BBAGEN-27641; No. of pages: 7; 4C: Biochimica et Biophysica Acta xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen
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Lena Dorothee Schütte a, Stefan Baumeister b, Benjamin Weis a, Christoph Hudemann c, Eva-Maria Hanschmann d, Christopher Horst Lillig d,⁎ a
Institute for Clinical Cytobiology and Cytopathology, Philipps-University Marburg, Germany Protein Analytics, Faculty for Biology, Philipps-University, Marburg Institute for Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University Marburg, Germany d Institute for Medical Biochemistry and Molecular Biology, Ernst-Moritz-Arndt University Greifswald, Germany b c
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Background: Glutaredoxins (Grxs) catalyze the reduction of protein disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism. These rapid, specific, and generally also reversible modifications are part of various signaling cascades regulating for instance cell proliferation, differentiation and apoptosis. Even though crucial functions of the conserved, mitochondrial Grx2a and the cytosolic/nuclear Grx2c isoforms have been proposed, only a few substrates have been identified in vitro or in vivo. The significance of redox signaling is emerging, yet a general lack of methods for the time-resolved analysis of these distinct and rapid modifications in vivo constitutes the biggest challenge in the redox signaling field. Methods and results: Here, we have identified potential interaction partners for Grx2 isoforms in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates are part of the cytoskeleton or act in protein folding, cellular signaling and metabolism. Part of these interactions were further verified by immunoprecipitation or a newly established 2-D redox blot. Conclusions: Our study demonstrates that Grx2 catalyzes both the specific oxidation and the reduction of cysteinyl residues in the same compartment at the same time and without affecting the global cellular thiolredox state. General significance: The knowledge of specific targets will be helpful in understanding the functions of Grx2. The 2-D redox blot may be useful for the analysis of the overall thiol-redox state of proteins with high molecular weight and numerous cysteinyl residues, that evaded analysis by previously described methods. © 2013 Published by Elsevier B.V.
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Article history: Received 25 October 2012 Received in revised form 2 July 2013 Accepted 8 July 2013 Available online xxxx
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Keywords: Glutaredoxin Protein disulfide Intermediate trapping Proteomics Redox state Redox blot
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Identification of potential protein dithiol-disulfide substrates of mammalian Grx2
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1. Introduction
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Glutaredoxins (Grxs) are oxidoreductases of the thioredoxin (Trx) family, which post-translationally regulate proteins via specific modifications at redox-sensitive cysteinyl residues (for an overview see Refs. [1–3]). These modifications constitute rapid, specific, and generally reversible events and are as parts of various signaling cascades essential for the integrity of the cell. Grxs catalyze the reduction of target disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism [4,5]. The dithiol mechanism depends on the active site motif Cys–X–X–Cys, i.e. the more Nterminal Cys performs a nucleophilic attack on the target disulfide, forming a Grx-protein-mixed disulfide. This intermediate is reduced by the more C-terminal Cys, releasing the reduced protein. The monothiol
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⁎ Corresponding author at: Institut für Biochemie und Molekularbiologie, Universitätsmedizin Greifswald KdöR, Ferdinand-Sauerbruch-Strasse (J.03-35), 17475 Greifswald, Germany. Tel.: +49 3834 865407; fax: +49 3834 865402. E-mail address:
[email protected] (C.H. Lillig).
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mechanism is initiated by a nucleophilic attack on a glutathionylated protein by the N-terminal Cys. In contrast to the dithiol mechanism, a Grx-GSH-mixed disulfide is formed and the protein substrate is instantly reduced and constitutes the leaving group of the reaction. The mixed glutathione disulfide also occurs when a disulfide in the Grx active site is reduced by one molecule of GSH. The Grx-GSH-mixed disulfide is subsequently reduced by a second GSH molecule yielding the reduced dithiol Grx and glutathione disulfide (GSSG) [6]. GSSG is reduced by glutathione reductase with electrons donated from NADPH. The awareness of the general significance of redox signaling and the impact of the Trx family proteins as key mediators of redox homeostasis are emerging. However, due to the rapid nature of redox reactions, the distinct and fragile chemistry of the numerous oxidative modifications and the general lack of specific in vivo techniques for a local and time-resolved analysis of redox changes, in particular confirmed substrates and signaling pathways, are rare. The mammalian Grx2 is characterized by the uncommon active site motif Cys–Ser–Tyr–Cys (instead of the regular dithiol consensus Cys– Pro–Tyr–Cys), the ability to receive electrons from Trx reductase and
0304-4165/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbagen.2013.07.009
Please cite this article as: L.D. Schütte, et al., Identification of potential protein dithiol-disulfide substrates of mammalian Grx2, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.07.009
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2. Materials and methods
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2.1. General methods
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Chemicals and enzymes were purchased from Sigma, unless otherwise stated, and of analytical grade or better. Antibodies detecting human and mouse Grx2c have been described before [19]. Antibodies detecting β-actin (sc-47778), α-tubulin (T9026), calcineurin B (C0581), G3P (G9545), HSP60 (sc-13115), PSMD9 (sc-100996), and RuvB like 1 (ab75826) were purchased from Abcam (Cambridge, UK), Santa Cruz Biotechnology Inc. (Santa Cruz, USA), and Sigma Aldrich (Steinheim, Germany), respectively. SDS–PAGE and Western blots were run using the Novex Mini-Cell (Invitrogen), pre-casted Precise gels (4–20%, Thermo Scientific), and PVDF or nitrocellulose membranes (Macherey & Nagel) according to the manufacturers' instructions. Horseradish peroxidase conjugated anti-rabbit and -mouse IgGs were obtained from Bio-Rad, and alkaline phosphatase-conjugated antibodies, from Sigma. Western blots were developed by enhanced chemiluminescence staining. For immunoprecipitation, the IgG fraction from 1 ml of a specific Grx2 serum [19] was purified using HisTrap protein A columns and the Äkta FPLC system as suggested by the manufacturer (GE Healthcare). Next, IgGs were coupled to 1 ml CNBr-activated Sepharose beads (GE Healthcare). These beads were incubated with protein extracts, spun down, and washed three times with PBS. Precipitated Grx2 and co-precipitated proteins were eluted by incubation of the beads with 2 volumes of 2fold SDS sample buffer containing 0.125 M Tris/HCl pH 6.8, 4 mM EDTA, 0.4% bromphenol blue, 20% glycerol and 2% SDS at 95 °C for 5 min. For the propagation of HeLa and HeLa-Grx2c cells [14], all media, fetal calf serum (FCS), antibiotics (penicillin, streptomycin), trypsin and phosphate buffered saline (PBS) were purchased from PAA (Cölbe, Germany), and disposable plastics, from Sarstedt (Nümbrecht, Germany). HeLa cells were propagated in 1 g/l glucose-containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and 100 U/ml penicillin and streptomycin.
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2.3. Preparation of cell and organ lysates for intermediate trapping
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Brain, liver, and testes were extracted from mice, and processed in the same way as cultured HeLa cells harvested by trypsinization. Fivefold the volume of lysis buffer (10 mM Tris/HCl pH 7.4, 0.1% NP40, 10 mM NaCl, 3 mM MgCl2, and 1-fold protease inhibitor cocktail (Roche, Mannheim, Germany)) was added to the samples or cell pellets and incubated at room temperature for 15 min. Tissues were homogenized with a pistil. Samples were flash-frozen in liquid nitrogen, gently thawed on ice, and clarified by centrifugation at 13,000 rpm for 10 min at 4 °C in a microcentrifuge. Total protein levels were determined using Bradford reagent (Bio-Rad, Hercules CA, USA).
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2.4. Intermediate trapping, 2-D gel electrophoresis, and mass spectrometry 162 Ten milligrams of purified Cys–Ser–Tyr–Ser mutants of human and mouse Grx2c were immobilized on CNBr-activated Sepharose (GE Healthcare) as suggested by the manufacturer. The columns were washed with excess PBS containing 10 mM DTT (PAA). Next, 100–200 mg extracts from mouse tissues or HeLa cells, re-buffered in PBS using PD-10 Sephadex G25 columns (GE Healthcare), were slowly applied to the column. The columns were washed with 50 volumes PBS and, subsequently, 50 mM Tris/HCl, 1 mM EDTA, 500 mM NaCl. Trapped candidate protein disulfide substrates of Grx2 were eluted with 20 mM GSH and/or DTT in PBS. Following a wash step with 100 volumes PBS, residual proteins bound to the Grx2 matrix were eluted with 100 mM acetic acid. Eluted proteins were precipitated with 12.5% trichloric acetic acid at 4 °C for 2 h or overnight. Precipitates were harvested by centrifugation at 13,000 rpm for 10 min at 4 °C in a microcentrifuge, washed with ice-cold acetone, centrifuged at 13,000 rpm for 10 min at 4 °C, and resuspended in 165 μl rehydration buffer (8 M urea, 1% NP40, 20 mM DTT, 0.5% ampholytes pH 3–10). 2-D gel electrophoresis was performed using the XOOM system (GE Healthcare) as instructed by the manufacturer; pH 3–10 stripes were used for isoelectric focusing, 4–12% Bis-Tris SDS gels for the second dimension. Protein thiols were alkylated with N-ethyl malemeide (NEM) during the procedure. Gels were stained with colloidal Coomassie Brilliant Blue (Fermentas). Mass spectrometry was performed in the proteomics core facility of the collaborative research center (SFB) 593, Philipps-University Marburg, as described in Ref. [21]. Peptide mass fingerprint peak lists were compared to the SwissProt database, restricted to mouse or human taxonomy, respectively, using the Mascot search engine (Matrix Science Ltd., London, UK). Peptide mixtures that yielded a score of at least 56 were regarded as positive identifications.
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2.5. 2-D redox blot
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For the thiol redox state-dependent modification of proteins' isoelectric points, 5-maleimido isophthalic acid (MIPA) was synthesized as described in Ref. [22]. The product purity and stability was confirmed by NMR spectroscopy. HeLa and HeLa-Grx2c cells were harvested by trypsinization, washed in PBS, and incubated for 15 min in PBS containing 100 mM NEM. Next, cells were lysed by addition of 2% CHAPS and incubated with 4 volumes of 100 mM sodium phosphate pH 7.2,
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The plasmids for the recombinant expression of human and mouse Grx2c in Escherichia coli were described before [7,12]. For the intermediate trapping experiments, the more C-terminal active site cysteinyl residues of human and mouse Grx2c were exchanged for seryl residues (changing the Cys–Ser–Tyr–Cys active sites to Cys–Ser–Tyr–Ser) by site directed mutagenesis as described in Ref. [20]. All Grx2 proteins were expressed in E. coli in a 5 l bioreactor and purified by immobilized metal affinity chromatography and FPLC as described in Ref. [18].
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2.2. Site directed mutagenesis, protein expression, and purification
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the coordination of a [2Fe2S] cluster [7–10]. Due to alternative splicing and transcription initiation, the GLRX2 gene does not only give rise to the well characterized, ubiquitous mitochondrial Grx2a, but moreover to some testis- and cancer-specific cytosolic/nuclear isoforms, in humans Grx2b and Grx2c, in mouse the ubiquitous Grx2c and the testis-specific Grx2d [11,12]. Silencing of Grx2a sensitizes HeLa cells to doxorubicin- and phenylarsine oxide-induced cell death [13], whereas the overexpression protects the cells from doxorubicin- and 2-deoxyD-glucose-induced apoptosis [14]. Grx2a catalyzes both reversible protein disulfide formation and protein de-glutathionylation, demonstrated, for instance, for proteins of the inner mitochondrial membrane such as the 75 kDa subunit of complex I [15,16]. In zebrafish, depletion of cytosolic Grx2 during embryonic development causes the loss of the ability to develop an axonal scaffold and the cell death of essentially all types of neurons. The function of the protein is thus essential for brain development [17]. Despite of these well established phenotypes, so far only the mitochondrial 2-Cys peroxiredoxin 3, another member of the Trx fold protein family regulating hydrogen peroxide concentrations, was confirmed as substrate of Grx2a in vitro and in vivo [18]. The lack of knowledge on substrates and targets of the different Grx2 isoforms prompted us to identify potential interaction partners in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates, functionally connected, for instance, to the cytoskeleton, chaperones, signaling, and cell metabolism, were further verified by immunoprecipitation and/or a newly established 2-D redox blot, which allows to analyze the overall thiol redox state, even of high molecular weight proteins with numerous cysteinyl residues.
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Please cite this article as: L.D. Schütte, et al., Identification of potential protein dithiol-disulfide substrates of mammalian Grx2, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.07.009
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3. Results
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3.1. Identification of potential substrates of Grx2
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We used an intermediate trapping approach to isolate potential protein disulfide substrates of Grx2 from tissue and cell extracts. Cys–Ser– Tyr–Ser active site mutants of human and mouse Grx2 were produced by site directed mutagenesis. These proteins lack the more C-terminal cysteinyl residue that resolves the intermediate disulfide formed between a Grx and a target protein during the dithiol catalytic cycle, i.e. when a disulfide between two cysteinyl residues in a target protein is reduced. The mutant Grxs were immobilized to a CNBr-activated Sepharose, reduced, and allowed to react with potential target proteins. In the case of mouse Grx2, potential substrates were isolated from tissue extracts from brain, liver, and testis, in the case of human Grx2 from HeLa cell extracts. Following a stringency washing step, proteins that formed a disulfide with the Grx2 mutants were eluted by switching to reducing conditions. Finally, residual proteins were eluted in a second step with acetic acid. Eluted proteins were concentrated by precipitation, separated by 2-D gel electrophoresis, isolated, and identified by mass spectrometry. Multiple runs of protein isolation from the same source yielded essentially identical spot patterns in the 2-D gel electrophoresis, demonstrating the specificity of the interaction and the reproducibility of this approach. An example is depicted in Supplementary Fig. 1. In total, we isolated and identified 50 different proteins in fractions eluted by reducing conditions, and two in fractions isolated only by denaturing conditions (Table 1). From these, 41 proteins are annotated as cytosolic and/or nuclear proteins in the UniProt database [23], eight as mitochondrial, and three as targeted to the secretory pathway. Only one protein identified, apolipoprotein A-1 isolated by denaturing conditions only, does not contain at least one cysteinyl residue. Functionally, the largest group of proteins identified here is related to the cytoskeleton (13 proteins), followed by chaperones (ten), proteins related to proteolysis, the proteasome or ubiquitination (eight), metabolic enzymes (seven), proteins involved in transcription or translation (five), redox enzymes (four), and some other proteins related to calcium signaling, transport processes, or DNA damage response (for details see Table 1).
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3.2. Confirmation of potential Grx2 interacting proteins
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At first, we analyzed potential interactions of human, cytosolic Grx2c with actin, tubulin, calcineurin B, glycerin aldehyde 3-phosphate dehydrogenase (G3P), 26 proteasome non-ATPase regulatory subunit 9 (PSMD9), and the mitochondrial HSP60 (mtHSP60) in vivo. Grx2 was isolated from both wildtype HeLa cells, which only express mitochondrial Grx2a, and Grx2c stable expressing HeLa cells (HeLa Grx2c) by immunoprecipitation. The eluate was blotted for β-actin, α-tubulin, calcineurin B, G3P, and mtHSP60 (Fig. 1). Confirming the results from the intermediate trapping experiments, both actin and tubulin were specifically isolated together with Grx2 from cells expressing both cytosolic Grx2c and the endogenous mitochondrial Grx2a, but not from
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4. Discussion
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Redox signaling is specific, spatially and timely controlled, and reversible. The specificity results from (a) the reactivity of the modified thiol group and (b) the specificity of the enzymes that catalyze the post-translational redox modifications. This specificity was well documented in our intermediate trapping approach. Repetitive trapping experiments from the same source yielded essentially identical spot patterns in subsequent 2-D PAGE analyses (see Supplementary Fig. 1). As additional control, we established a redox DIGE assay and compared the thiol redox state of proteins isolated from control HeLa cells with extracts isolated from cells (a) expressing the cytosolic isoform Grx2c, or (b) with siRNA-silenced expression of mitochondrial Grx2a. Notably, neither the silencing of Grx2a, nor the expression of Grx2c, altered the
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wildtype cells containing only the mitochondrial isoform. Significantly more calcineurin B and G3P were also associated with Grx2 from HeLa Grx2c cells (Fig. 1). Mitochondrial HSP60 was associated with Grx2 in both HeLa cell lines. In contrast, no co-precipitation of PSMD9 with Grx2 could be demonstrated in this approach. Besides analyzing general protein interactions, it would be most desirable to determine the influence of Grx2c, or any other given redox protein, on the thiol redox state of specific proteins in vivo. Currently available methods, however, only allow to analyze the redox state of small proteins with few cysteinyl residues by electrophoretic mass shift assays following alkylation with compounds of significant molecular weight, e.g. Ref. [24]. We sought to develop a method that would also allow the analysis of proteins with high molecular weight, with numerous cysteinyl residues, and other post-translational modifications, by enabling the use of for instance phosphorylation-specific antibodies. We therefore synthesized MIPA, a malemeide compound that contains two carboxyl groups. The success and efficiency of the synthesis and the long term stability of MIPA were controlled by NMR analysis (see Supplementary Fig. 2). To analyze protein redox states, all (reduced) thiol groups of the proteome were blocked by alkylation with NEM before and during protein isolation under denaturing conditions. In the second step, the originally in vivo oxidized cysteinyl residues were reduced with TCEP and alkylated with MIPA, thus adding two carboxyl groups to the protein per originally oxidized cysteinyl residue. Hence, isoelectric focusing separates the originally reduced and oxidized forms of any given protein. These electrophoretic shifts were documented by 2-D gel electrophoresis (isoelectric focusing followed by SDS– PAGE) and Western blotting with specific antibodies targeted against the protein of interest. For a scheme of the procedure, see Supplementary Fig. 3. We have thoroughly evaluated and optimized the procedure ensuring (a) the efficiency of the initial blocking step with NEM to avoid the oxidation of cysteinyl residues during cell lysis and (b) the efficiency of MIPA as thiol-specific alkylator. To ensure that the 2-D redox blots from two samples were aligned properly for comparison, we included a cytochrome C-based IEF marker in the samples, that was detected by a second staining of the Western blot with a cytochrome Cspecific antibody. Fig. 2 summarizes the analysis of the redox state of PSMD9, RuvBlike 1 (RUVB1), and collapsin response-mediator protein 2/dihydropyrimidinase-related protein 2 (CRMP2/DPYL2) that were identified in our intermediate trapping screening as potential substrates of cytosolic Grx2c, but – in case of PSMD9 and RUVB1 – whose interaction could not be confirmed by co-immunoprecipitation. As depicted, the electrophoretic mobility of both PSMD9 and RUVB1 was shifted to more basic conditions in IEF. Thus, for both proteins more thiols were modified with MIPA, indicating more oxidized cysteinyl residues for both proteins in HeLa cells that express Grx2c compared to cells that do not express this oxidoreductase in the cytosol. In contrast and confirming previous results in developing zebrafish [17], CRMP2/ DPYL2 signals shifted towards more acidic conditions, suggesting the reduction of CRMP2 cysteinyl residues in the presence of Grx2c.
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150 mM NaCl, 5 mM EDTA, 8 M urea and 100 mM NEM in an anaerobic chamber for 20 min. The crude extracts were clarified by centrifugation (20,000 g, 4 °C in a microcentrifuge). To remove excess of NEM, proteins were precipitated by addition of 12% TCA and resuspended in 8 M urea, 100 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA, pH 7.2. Originally oxidized thiols were reduced by addition of 10 mM Tris(2-carboxyethyl)phosphine (TCEP) for 30 min and modified by 20 mM MIPA for another 30 min at room temperature. NEM- and MIPA-modified proteins were precipitated again by addition of 12% TCA, resuspended in rehydration buffer, and separated by 2-D gel electrophoresis (see above). Gels were blotted on PVDF membranes, stained with target-specific antibodies and were developed using the enhanced chemiluminescence method.
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Please cite this article as: L.D. Schütte, et al., Identification of potential protein dithiol-disulfide substrates of mammalian Grx2, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.07.009
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No.
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6/6 6/6 2/2 3/3 2/2 7/6 8/8 5/3 8/8 6/6 5/5 4/4 1/1 7/8 28/24 11/8 17/17 13/13 9/9 4/4 4/4 7/7 3/3 3/3 2/2 55/54 6/6 14/13 6/4
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Cytoskeleton Cytoskeleton Cytoskeleton Cytoskeleton Cytoskeleton Cytoskeleton Chaperone Chaperone Chaperone Ubiquitination Cytoskeleton Ubiquitination
12/12 12/12 7/7 8/8 8/8 8/8 6/6 8/8 10/10 12/12 1/1 22/24
Mitochondrion CH60 Hspd1 protein, heat shock protein 60 (mito) ECHM Enoyl CoA hydratase EFTU Elongation factor Tu, mitochondrial precursor GLRX2 Glutaredoxin 2, mitochondrial precursor GRP75 Heat shock 70kD protein, mitochondrial precursor GRPE1 Human mitochondrial GrpE like 1 SUOX Sulfite oxidase TI17B Mitochondrial import inner membrane translocase Tim17-B
Chaperone Metabolism Transcription/-lation Redox Chaperone Chaperone Redox Transport
3/3 7/8 7/6 4/4 5/5 4/4 9/9 3/3
Secretory pathway APOA1 Apolipoprotein A-1 GALT7 UDP-N-acetyl-α-D-galactosaminyltransferase 7 GRP78 Heat shock 70 kD protein 5 (Grp78)
Transport Metabolism Chaperone
0/0 12/12 2/2
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t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49 t1:50 t1:51 t1:52 t1:53 t1:54 t1:55 t1:56 t1:57 t1:58 t1:59 t1:60 t1:61 t1:62 t1:63 t1:64
“global redox state” of the proteome (Supplementary Fig. 4 and Table 1), but rather modified specific protein targets, confirming earlier results [13,17,18] and demonstrating the specificity of compartmentalized redox signaling events. Although Grx2 is an efficient catalyst of dithiol mechanism reactions, i.e. a reductase of protein disulfides, it also catalyzes the reversible (de-) glutathionylation of proteins and small molecular weight compounds [9,25]. Our proteomic approach favored the identification of protein disulfide substrates, because reaction of the immobilized Grx2 Cys–Ser– Tyr–Ser mutant with glutathionylated substrates would only lead to
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Cytoskeleton Cytoskeleton Cytoskeleton Metabolism Calcium signaling Protease/-asome Cytoskeleton Metabolism Metabolism Transcription/-lation Chaperone Chaperone Chaperone Metabolism Cytoskeleton Calcium signaling Cytoskeleton DNA damage (?) Transcription/-lation Redox Redox Protease/-asome Protease/-asome Protease/-asome Protease/-asome Ubiquitination Transcription/-lation Metabolism Transcription/-lation
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Entry Cytosol/nucleus ACTB β-Actin ACTC α-Actin cardiac muscle 1 ACTY β-Centractin, actin related protein 1B ARGI1 Arginase CANB1 Protein phosphatase 3, subunit B α, calcineurin B type I DPP3 Dipeptidyl peptidase III DPYL2 Dihydropyrimidinase-related protein 2 G3P Glyceraldehyde-3-phosphate dehydrogenase GNMT Glycine N-methyltransferase HNRPF Heterogeneous nuclear ribonucleoprotein F HSP72 Heat shock-related 70 kDa protein 70.2 HSP7C Heat shock protein 8 HSPB1 Heat-shock protein β-1 IMDH2 Inosine-5′-monophosphate dehydrogenase 2 KI20B Kinesin-like protein KIF20B KKCC1 Calcium/calmodulin-dependent protein kinase kinase 1 α KLH20 Klhl20 protein LRC42 Leucine-rich repeat-containing protein 42 PCBP1 Poly(rC)-binding protein 1 (α-CP1) (hnRNP-E1) PRDX1 Peroxiredoxin 1 PRDX4 Peroxiredoxin 4 PRS7 26S Protease regulatory subunit 7 PRS8 26S Protease regulatory subunit 8 PSMD9 26S Proteasome non-ATPase regulatory subunit 9 PSME3 Proteasome activator complex subunit 3 RNF31 E3 ubiquitin-protein ligase RNF31 RUVB1 RuvB-like 1 SERA D-3-phosphoglycerate dehydrogenase TAF1A TATA box-binding protein-associated factor RNA polymerase I sub. A TBA1B α-Tubulin (α-1b) TBA3 α-Tubulin (α-1a) TBB2A β-Tubulin (β-2a) TBB3 β-Tubulin (β-3) TBB4A β-Tubulin (β-4a) TBB4B β-Tubulin (β-4b) TCPB T-Complex protein 1 subunit β TCPE T-Complex protein 1 subunit ε TCPQ T-Complex protein 1 subunit θ TERA Transitional endoplasmic reticulum ATPase TPM1 Tropomyosin-1 α chain VCIP1 Deubiquitinating protein VCIP135
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t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37
Cys
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Table 1 Potential disulfide substrates of mammalian glutaredoxin 2 identified by intermediate trapping, 2-D gel electrophoresis, and mass spectrometry. Cell lysates of mouse brain (B), liver (L) and testes (T) as well as human HeLa cells (H) were analyzed for protein interaction with Grx2 using the intermediate trapping approach. The data from the analysis are enlisted alphabetically with emphasis to the intracellular location, stating the official UniProt entry, the protein name and the approximate function. The number of cysteinyl residues in each protein is indicated for mouse and human (m/h) proteins, as well as the elution conditions, i.e. by a reductant (R) or acetic acid (A). Trapped interactions are shown by a dot and the Mascot score.
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t1:1 t1:2 t1:3 t1:4 t1:5
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R
Mascot A
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112 67 68 106 103 63 105 72 71 93 141 83 102 149 64 62 59 58 76 79 117 82 99 77 130 60 73 66 64
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67 85 134 110 98 72 88 106 70 110 MS/MS 62
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80 81 184 MS/MS 74 86 113 58 ● ●
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Score
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99 87 136
glutathionylated Grx2 and the liberation of the target protein. From the 50, in this screening identified, protein dithiol-disulfide substrates of the oxidoreductase, nine have previously been demonstrated to be susceptible to glutathionylation. In the cytosol are the following: beta-actin [26], glyceraldehyde-3-phosphate dehydrogenase [27,28], Peroxiredoxin 1 and 4 [29,30], T-complex protein 1 subunit beta [31], alpha-tubulin [32]; in mitochondria: enoyl CoA hydratase [33], the HSP70 chaperone GRP75 [31], and the chaperonin HSP60 [30,31]. However, this does not necessarily mean that these proteins were trapped in their glutathionylated form here, at least some of them have also been
Please cite this article as: L.D. Schütte, et al., Identification of potential protein dithiol-disulfide substrates of mammalian Grx2, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.07.009
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reported to form intra- or intermolecular disulfides: beta-actin for instance can form intermolecular disulfides [34,35], tubulin contains intermolecular disulfides between the subunits of the alpha-beta heterodimer [36], 2-Cys Prxs form intermolecular disulfides during their catalytic cycle [37], and GRP75 and HSP60 were characterized as disulfide targets of Trx2 [38]. Here, we trapped two 2-Cys Prxs as potential substrates of nonmitochondrial Grx2: cytosolic Prx1 and Prx4, that is targeted to the secretory pathway. We have previously shown that the mitochondrial 2-Cys Prx3 can be reduced by Grx2a in vitro and in vivo [18]. In this function, Grx2a and Trx2 complement each other. Moreover, both of these redoxins are substrates for thioredoxin reductase [39,9]. Engelhard
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Fig. 1. Co-immunoprecipitation of selected potential target proteins of Grx2 from wildtype and HeLa cells expressing cytosolic Grx2c. Affinity-purified glutaredoxin 2 (Grx2) antibodies were immobilized on CNBr-activated Sepharose and were incubated with wildtype and Grx2 expressing HeLa cell lysates. Unbound proteins (input), as well as bound proteins, eluted with acetic acid (IP) were analyzed for beta actin (ACTB), alpha tubulin (TUBA), calcineurin subunit B type 1 (CANB1), glyceraldehyde-3-phosphate dehydrogenase (G3P), 26 S proteasome non-ATPase regulatory subunit 9 (PSMD9), heat shock protein 60 (CH60) and glutaredoxin 2 by Western blot using specific antibodies.
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et al. have recently identified a number of potential Trx2 substrates by an in situ intermediate trapping approach similar to the strategy applied here. Among the proteins identified, three more mitochondrial proteins reacted with Trx2 in thiol-disulfide exchange reactions: elongation factor Tu, the HSP70 chaperone GRP75, and the chaperonin HSP60 [38]. These proteins were also identified as substrates for Grx2, in our approach, emphasizing the overlapping functions of the two mitochondrial oxidoreductases. It is worth mentioning that both studies also gave rise to a set of individual interaction partners for Trx2 and Grx2 and therefore distinct functions and specific redox circuits. In parallel to this study, we demonstrated that cytosolic Grx2, which lacks a mitochondrial transit sequence and is closely related to human Grx2c, is essential for brain development in zebrafish [17]. Fish lacking Grx2 during embryonic development fail to develop axonal scaffolds and almost all types of neurons undergo apoptosis. In the screening presented here, we have identified numerous constituents of the cytoskeleton as potential targets of Grx2c, among them collapsin response mediator protein 2. In fact, applying our newly developed 2-D redox blot, we could demonstrate that in the absence of Grx2, the thiol redox state of CRMP2 is more oxidized in zebrafish embryos. The original fish phenotype was rescued by expression of wildtype Grx2, but not by expression of the Cys–X–X–Ser mutant. This developmental function of Grx2 is thus dependent on the dithiol mechanism, suggesting the reduction of a specific protein disulfide in CRMP2. Here, we were now able to confirm the reduction of CRMP2 thiols in the presence of Grx2c in the HeLa cell culture model (Fig. 2). Thiol-disulfide exchanges are reversible. In vitro, the direction of these reactions is determined by thermodynamic restrictions, i.e. the redox potentials of the reactants. In vivo, however, reactants are rarely in equilibrium due to the flux of metabolites or electrons through distinct pathways. This flux is controlled by the activity of enzymes, such as the oxidoreductases of the Trx family. Grxs catalyze thiol-disulfide reactions in both directions. Grx2, for instance, can be reduced by GSH to catalyze the reduction of protein disulfides [40], and it can be oxidized by GSSG to be re-reduced by thioredoxin reductase [9]. It is therefore not contradictory to find some targets more reduced in vivo, e.g. CRMP2 [17], while the cysteinyl residues of others (PSMD9, RUVB1) appear to be more oxidized in the presence of Grx2c. Corroboratively, Beer et al. reported the catalysis of both protein disulfide formation and glutathionylation of mitochondrial proteins by Grx2a [15]. Hence, it is not only essential to identify new substrates, but rather to determine the specific post-translational modification(s), in order to understand the mechanism of redox regulation of the individual protein and also the functional pathway it is part of.
Fig. 2. 2-D redox blots of 26 proteasome non-ATPase regulatory subunit 9 (PSMD9), Ruv-like 1 (RUVB1), and collapsin response-mediating protein 2 (CRMP2/DPYL2) in wildtype and HeLa cells expressing cytosolic Grx2c. Free thiol groups were alkylated with NEM during denaturing lysis of HeLa wildtype and Grx2c expressing HeLa cells. Initially oxidized cysteinyl residues were reduced with TCEP and alkylated with MIPA, a compound comprising two carboxyl groups. Therefore, the isoelectric point of proteins shifts, depending on their original redox state. Samples were analyzed by 2-D gel electrophoresis and Western blot, using specific antibodies and the enhanced chemiluminescence method (upper panel). Cytochrome C staining was used for correct alignment of the blots (see Materials and methods section). Band intensities were analyzed densiometrically using ImageJ (lower panels).
Please cite this article as: L.D. Schütte, et al., Identification of potential protein dithiol-disulfide substrates of mammalian Grx2, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.07.009
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Appendix A. Supplementary data
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References
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The authors gratefully acknowledge the excellent technical assistance of Sabrina Oesteritz and the financial support by the Deutsche Forschungsgemeinschaft (SFB593-N01), the Kempkes Foundation, the Karolinska Institute, and the von Behring-Röntgen Foundation.
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Until recently, the progress in understanding redox signaling events was largely hindered by the lack of methods to (a) analyze redox events non-disruptively in vivo and (b) to trap and monitor specific redox modifications of specific proteins in vivo. In respect to the general lack of methods, the largest progress has been made in form of the development of redox sensitive fluorescent probes that specifically react to the production of either hydrogen peroxide or GSSG [41]. While new probes, such as sulfenic acid-specific compounds or the oxicat approach, already allow to quantitatively analyze redox modifications on a proteomic level, e.g. Refs. [42,43], the detection of redox modifications on specific proteins remains challenging. Available assays, based on the induction of electrophoretic mass shifts and Western blotting, e.g. Ref. [44], are due to the limited resolution of PAGE restricted to proteins of low molecular weight and few cysteinyl residues. In this study, we aimed at overcoming some of these problems by using a thiol-specific probe that alters the isoelectric point of proteins. Although this requires the separation of extracts by 2-D PAGE, it is independent of the molecular weight of the protein of interest, allows the analysis of proteins with multiple thiol groups, and other post-translational modifications (if specific antibodies are available). The major disadvantages are the rather complex procedure and the restriction to proteins with (unmodified) isoelectric points in the range of approximately pH 4–9. Nevertheless, if specific “difficult” proteins are to be analyzed, the protocol may be the method of choice over costly quantitative proteomic approaches. The knowledge of specific targets already helped and will certainly continue to contribute in understanding the functions of redoxins, such as the isoforms of Grx2, and the importance of redox signaling in general. New methods are urgently needed to analyze volatile and transient post-translational redox modifications. We are convinced that the 2-D redox blot could be helpful for the analysis of various proteins, in particular of proteins with high molecular weight and a large number of cysteinyl residues. Our study demonstrates that a single thioldisulfide oxidoreductase can catalyze the specific oxidation of protein thiols on some proteins, while reducing cysteinyl residues on other proteins — in the same compartment and without affecting the global thiol-redox state.
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