Article pubs.acs.org/jpr
First Proteomic Study of S‑Glutathionylation in Cyanobacteria Solenne Chardonnet,† Samer Sakr,‡ Corinne Cassier-Chauvat,‡ Pierre Le Maréchal,† Franck Chauvat,‡ Stéphane D. Lemaire,§,∥ and Paulette Decottignies*,† †
IBBMC, CNRS UMR 8619, Univ Paris-Sud, Orsay, France iBiTec-S, CEA, CNRS UMR8221, Univ Paris-Sud, CEA-Saclay, Gif sur Yvette, France § IBPC, CNRS UMR8226, Paris, France ∥ Sorbonne Universités, UPMC Univ Paris 06, UMR8226, Paris, France ‡
S Supporting Information *
ABSTRACT: Glutathionylation, the reversible post-translational formation of a mixed disulfide between a cysteine residue and glutathione (GSH), is a crucial mechanism for signal transduction and regulation of protein function. Until now this reversible redox modification was studied mainly in eukaryotic cells. Here we report a large-scale proteomic analysis of glutathionylation in a photosynthetic prokaryote, the model cyanobacterium Synechocystis sp. PCC6803. Treatment of acellular extracts with N,Nbiotinyl glutathione disulfide (BioGSSG) induced glutathionylation of numerous proteins, which were subsequently isolated by affinity chromatography on streptavidin columns and identified by nano LC−MS/MS analysis. Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and mass spectrometry analysis. Taken together the two approaches allowed the identification of 383 glutathionylatable proteins that participate in a wide range of cellular processes and metabolic pathways such as carbon and nitrogen metabolisms, cell division, stress responses, and H2 production. In addition, the glutathionylation of two putative targets, namely, peroxiredoxin (Sll1621) involved in oxidative stress tolerance and 3-phosphoglycerate dehydrogenase (Sll1908) acting on amino acids metabolism, was confirmed by biochemical studies on the purified recombinant proteins. These results suggest that glutathionylation constitutes a major mechanism of global regulation of the cyanobacterial metabolism under oxidative stress conditions. KEYWORDS: carbon metabolism, cyanobacteria, cysteine modification, glutathionylation, mixed-disulfide bridge, nitrogen metabolism, proteomics, redox regulation, Synechocystis
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INTRODUCTION
by reaction with activated thiols such as sulfenic acids or Snitrosylated thiols.6 On the other hand, glutathionylation could be catalyzed by specific enzymes. The reverse reaction, deglutathionylation, is catalyzed by the thiol−disulfide oxidoreductase glutaredoxins (Grx).7 Alternatively, thioredoxins (Trx) have been also proposed to act as deglutathionylase enzymes, as described in yeast8 or for cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from A. thaliana.9 Several large-scale proteomic methods have been developed to identify proteins undergoing glutathionylation either in vitro or in vivo (reviewed in ref 10). A 35S-cysteine labeling strategy in the presence of protein synthesis inhibitors has been reported for oxidatively stressed human T-lymphocytes,11 as well as glutathione S-transferase overlay in rat tissues.12 The use of biotinylated-oxidized glutathione (BioGSSG)13 or its ethyl
Photosynthetic organisms are continuously challenged with toxic reactive oxygen species (ROS) generated by numerous metabolic pathways, especially photosynthesis and respiration. These ROS are handled by cellular antioxidant defense systems that, among other processes, maintain the redox homeostasis of cellular thiols.1 Glutathione, a highly abundant (from 0.1 to 10 mM) tripeptide (γ-Glu-Cys-Gly), occurring mostly in its reduced form (GSH), is one of the major cellular antioxidant redox buffers in most organisms.2 Moreover, glutathione can form a mixed-disulfide bridge between the thiol group of its cysteine and an accessible free thiol on a protein, a reaction termed as protein S-glutathionylation.3,4 As this post-translational modification is reversible and seems to occur mostly under oxidative or nitrosative stress conditions, it constitutes an important mechanism of redox signaling by protecting specific cysteine residues or by modulating protein activity.3,5 However, its mechanism is not yet completely understood. Glutathionylation may occur spontaneously by thiol−disulfide exchange or © 2014 American Chemical Society
Special Issue: Environmental Impact on Health Received: June 23, 2014 Published: September 11, 2014 59
dx.doi.org/10.1021/pr500625a | J. Proteome Res. 2015, 14, 59−71
Journal of Proteome Research
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ester (BioGEE)14 in combination with avidin-affinity selection has been also described. Another strategy applied to endothelial-like cells15 and the malaria parasite16 consisted in deglutathionylation of mixed disulfides by specific Grx followed by their derivatization with N-ethylmaleimide and affinity purification of tagged proteins. However, these proteomic studies have been mostly directed to animal cells or tissues, while little is known in photosynthetic organisms. 4,17 Thiolation targets emerging in vivo in A. thaliana18 or the eukaryotic green algae Chlamydomonas reinhardtii19 after oxidative stress (induced by tert-butyl hydroperoxide or diamide, respectively) have been identified using a 35S-cysteine labeling approach. Recently we pursued the latter work by performing a large-scale in vitro study of the “glutathionylome” of C. reinhardtii, using biotinylated-oxidized glutathione.20 Numerous proteins were identified, highlighting the sensitivity of biotin-based approaches. This study suggested a crucial role of glutathionylation for regulation of carbon assimilation, especially by modulating the activity of Calvin−Benson cycle enzymes. While glutathione is widely distributed in eukaryotes, its production in prokaryotes is restricted to cyanobacteria, proteobacteria, and a few strains of Gram-positive bacteria.21,22 Cyanobacteria, formerly termed blue-green algae, are the only prokaryotes that perform oxygen-evolving photosynthesis. These ancient microorganisms (∼2.6 billion years) are regarded as the progenitors of the oxygen-rich atmosphere of our planet23 that enabled the evolution of the vital respiration pathway, as well as the ancestors of the plant chloroplast.24 In colonizing most waters (fresh, brackish, and marine) and terrestrial (including deserts) environments, cyanobacteria have evolved as the largest and most diverse group of bacteria.25 They play important roles in global biogeochemical cycles and make up organic assimilates essential to the food chain. Hence, cyanobacteria are regarded as promising “low-cost” microbial cell factories for the ecologically responsible production of natural products, secondary metabolites, and biofuels.26,27 Glutathionylation has been poorly studied in cyanobacteria so far. Recently glutathionylation was reported to regulate the activity of two proteins from the cyanobacterium Synechocystis sp. PCC6803.28,29 This organism, the best studied unicellular cyanobacterium, harbors a small sequenced and fully annotated genome,30 which facilitates proteomic approaches. Synechocystis sp. PCC6803 (hereafter Synechocystis) possesses only one monothiol31 and two dithiol Grxs, which have been shown to specifically interact with various protein partners operating in the tolerance to toxic metals such as arsenate,32 mercury,28 selenate,33 uranyl,28 and various other stresses.34 In addition, several targets of one dithiol Grx (Ssr2061) from Synechocystis have been identified by proteomic methods.35 In this paper, we report the first large-scale proteomic study of glutathionylation in Synechocystis. About 350 proteins were identified as glutathionylatable proteins, and the site of modification was determined for 125 targets. In addition, the glutathionylation of two of these targets, namely, peroxiredoxin (PrxII, Sll1621) and 3-phosphoglycerate dehydrogenase (PGDH, Sll1908), has been validated in vitro using recombinant proteins.
Article
EXPERIMENTAL SECTION
Materials
Modified trypsin was purchased from Promega (Madison, WI, USA). EZ link Sulfo-NHS-Biotin was from Perbio Science (Pierce, Cramlington, U.K.). Streptavidin-agarose, oxidized Lglutathione (GSSG) and D-3-phosphoglycerate were from Sigma-Aldrich (St. Louis, MO, USA). Electrophoresis reagents were obtained from Bio-Rad (Bio-Rad, Hercules, CA, USA). Synechocystis Cultures and Total Protein Extraction
Synechocystis sp. PCC6803 was grown at 30 °C under continuous white light of standard fluence (2500 lx, i.e., 31.25 μE m−2 s−1), on BG11 medium enriched with 3.78 mM Na2CO3.36 Mid-log phase cultures (OD580nm = 0.5, i.e., 2.5 × 107 cells mL−1) grown under standard conditions were harvested by centrifugation (10,000g) for 10 min. Cells were resuspended in 30 mM Tris-HCl, pH 8.0, immediately frozen in an Eaton press chamber cooled in a dry ice/ethanol bath, and disrupted (250 MPa, 6 T). Lysate was clarified by centrifugation for 20 min (13,000g) at 4 °C, and protein concentration was determined using the Bradford reagent (BioRad). Synthesis of BioGSSG
EZ link Sulfo-NHS-Biotin, a soluble biotinylation reagent, was used to couple biotin to the primary amino groups of glutathione disulfide under mild alkaline conditions. Specifically, the biotinylation reagent (50 μL, 64 mM) was added to GSSG (50 μL, 32 mM) in 100 mM potassium phosphate buffer, pH 8.0, and the mixture (100 μL) was left to derivatize for 1 h at room temperature. After incubation, untreated biotin was quenched by adding 35 μL of 0.6 M ammonium bicarbonate buffer. Glutathionylation of Protein Extract and Separation by SDS-Gel Electrophoresis (Supplemental Figure 1)
Proteins (250 μL, 6 mg) were incubated with or without (control experiment) freshly synthesized BioGSSG (100 mL) for 1 h at room temperature in a final volume of 400 μL (estimated BioGSSG concentration = 2.5 mM). After removal of BioGSSG and excess reagents on G-25 gel filtration columns (HiTrap desalting, 5 mL, GE Healthcare), protein extracts were loaded on streptavidin-agarose columns (1 mL). After 6 washes with 30 mM Tris-HCl pH 7.9 containing 600 mM NaCl, 0.25% Triton, and 1 mM EDTA, followed by one wash without NaCl, retained proteins were eluted with 20 mM DTT in 10 mM ammonium bicarbonate. They were then concentrated by evaporation using a SpeedVac concentrator, separated by SDSPAGE on 10% or 15% polyacrylamide gels with a Protean II XL system (Bio-Rad), and stained with Coomassie Brilliant Blue R250. Western Blot
After G-25 columns, BioGSSG-treated proteins were submitted to non-reducing SDS-PAGE and then transferred on nitrocellulose membrane for 1 h at 100 V in 20 mM Tris, 150 mM glycine, 10% ethanol, 0.05% SDS. Mouse anti-biotin primary antibodies (Sigma) were applied for 1 h at room temperature followed by secondary antibodies (anti-mouse IRDye 680conjugated) diluted 1/10000. The fluorescent signal was scanned using the Odyssey infrared imaging system (Li-Cor, Lincoln, NE, USA) operating at 685 nm excitation wavelength and 710 nm for emission. 60
dx.doi.org/10.1021/pr500625a | J. Proteome Res. 2015, 14, 59−71
Journal of Proteome Research
Article
with 100 mg mL−1 ampicillin at 37 °C. After 4 h of incubation at 37 °C, cells were harvested by centrifugation at 7,000 rpm for 5 min at 4 °C, resuspended in buffer A (30 mM Tris-HCl, pH 7.5, 400 mM NaCl, 20 mM imidazole) containing 2 mg mL−1 lysozyme (Sigma) and incubated on ice for 1 h. The suspension was then sonicated using Ultrasonic Cell Disruptor, Microson on ice (6 pulses, 20 s each). Cell lysates were clarified by centrifugation at 13,000 rpm for 30 min at 4 °C and loaded onto a 1 mL Ni-NTA agarose column (Qiagen), preequilibrated with buffer A. After washing with 10 bed volumes of buffer A supplemented with 50 mM of imidazole, following the manufacturer’s protocol, proteins were eluted with 300 mM imidazole in buffer A. Aliquots of protein fractions were analyzed by SDS-PAGE, and selected fractions were pooled and concentrated using Amicon Ultra centrifugal filter devices (Millipore). Purified proteins were stored in 30 mM Tris, pH 8, 400 mM NaCl at −80 °C. Protein concentration was determined by the Bradford assay (Bio-Rad protein assay) using bovine serum albumin as the standard or with NanoDrop 2000 Spectrophotometer (Thermo Scientific) using the molar extinction coefficient (εM for PrxII = 22460 M−1 cm−1; εM for PGDH = 18910 M−1 cm−1).
In-Gel Digestion and MS/MS Mass Spectrometry
Following SDS-PAGE, bands of interest were excised, destained with ammonium bicarbonate and acetonitrile, and then submitted to in-gel digestion after reduction and carbamidomethylation using the automated system Digest Pro96 (Intavis AG, Bremen, Germany) as described.37 Peptides were redissolved in 12 μL of 30% acetonitrile containing 0.1% v/v formic acid and then diluted to a final concentration of 5% acetonitrile. Mass spectra were acquired on an Agilent 1200 nanoflow LC system coupled to a 6330 Ion Trap equipped with the Chip Cube orthogonal ionization system (Agilent Technologies, Santa Clara, CA, USA) as previously described.38 Proteins were identified with the search program X!tandem (http://prowl.rockefeller.edu), using X!tandem pipeline (http://pappso.inra.fr/bioinfo/xtandempipeline/) using the Synechocystis database from UniprotKB (release 20110524, 3507 entries). Mass accuracy tolerance was set to 100 ppm on the parent ion mass and 0.5 Da in MS/MS mode. One possible missing cleavage site per peptide, carbamidomethylation for Cys as fixed modification and oxidation for Met as variable modification were considered in searches. Identifications were validated according to the established guidelines for proteomic data publication39 and notably protein identification was validated only if at least two different sequences were identified with high quality mass spectra (peptide E value
