmingdale, NY, USA), anti-PRDX1 (NBP137095; Novus, Littleton, CO, USA), anti-PRDX2 (H00007001-M01; Abnova, Taipei City, Taiwan) and anti-PRDX4 (ab16943; Abcam). Secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. All other reagents were from Sigma-Aldrich (St. Louis, MO, USA).

Protein sample preparation The labeling protocol is outlined in Figure 1. At 3 h prior to experimental treatments, cells were washed with warm PBS and medium was changed to OPTI-MEM buffer (Invitrogen). For DTT and H2O2 treatments, HEK293 cells were incubated with either 1 mM DTT or 5 µM H2O2 in medium and incubated for 5 min prior to sample preparation. After treatments, medium was removed and cell proteins were precipitated with 10% w/v TCA in acetone to protonate all redox-active thiolate anions and halt thiol-disulfide exchanges by denaturing and inactivating proteins21. Samples were centrifuged and cell pellets collected and sonicated in a Bioruptor ultrasonicator under cold water (UCD-200TM; Cosmobio, Tokyo,


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Japan). These samples were centrifuged and supernatants were removed. Pellets were sonicated again under cold water with acetone, and this was repeated two times. Equivalent volumes of each sample suspension were collected and dissolved in Tris-denaturing buffer (200 mM Tris pH 8, 2% (w/v) SDS, 7M Urea, 5mM EDTA, 1mM neocuproine). Protein concentrations were measured in these aliquots by the BCA method following the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). Based on analysis of these solubilized aliquots, additional aliquots (300 µg protein per sample) were then collected from the precipitated samples in acetone, centrifuged and pellets dissolved in Tris-denaturing buffer containing 1 mM iodoTMT reagent. Samples were sonicated briefly and incubated at room temperature (RT) overnight in the dark. After incubation, the mixtures were precipitated with methanol/chloroform and then washed three times in ice-cold acetone, as described previously22. The iodoTMTlabeled proteins were redissolved in Tris/SDS buffer (200 mM Tris pH 8, 2% (w/v) SDS, 5 mM EDTA) and reduced with 50 mM dithiothreitol (DTT) at 37 °C for 1 h. After methanol/chloroform precipitation, pellets were washed with acetone and free thiols labeled by dissolving in Tris/SDS buffer containing 1 mM iodoTMT reagent and incubating at RT overnight in the dark. After methanol/chloroform precipitation, the protein pellet was resuspended with 200 mM Tris pH 8, 8 M urea, 1 M ammonium bicarbonate and 0.01% decyl βD-glucopyranoside (DG) containing lysyl endoprotease (LysC). After incubating at 37 °C for 3 h, samples were diluted 8-fold in 100 mM Tris (pH 8) and 0.01% DG buffer containing trypsin, and digested at 37 ° C for 3 h. Undigested proteins were removed by centrifugation at 15000g at RT for 30 min. The peptides were then diluted 5-fold with Tris-buffered saline Triton (TBST) buffer (25 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.01% Triton X-100). The peptide samples were incubated with 200 µl anti-TMT antibody resin overnight at 4 °C with end-over-end rocking.


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After the supernatant was removed, the resin was washed four times with one column volume of TBST and three times with one column volume of Milli-Q water. Peptides were finally eluted with four column volumes of elution buffer (2% acetonitrile (ACN), 0.1% trifluoroacetic (TFA), pH ~2, estimated with a pH indicator) and purified on a C18 spin column. For peptide enrichment, the above samples were lyophilized and redissolved in 20 µl 50 mM ammonium bicarbonate buffer (pH 8.8) containing 1.4 M guanidine hydrochloride. The resulting peptides were analyzed by nanoscale liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). In order to assure reproducibility and reliability, four independent preparations were performed and each sample was analyzed three times by LC/MS/MS.

LC-MS/MS Samples were analyzed with a custom-made nanopump system (LC-Assist, Tokyo, Japan) coupled to a TripleTOF 5600 MS/MS (AB Sciex, Framingham, MA, USA). Samples were loaded on a custom-made C18 column (150 µm × 500 mm) packed with Mightysil RP-18 GP (3 µm) (Kanto Chemical, Tokyo, Japan), operated at a flow rate of 1 µl/min−1 for 30 min during sample injection. For elution, mobile phase A (H2O and 0.1% formic acid (FA)) and mobile base B (0.1% FA in ACN) were used with a 120 min linear gradient from 0 % to 40% B at a flow rate of 100 nL/min−1. Eluted peptides from the reversed-phase chromatography were directly loaded on the nESI source in positive ionization and high sensitivity modes. The MS survey spectrum was acquired in the range of 400 to 1250 m/z in 250 ms. For information-dependent acquisition (IDA), the 25 highest intensity precursor ions above 50 counts threshold with charge states +2 and +3 were selected for MS/MS scans. Each MS/MS experiment set the precursor m/z on a 12 s


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dynamic exclusion and scan range was 100 to 1500 m/z in 100 ms. The relative collision energy (CE) used for MS/MS for each peptide precursor was determined by the following equation: CE = slope x (m/z) + intercept. And parameters of slope and intercept were as follows: (Charge state, Slope, Intercept) = (Unknown, 0.0575, 9), (1, 0.0575, 9), (2, 0.0625, -3), (3, 0.0625, -5), (4, 0.0625, -6), or (5, 0.0625, -6). Maximum allowed CE was set at 80 V. After every six samples (at 24 h intervals), auto calibration was performed to maintain high mass accuracy in both MS and MS/MS acquisitions.

Protein identification and quantification Protein identifications were performed with the Mascot Server v.2.3 search engine (Matrix Science, Inc., Boston, MA, USA) using the NCBI non-redundant human protein dataset (NCBInr RefSeq Release 71, containing 179460 entries) after transferring the original MS/MS file data (*.wiff) to the *.mgf format. The search was set up for full tryptic peptides with a maximum of 2 missed cleavage sites. Charge states of peptides were set to +2 and +3. IodoTMT6plex at cysteine was included as variable modification. The false discovery rate (FDR) for peptide identification was set to 5% in all analyses using Mascot Percolator. Additionally, iodoTMT6plex plus oxygen was manually implemented as variable modification, to investigate direct iodoTMT modification to cysteine sulfenic acid. The other search parameters were set as follows: MS/MS fragment ion mass tolerance of ±0.25 Da and peptide tolerance of ±250 ppm. The relaxed tolerance setting was selected based on the better yields of FDR. Exclusions of peptide events were as follows: peptide was not modified with an iodoTMT label and any reporter ion intensities did not exceed 30 counts (threshold). For relative quantification of the


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reporter ion intensities of TMT-labeled cysteines, raw ion intensities for each reporter ion from each individual MS/MS spectrum were extracted (e.g. Supplemental Figure S-1). With the use of the raw intensities for each reporter, the following ratios were generated: 129/(126+129)*100 (percentage reversible oxidation in control, control group), 130/(127+130)*100 (percentage reversible oxidation in DTT treated cells, DTT group) and 131/(128+131)*100 (total available cysteine in H2O2 treated cells, H2O2 group). The average ratios were determined by at least three spectra, irrespective of their charge states. Fold changes in oxidation were determined using the ratio of percentage reversible oxidations in the DTT group or and in the H2O2 group, over those in the control group, for each peptide.

Cell Culture and Treatment Human embryonic kidney (HEK239T) cells (ATCC, Manassas, VA, USA) were cultured in DMEM containing 10% (v/v) heat-inactivated FBS at 37 °C in a saturated humidity atmosphere containing 95% (v/v) air and 5% (v/v) CO2.

Constructs and stable cell lines To construct plasmids for stable expression of Grx1-roGFP2 and TXN in mammalian cells, the cDNA encoding full-length Grx1-roGFP2 was PCR amplified and transferred to the vector pMXs-GW-IP, which was constructed from Gateway Vector System (Invitrogen, Waltham, MA, USA) and pMXs-GW (Addgene, Cambridge, MA, USA) and included insertion of puromycin resistance gene after the IRES2 region19. All constructs were verified by sequencing.


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Retroviruses were produced using the PlatE packaging cell as described previously20. Stably expressing HEK293T cell lines were created by infection with the pMXs-GW-IP retroviral vector. Cells resistant to puromycin (1 µg/ml) were selected and were maintained in the incubator as described above. HEK239T cells stably expressing Grx1-roGFP2 were used only for assessment of redox responses under DTT and H2O2 treatments (Figure 2).

Measurements of redox states of redox-active proteins and Grx1-roGFP2 in cells Protein samples were prepared as described above. Briefly, HEK293T cells cultured in OPTIMEM buffer were treated with either 1 mM DTT or 5 µM H2O2 in medium and incubated as indicated in the Figure (0, 1, 2, 5, 10 or 20 min). After incubation, the medium was changed to 10% w/v TCA in acetone buffer to prevent further thiol–disulfide exchange. After cell pellets were collected by scraping, these samples were sonicated with a Bioruptor ultrasonicator briefly under cold water (UCD-200TM; Cosmobio)23. Samples were then centrifuged, supernatants were removed and pellets were again sonicated under cold water with acetone. Equivalent protein quantities of each sample were collected by centrifugation and dissolved in Tris-alkylation buffer (100 mM Tris-HCl, pH 6.8, 2% SDS) containing 5 mM methoxypolyethylene glycol (mean MW 2,000)-maleimide (mPEG2000-mal, Sunbright ME-020MA; NOF Corporation, Tokyo, Japan). The mixture was incubated at 25 °C for 2 h to alkylate the free cysteine sulfhydryl groups. Proteins from the resulting lysates were separated by SDS-PAGE, blotted onto an Immobilon-P membrane (Millipore, Darmstadt, Germany), incubated with appropriate primary followed by secondary antibodies and labeled bands were detected with ECL Select Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) and LAS-3000 mini (Fujifilm, Tokyo,


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Japan) to visualize the oxidation levels of redox-active proteins. In parallel with this analysis, after the treatments with DTT or H2O2, HEK239T cells stably expressing Grx1-roGFP2 were collected at each time point in PBS buffer containing 20 mM NEM and incubated on ice for 10 min. After brief centrifugation, cell pellets were lysed with the lysis buffer and supernatants were transferred to a 96-well plate. Fluorescence was read using a Spectra MAX M5 (Molecular devices, Sunnyvale, CA, USA) with excitation at 405 and 488 nm and emission at 530 nm. This analysis was performed in triplicate and average data were plotted.

Protein immunoprecipitation analysis HEK293T cells stably expressing TXN-FLAG were washed once with PBS containing 20 mM NEM and lysed with lysis buffer (50 mM Hepes-NaOH, pH 7.5, containing 150 mM NaCl, 0.25% Triton X-100, 20 mM NEM and protease inhibitors). The resulting supernatant was mixed with anti-FLAG M2-agarose beads (Sigma-Aldrich, MO, USA) for 1 h and the beads were washed twice with wash buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl and 0.1% Triton X-100). After elution with FLAG peptide (0.5 mg/ml, Sigma-Aldrich), samples were further analyzed by immunoblotting as mentioned above.

Statistical analysis and annotation of subcellular localizations Statistical significance and p values for each iodoTMT modification site were calculated by a t-test (two-tailed, equal variances). For quantitative changes, a 1.2-fold cutoff was set to determine redox-reversible proteins, with a p-value of

Redox Sensitivities of Global Cellular Cysteine Residues under

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