Proteomic Profiling of Perturbed Protein Sulfenation in Renal Medulla of the Spontaneously Hypertensive Rat Raymond Tyther,† Ahmad Ahmeda,‡ Edward Johns,‡ Brian McDonagh,§ and David Sheehan*,† Proteomics Research Group, Department of Biochemistry, University College Cork, Ireland, Department of Physiology, University College Cork, Ireland, and Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de Co´rdoba, Spain Received February 26, 2010
Protein sulfenic acids have been proposed as potential biochemical switches for redox signaling. This post-translational modification (PTM) is readily reversible, in contrast to some other types of oxidative PTM. Enhanced oxidative stress has been reported as a feature of hypertension, and renal function has been implicated in the development and progression of the disease in animal models such as the spontaneously hypertensive rat (SHR). However, reactive oxygen species (ROS) are also signaling molecules and may play a role in vascular function. To investigate protein sulfenation under hypertensive conditions, we examined protein extracts of SHR kidney medulla in comparison to medulla from normotensive Wistar rats. Total free thiol content of the SHR medulla was significantly lower than that of Wistar medulla, indicating enhanced oxidation of sulfhydryls. Protein sulfenation was also significantly greater in the medulla of hypertensive animals. Thioredoxin reductase activity was also reduced in SHR medulla and this may account, in part, for enhanced protein sulfenation. Purification of sulfenated proteins from SHR medulla revealed several proteins involved in processes such as metabolism, antioxidant defense, and regulation of nitric oxide synthase. Enhanced sulfenation may represent perturbed redox signaling in SHR medulla, or simply enhanced ROS generation. Keywords: sulfenation • two-dimensional electrophoresis • kidney • hypertension • rat • carbonylation
Introduction Cysteine residues in proteins are subject to various oxidative modifications. Disulfide bridge formation at these residues has long been of interest but, more recently, attention has also centered on other post-translational modifications (PTM) such as S-nitrosylation, S-glutathionylation and sulfenation.1 Proteomic scrutiny of sulfenation has been made possible by innovative studies by Saurin et al.2 Through exploiting the ability of arsenite specifically to reduce protein sulfenic acids, and employing a “biotin switch”-style methodology, Saurin et al. explored sulfenation in tissues exposed to hydrogen peroxide. These changes were examined in an experimentally induced state of enhanced oxidative stress, but the methodology is readily applicable in situations where endogenous generation of reactive oxygen species (ROS) is enhanced such as in hypertension.3,4 The interaction of ROS with reduced protein thiols can generate protein sulfenic (-SOH), sulfinic (-SO2H) or sulfonic (-SO3H) acids.5,6 Sulfenic acid formation is readily reversible and can serve as a reaction intermediate for a more stable * To whom correspondence should be addressed. Prof. David Sheehan, Proteomics Research Group, Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Mardyke, Cork, Ireland. Phone: 353 21 4904207. Fax: 353 21 4274034. E-mail:
[email protected].. † Department of Biochemistry, University College Cork. ‡ Department of Physiology, University College Cork. § Universidad de Co´rdoba.
2678 Journal of Proteome Research 2010, 9, 2678–2687 Published on Web 04/01/2010
reduced or oxidized state. However, evidence for more enduring protein sulfenic acids exist, such as the redox sensitive Escherichia coli transcription factor OxyR7 and the Bacillus subtilis organic peroxide sensor OhrR.8 In contrast, the sulfinic and sulfonic acid forms are thought to be irreversible, “hyperoxidized” states, with possible deleterious consequences for protein function. This has not proved to be exclusively the case, at least with respect to protein sulfinic acids. Sulfinic acids are known to form in the antioxidant peroxiredoxins,9 which can be reduced by a specific sulfiredoxin.10 Sulfinic acid formation has also been demonstrated to be necessary for the activation of the neuronal chaperone DJ-1.11,12 The readily reversible nature of the sulfenic acid PTM has made it an attractive candidate for investigation into redox signaling. Provided that cellular antioxidant capacity has not been compromised, ROS may act, not as agents of oxidative damage, but as signaling molecules, a phenomenon that has already been observed in mitosis.13 In addition to intracellular signaling,14 several discrete lines of evidence also suggest a physiological role for ROS.15-20 This observation is of particular importance with regard to renal function, not least because of the singular nature of the medulla, one of the most hypoxic regions in the body.21 Although nitric oxide (NO) mediated signaling appears more significant in the medulla with respect to the more normoxic cortex,22,23 ROS may also have a role in regulation of renal oxygenation.24 However, the relatively hypoxic state of the 10.1021/pr1001719
2010 American Chemical Society
Protein Sulfenation in Kidney of Spontaneously Hypertensive Rat medulla may also render it more susceptible to oxidative damage.25 A delicate equilibrium must be maintained in the medulla to ensure normal renal function, and the enhanced ROS generation seen in hypertension may lead to an imbalance,26 possibly through inhibiting NO signaling.26,27 The spontaneously hypertensive rat (SHR) model has the potential to give special insights in this regard and has been previously used in proteomic investigations of hypertension.28,29 In this study, we examined protein sulfenic acid formation in the kidney medulla of an established animal model of hypertension, the SHR, and compared them to normotensive Wistar rats. In addition, we investigated thiol redox status within the tissues and identified a subset of sulfenated proteins within the SHR medulla proteome. This population of sulfenated proteins may be targets of enhanced ROS generation and thus be redox-regulated.
Methods Animals and Tissue Preparation. Rats were obtained from Harlan (U.K.) and maintained in the Biological Services Unit (University College Cork) for at least 1 week prior to use. Animals received regular laboratory diet and tap water ad libitum. All procedures were performed in accordance with national guidelines and the European Community Directive 86/ 609/EC and approved by the Animal Experimentation Ethical Committee of University College Cork. Male Wistar and SHR weighing 250-300 g were anesthetized with 0.75-1.0 mL of a chloralose/urethane mixture (16.5/250 mg/mL, respectively). Kidneys were exposed via retroperitoneal incision, quickly removed and placed on ice. Cortex was dissected from medulla, and tissues were weighed, diluted to 25% with homogenization buffer (250 mM Hepes, pH 7.7/1 mM EDTA/0.1 mM neocuproine), and homogenized with a Polytron PCU2 Tissue Homogenizer (Kinematica, Switzerland). The homogenate was centrifuged at 20 000g at 4 °C for 20 min, and the supernatant was collected. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Gmbh, Germany) and fractions stored at -70 °C until use. Labeling of Sulfenated Proteins. Sulfenated proteins were detected and purified as described in Saurin et al.2 For Western blot studies, rat medulla homogenate containing 300 µg of protein was incubated with blocking buffer (100 mM Tris-HCl/ 100 mM maleimide/1% SDS, pH 7; all Sigma) for 2 h at room temperature, with rotation, in the dark. Excess maleimide was removed using Zebra desalt spin columns (Pierce) and the solution was then treated with 20 mM sodium arsenite (Sigma) and 0.1 mM biotin-maleimide (Sigma) for 30 min with rotation specifically to reduce and label sulfenated residues. For negative controls, sample was treated with 5 mM dimedone for 30 min prior to the addition of sodium arsenite and biotinmaleimide (BIAM). Unbound BIAM was removed by passing the solution through Zeba Desalt spin columns (Pierce) and the sample was reconstituted in nonreducing SDS polyacrylamide gel electrophoresis (SDS-PAGE) buffer. Detection and Purification of Sulfenated Proteins. Labeled protein (40 µg) was subjected to SDS-PAGE30 on 12% polyacrylamide gels on an Atto AE-6450 mini PAGE system (Atto, Japan). Proteins were then transferred (100 mA per blot, 55 min) to Protran nitrocellulose (0.2 µM) membranes (Whatman, Germany) using an AE-6677 HorizBlot (Atto, Japan) and equivalent protein loading was confirmed by staining with Ponceau S (0.2%) in 5% acetic acid. Membranes were blocked overnight at 4 °C with 1% BSA in PBS containing 0.05% Tween
research articles
(PBST). Membranes were subsequently incubated for 1 h at room temperature with streptavidin-HRP (Sigma) at a 1/5000 dilution in PBST, and were washed 2 × 15 min with PBST, before sulfenated proteins were detected on X-OMAT film (Sigma) using the SuperSignal West Pico chemiluminescence kit (Pierce). Blot images were acquired using an image scanner (GS-800 calibrated densitometer, BioRad) and protein sulfenation was quantified by densitometric analysis using Quantity One 4.5.2 analysis software (BioRad, CA). For purification studies, 5 mg of protein was treated as described above, and diluted 1 in 10 with 100 mM Tris-HCl/ 0.1% Triton X-100/50 µL protease inhibitor cocktail (Sigma) prior to overnight incubation with EZview Red streptavidin affinity gel (Sigma) at 4 °C. The beads were then washed 7 times with 100 mM Tris-HCl pH 7/0.1% SDS/0.1% triton X-100/100 mM NaCl. Sulfenated proteins were eluted from the beads by boiling for 5 min in SDS sample buffer containing 0.4 M urea, and prepared for two-dimensional SDS-PAGE (2D SDS-PAGE). Protein Preparation and Electrophoresis Methods. Purified proteins were rehydrated in buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 4% carrier ampholyte (Pharmalyte 3-10, Amersham-Pharmacia Biotech, U.K.), 1% DeStreak reagent (Amersham-Pharmacia Biotech, U.K.) and a trace amount of bromophenol blue. Final volumes of 125 µL were loaded on 7 cm pH 3-10 nonlinear immobilized pH gradient (IPG) strips (BioRad, CA) and rehydrated overnight for at least 15 h. IPG strips were focused on a Protean isoelectric focusing (IEF) Cell (BioRad) with linear voltage increases: 250 V for 15 min; 4000 V for 2 h; then up to 20 000 Vh. Following IEF, strips were equilibrated (20 min) in equilibration buffer (6 M urea, 0.375 M Tris, pH 8.8, 2% SDS, and 20% glycerol) containing 2% DTT, and then for 20 min in equilibration buffer containing 2.5% iodoacetamide. Equilibrated strips were electrophoresed on 12% SDS-PAGE gels at a constant voltage (150 V) at 4 °C using an Atto AE-6450 mini PAGE system (Atto, Japan). Purified proteins were visualized by silver staining31 and analytical gels underwent IEF, 2D SDS-PAGE, and colloidal Coomassie Blue G250 staining as described previously.32 Total Free Thiol Concentration. Total free thiol concentration was measured as described by Allanore et al.33 Briefly, 40 µL of supernatant was added to 1.0 mL of 0.1 M Tris, 10 mM EDTA, pH 8.2. Absorbance at 412 nm was measured prior to the addition of 40 µL of 10 mM (5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Sigma) in methanol to the sample. The absorbance prior to addition of DTNB was subtracted from that obtained post incubation. A control containing DTNB only was included, and the concentration of thiol groups was calculated using a molar extinction coefficient of 14 150 M-1 cm-1 at 412 nm. Thioredoxin Reductase Activity. Thioredoxin reductase (TrxR) activity was measured using a commercially available thioredoxin reductase assay kit (Sigma). TrxR activity was determined by following the reduction of 5,5′-dithiobis(2nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB) and measuring the strong yellow color produced at 412 nm. Determinations were carried out in triplicate in 96-well plates according to the manufacturer’s instructions. Spot Excision, Tryptic Digestion, and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Following 2D SDS-PAGE, proteins were visualized using colloidal Coomassie Brilliant Blue G-250, and spots of interest excised from the gel. Proteins were in-gel digested with trypsin (sequencing grade, modified; Promega U.K.) using an Investigator ProGest Journal of Proteome Research • Vol. 9, No. 5, 2010 2679
research articles robotic workstation (Genomic Solutions Ltd., Huntingdon, U.K.). Briefly, proteins were reduced with DTT (60 °C, 20 min), S-alkylated with iodoacetamide (25 °C, 10 min), and then digested with trypsin (37 °C, 8 h). The resulting tryptic peptide extract was dried by rotary evaporation (SC110 Speedvac; Savant Instruments, NY) and dissolved in 0.1% formic acid for LC-MS/MS analysis. Peptide solutions were analyzed using an HCTultra PTM Discovery System (Bruker Daltonics Ltd., U.K.) coupled to an UltiMate 3000 LC System (Dionex Ltd., U.K.). Peptides were separated on a Monolithic Capillary Column (200 µm i.d. × 5 cm; Dionex part no. 161409). Eluent A was 3% acetonitrile in water containing 0.05% formic acid; eluent B was 80% acetonitrile in water containing 0.04% formic acid with a gradient of 3-45% B in 12 min at a flow rate of 2.5 µL/min. Peptide fragment mass spectra were acquired in data-dependent AutoMS (2) mode with a scan range of 300-1500 m/z, 3 averages, and up to 3 precursor ions selected from the MS scan 100-2200 m/z). Precursors were actively excluded within a 1.0 min window, and all singly charged ions were excluded. Peptide peaks were detected and deconvoluted automatically using DataAnalysis version 3.2 (Bruker). Mass lists in the form of Mascot generic files were created automatically and used as the input for Mascot MS/MS Ions searches of the National Center for Biotechnology Information nonredundant (NCBI nr) database 20070926 (5 519 594 sequences; 1 911 975 371 residues; date 5/10/2007) using the Matrix Science Web server Mascot version 2.2 - timestamp: 5 Oct 2007 at 09:00:25 GMT (www.matrixscience.com). Default search parameters used were the following: Enzyme ) trypsin, max. missed cleavages )1; fixed modifications ) carbamidomethyl (C); variable modifications ) oxidation (M); peptide tolerance (1.5 Da; MS/ MS tolerance (0.5 Da; peptide charge ) 2+ and 3+; instrument ) ESI-TRAP. The taxonomy was restricted to Rattus (70 300 sequences), to improve the speed of the search and because all proteomic analyses were carried out in rat tissue no significant contamination would have arisen. When the search was repeated in the absence of species restriction, the optimal protein identifications faithfully matched those obtained from the Rattus search. In addition, Mascot searches of all spectra were performed against the decoy database available at the Matrix Science Web site using the same parameters as in the principal search. This helped obtain an estimate of the false positive peptide matches above identity threshold, and did not return a false discovery rate of >0.00% in any case. For protein identification, the protein sequence containing the highest number of rank 1 peptides with ion scores g39 (P > 0.05) was chosen. An ion score g39 indicates significant identity according to the Mascot MS/MS ion search, and the significance threshold is set at the 95% confidence level, thus, making it a reliable and comparable indicator of the significance of the database search. When issues of ambiguous identification arose, the proteins were further assessed in terms of how well their theoretical MW and pI matched the corresponding spot on the 2D-gel, which MS data peptide matches demonstrated greatest significance, and which entries were most-recent/best-annotated. Where after further inspection it was still not feasible to definitively resolve any ambiguity, all the top protein matches were reported in the MS data table. Statistical Analysis. For blot, enzyme activity, and free thiol content analyses, all data are mean ( SD, and significance (P < 0.05) was investigated using unpaired, two-sample unequal variance Student’s t-test. 2680
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Figure 1. (A) Representative blot of protein cysteine sulfenation from Wistar and SHR rat kidney medulla. Treatment with dimedone abolished the arsenite-dependent detection of sulfenic acids. Ponceau S staining of blots after transfer revealed equivalent loading of total protein. Bands obtained from four experiments were analyzed by densitometry. (B) Mean optical density of total bands in lane from four independent experiments. Data are mean ( SD n ) 4. *P < 0.05.
Results Relative Protein Sulfenation Levels in Hypertensive and Normotensive Rat Kidney Medulla. To investigate whether the SHR exhibited enhanced protein sulfenation with respect to normotensive Wistar, medulla extracts were subjected to the arsenite-reduction/BIAM labeling procedure.2 Sulfenation was detected with streptavidin-HRP on Western blots after onedimensional nonreducing SDS-PAGE (Figure 1A). Pretreatment of SHR medulla extract with the sulfenic acid-specific reagent dimedone34 prior to the arsenite reduction step abolished any streptavidin-HRP signal (Figure 1A). Quantification via densitometry revealed significantly greater sulfenation in SHR medulla with respect to normotensive Wistar controls (Figure 1B). Purification and 2D SDS-PAGE of Sulfenated Proteins. Sulfenated proteins purified from SHR medulla (n ) 3) were further analyzed by 2D SDS-PAGE using pH 3-10 NL gradients (Figure 2), and visualized via silver-staining. No nonspecific contaminating proteins were detected in silver-stained31 2-D SDS-PAGE gels of streptavidin-agarose bead washes. Protein spots common to all gels generated from affinity purified samples were matched against Coomassie-stained 2D SDSPAGE gels generated from total tissue extract, and matched spots were excised for identification by LC-MS/MS.
Protein Sulfenation in Kidney of Spontaneously Hypertensive Rat
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Figure 2. Representative 2D SDS-PAGE gels of sulfenated proteins purified from Wistar medulla (A) and SHR medulla (B). Spot numbers correspond to those in Table 1.
Figure 3. Total free thiol content. Total free thiol content was compared in the medulla and cortex of both SHR and Wistar rats. The histogram represents mean free thiol content from two independent experiments. Data are mean ( SD n ) 4. *P < 0.05. (SHR vs Wistar). †P < 0.05 (Cortex vs Medulla).
Thirty-two distinct proteins were successfully identified (see Supporting Information Figure SI for a complete list of peptides identified) which were categorized according to function as reported in the current literature (Table 1). The proteins are principally involved in processes such as protein folding, apoptosis, metabolism, cytoskeleton organization and cell motility, and protection against oxidative stress. In contrast, relatively few sulfenated protein spots were visible in Wistar medulla, which was also the case in one-dimensional SDSPAGE blots. Tissue Total Free Thiol Content. Free thiols are the main index of total antioxidant capacity of cellular homogenates.35 Total free thiol content was measured colorimetrically and compared in the medulla and cortex of both SHR and Wistar kidneys. Free thiol content in the medulla was significantly greater than in the cortex in both animal groups (Figure 3). This is not surprising given that the cortex has both a higher blood flow rate and oxygen tension (PO2) value, whereas the hypoxic nature of the medulla may favor a more reduced state. However, the free thiol content of the SHR medulla was significantly less than that of Wistar medulla (Figure 3), suggesting that a greater proportion of thiols may be oxidized. Total free thiol content includes contributions from both reduced glutathione (GSH) and protein thiols.36 Although the GSH/GSSG system represents the major redox buffer of the
Figure 4. Tissue thioredoxin reductase activity. TrxR activity was compared in the medulla and cortex of both SHR and Wistar rats. The histogram represents mean TrxR activity from two independent experiments. Data are mean ( SD n ) 4. *P < 0.05. (SHR vs Wistar). †P < 0.05 (Cortex vs Medulla).
cell,37 the concentration of SH groups associated with protein is much greater than that of GSH.38 Tissue Thioredoxin Reductase Activity. Protein sulfenic acids can be recycled back to the thiol state by reductants such as thioredoxin, and GSH. However, the thioredoxin system is the best-characterized of these and has been implicated in regulation of several proteins.39-41 TrxR activity was significantly greater in the medulla than in the cortex of Wistar, but no such difference was observed in the SHR (Figure 4). However, TrxR activity in the SHR medulla was significantly lower than in Wistar medulla (Figure 4), which could have implications for the ability of the Trx system to modulate protein thiol status and sulfenation.
Discussion The principal finding of our study is that protein sulfenation is enhanced in the kidney medulla of SHR with respect to normotensive Wistar rats. To our knowledge, this is the first demonstration of altered protein sulfenation in a physiological, or at least pathophysiological, context. This finding is supported by the observation that thiol oxidation is globally enhanced within SHR medulla as indicated by reduction in free thiol concentration. However, this analysis does not differentiate between different types of thiol oxidation, and could also be Journal of Proteome Research • Vol. 9, No. 5, 2010 2681
2682
protein name
Albumin Serum albumin precursor 2 CNDP dipeptidase 2 3 Un-named protein product Serine (or cysteine) peptidase inhibitor, clade A, member 3K [Rattus norvegicus] 4 ARP3 actin-related protein 3 homologue 5 Glutathione synthetase Glutathione synthetase, isoform CRA_c [Rattus norvegicus] 6 Serine protease inhibitor alpha 1 7 ATP synthase beta subunit ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide, isoform CRA_a 8 Albumin Serum albumin precursor 9 Creatine kinase Ckb protein Creatine kinase B-type 10 Isocitrate dehydrogenase 3 (NAD+) alpha Isocitrate dehydrogenase 3 (NAD+) alpha, isoform CRA_a Isocitrate dehydrogenase 3 (NAD+) alpha, isoform CRA_d Isocitrate dehydrogenase 3 (NAD+) alpha, isoform CRA_f 11 Zinc binding alcohol dehydrogenase, domain containing 1 12 Pregnancy zone protein Alpha-1-macroglobulin precursor (Alpha-1-M) 13 β-Actin 14 Dimethylarginine dimethylaminohydrolase 1 15 3-Hydroxyanthranilate 3,4-dioxygenase 16 Lactate dehydrogenase B 17,19 Glutamate cysteine ligase, modifier subunit 18 Protease (prosome, macropain) 28 subunit, alpha 20 Peroxiredoxin 6 21 Catechol-O-methyltransferase 22 Peroxiredoxin 3 23 Peroxiredoxin 2 24 Adenine phosphoribosyl transferase Adenine phosphoribosyl transferase (predicted), isoform CRA_a [Rattus norvegicus] 25 UMP-CMP kinase 26 WD repeat domain 1 27, 28 Glyceraldehyde 3-phosphate-dehydrogenase 29 Hydroxyacid oxidase (medium-chain)
1
spot no.
Table 1. Sulfenated Proteins Purified from SHR Kidney Medulla
70.6/6.09 70.6/6.09 53/5.43 46.7/5.31 45.7/5.24 47.8/5.61 52.5/5.48 57.7/5.19 46.2/5.7 51.1/4.92 56.3/5.19 56.3/5.14 70.7/6.09 70.6/6.09 42.9/5.33 45.5/5.4 42.9/5.39 40/6.47 41.6/6.47 40.8/6.27 39.4/5.87 30.2/4.9 168.4/6.46 168.3/6.46 31.9/5.24 31.8/7.75 32.8/5.46 36.8/5.70 27.8/7.6 28.7/5.63 24.8/5.64 29.8/5.41 28.5/7.14 21.9/5.34 19.7/6.17 20.1/6.32 22.3/5.66 66.8/6.15 36/8.14 39.6/6.44
BAF94208 NP_037094 EDL85910 P17475 AAB02288 NP_599191 EDL84889 NP_599153 P02770 AAA40932 AAH87656 P07335 NP_446090 EDL95540 EDL95543 EDL95545 NP_001015009 NP_665722 Q63041 ABM16832 NP_071633 AAH85739 NP_036727 NP_059001 NP_058960 NP_446028 NP_036663 NP_071985 NP_058865 NP_001013079 EDL92756 Q4KM73 NP_001014157 AAH87743 NP_114471
predicted
NP_599153 P02770 NP_001010920 CAA28958 AAH62236
accession no.
Journal of Proteome Research • Vol. 9, No. 5, 2010 23.4/5.25 57.4/6.42 38.3/8.1 39.6/7.49
42.7/5.62 42.7/5.62 42.9/5.33 42.9/5.33 42.9/5.33 37.9/5.22 37.9/5.22 37.9/5.22 37.9/5.22 39.1/5.31 39/5.22 39/5.22 37.5/5.15 37.4/5.24 36.5/5.3 36.8/5.17 28.7/5.15 28.2/5.15 24.1/5.21 23.8/4.95 23.7/5.89 22.3/5.28 22/5.53 22/5.53
51.3/4.99
57.8/5.66 52/5.48 52/5.48 55/4.92 51.3/4.99 51.3/4.99
70.6/5.8 70.6/5.8 61/5.26 59.8/4.9 59.8/4.9
observed
molecular weight (kDa)/pI
4 3 10 11
6 7 10 10 10 5 5 5 5 1 6 6 7 2 11 15 4 5 9 1 4 5 4 4
17
3 16 15 9 12 18
30 26 10 14 14
peptides matched
206 195 589 792
446 446 546 546 546 230 230 230 230 44 350 350 284 204 378 817 382 190 333 52 178 183 174 174
1100
259 877 877 321 1100 1100
1598 1598 507 840 840
Mascot score
31% 19% 46% 48%
20% 20% 50% 47% 50% 25% 25% 25% 26% 7% 8% 8% 52% 42% 69% 63% 36% 32% 64% 4% 24% 43% 31% 30%
55%
16% 50% 45% 46% 61% 55%
64% 64% 42% 47% 48%
% coverage protein function
Nucleic acid synthesis Signal transduction/cytoskeleton assembly Metabolism R-hydroxyacid oxidation
Structural NOS regulation Nicotinic acid biosynthesis Metabolism Glutathione biosynthesis Proteolysis Antioxidant defense Cathecholamine degradation Antioxidant defense Antioxidant defense Nucleic acid synthesis
Putative NADP-dependent oxidoreductase Protease inhibition
Metabolism
Metabolism
Transport/homeostasis
Proteolysis Energy production
Structural Glutathione biosynthesis
Metallopeptidase Serpin-like
Transport/homeostasis
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Small Heat Shock protein Antioxidant defense
Antioxidant defense
34 35
33
31 32
protein function
Metabolism Proteolysis
8% 8% 21% 15% 15% 16% 40% 40% 48% 72% 36% 106 106 92 154 154 154 117 117 117 288 242 2 2 3 2 2 2 4 4 4 6 4 37.2/7.14 37/6.56 27.8/7.6 28/8.6 28/8.6 26.4/8.87 22.5/8.94 22.5/8.94 18.9/9.22 19.9/6.84 15.8/5.88 1GVE_A 1GVE_B NP_001004220 NP_001008218 NP_001008218 EDL88835 NP_446062 AAH78771 EDM12620 CAA42911 AAA40996
39.9/6.83 39.9/6.83 27.8/7.6 28/8.6 28/8.6 28/8.6 18.9/6.47 18.9/6.47 18.9/6.47 22.6/6.84 15.8/5.88
8% 8% 106 106 2 2 39.9/6.83 39.9/6.83 37/6.83 37.1/6.79 CAA52740 NP_037347
Aflatoxin B1 aldehyde reductase Aldo-keto reductase family 7, member A3 (aflatoxin aldehyde reductase) Chain A, Aflatoxin Aldehyde Reductase (Akr7a1) From Rat Liver Chain B, Aflatoxin Aldehyde Reductase (Akr7a1) From Rat Liver Electron-transfer-flavoprotein, beta polypeptide PREDICTED: similar to Proteasome subunit alpha type 7-like Proteasome (prosome, macropain) subunit, alpha type 7 rCG38543, isoform CRA_b Peroxiredoxin 5 precursor Peroxiredoxin 5 Peroxiredoxin 5, isoform CRA_b Alpha B-Crystallin Cu-Zn superoxide dismutase 30
% coverage Mascot score peptides matched observed predicted spot no.
protein name
accession no.
molecular weight (kDa)/pI
Table 1 Continued
Antioxidant defense
Protein Sulfenation in Kidney of Spontaneously Hypertensive Rat
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attributed to increased S-nitrosylation, S-thiolation, glutathionylation, disulfide-bridge, or “hyperoxidation” of thiols. In addition, we observed decreased TrxR activity in medulla which would result in less efficient restoration of sulfenated cysteines to the reduced state. Moreover, impairment of TrxR activity has been implicated in other pathologies,42 and TRX expression has been demonstrated to be deficient in SHR compared to normotensive control animals.43 TRX may also fulfill a distinct role in the kidney medulla under oxidative stress conditions, because it is retained in the medulla cytosol during ischemiareperfusion injury, but is depleted from the cortex cytosol.44 Protein sulfenation may arise through a number of mechanisms,45 so it is interesting to consider how they may account for the increased sulfenation observed in this instance. The primary mechanism of sulfenation is considered to be the reaction of H2O2 with cysteine thiol(ates),46 but hydrolysis of S-nitrosothiols may also create sulfenic acids.47-49 This latter route may be significant in the medulla given the prominent role of NO metabolism in this tissue.22,23 Studies by Eaton’s group may undermine S-nitrosothiols as contributors to sulfenation, as they found introducing the NO donor S-nitroso-Nacetylpenicillamine (SNAP) did not lead to increased sulfenation.50 Alternatively, disulfide bonds can be enzymatically hydrolyzed to generate a thiol and accompanying sulfenic acid as has been posited for Angiostatin fragments.51 Once again though, whether disulfide bonds contribute significantly to the protein sulfenation in vivo remains unclear, because Charles et al.50 found no correlation between sulfenation and treatment of myocytes with diamide. Sulfenic acids may also form thiyl radicals in the presence of hydroxyl radicals. This route might be dismissed in terms of redox signaling, given the highly reactive and short-lived nature of hydroxyl radicals, but could represent a biomarker of oxidative stress insult in our study. Interestingly, peroxynitrite (ONOO-) may also promote sulfenation,53 and this is believed to be the major toxic byproduct when superoxide impedes NO signaling in the medulla.54,55 To definitively establish the principal mechanism of sulfenation in our model, a more complete understanding of the interaction of ROS such as superoxide, NO, H2O2 and ONOOthan is presently available will be necessary. Various candidates have been proposed as the principal endogenous source of ROS in hypertension,56 such as xanthine oxidases, uncoupled nitric oxide synthases, leakage from the mitochondrial electron transport chain, and nonphagocytic NAD(P)H oxidases. Superoxide is the main ROS formed in each case and this in turn should be converted into H2O2 by endogenous superoxide dismutase (SOD). The importance of SOD in negating hypertension has been confirmed by renal studies involving the SOD mimetic TEMPOL55 but, should the H2O2 generated exceed the antioxidant capacity of the catalase present, or should that catalase be defective, enhanced sulfenation may result. Catalase activity has been variously described as being unchanged57,58 or diminished59 in different models of renopathy, but the antioxidant capacity of the medulla is known to be inferior to that of the cortex60 and excess H2O2 can abolish any amelioration of medullary blood flow brought about by TEMPOL.61 Aside from considerations of the route to formation, the nature of the cysteine “targeted” for sulfenation is also important. The uncatalyzed reaction of hydroperoxides with thiols is relatively slow, but the ionized form, the thiolate, reacts faster.62 The majority of protein cysteine residue -SH groups have a pKa close to 8.2, so they should rarely form thiolates. Journal of Proteome Research • Vol. 9, No. 5, 2010 2683
research articles However, those with lower pKa, such as cysteines flanked by basic amino acids, are more susceptible to oxidation.63 Perhaps the most striking finding in the follow-up study by Charles et al.50 was that sulfenation in the oxygen-sensing element responded to changes in PO2. In terms of oxygensensing, our detection of enhanced sulfenation in the SHR medulla has particularly interesting implications for the kidney because the hypoxic nature of the medulla is a necessary consequence of proper renal function. The low medullary PO2 of 10-20 mmHg arises because of the counter-flow arrangement of microvessels and nephrons required to accomplish urinary concentration. Furthermore, a chronic pro-oxidant state has been implicated in the renal fibrosis characteristic of the progression of hypertension,64 through induction of mRNA expression for collagens I, III and IV, and the fibrogenic cytokine, TGF-beta1.65 Many of the sulfenated proteins identified in the SHR medulla have been recognized as being redox sensitive in previous studies.2,50,66,67 Although this raises the possibility that sulfenation may act as a regulatory mechanism, the impact of this PTM on protein function has not been characterized in many cases. Whether our sample preparation and methodology provides a good representation of the population of sulfenated proteins depends on a number of factors. First, cysteine sulfenic acids are inherently reactive moieties5 and, consequently, sulfenic acids are often intermediates en route to more stable oxidation states. In such cases, sulfenation is simply a priming event and is of far less significance for protein function than the ultimate oxidation state. The proportion of protein cysteine residues converted to sulfenic acids in vivo most likely depends on the location of the cysteine within the protein structure, the cellular environment of the protein and the nature and concentration of the oxidant involved.62 For example, in two protein classes containing low pKa catalytic cysteines, second-order reaction rates with H2O2 vary from 105-108 M-1 s-1 in the case of peroxiredoxins68,69 to 10-160 M-1 s-1 in the case of protein tyrosine phosphatases.70-72 Therefore, proteins with lower sulfenation rates may escape detection. Our use of denaturing SDS to ensure complete blockage of free thiol sites may also have the unintended consequence of eliminating less stable sulfenic acids, because protein structure is involved in sulfenic acid stabilization.63 Blocking and specific labeling also occur after tissue homogenization so protein sulfenation becomes decoupled from subcellular location. Sulfenated proteins may be more common in the oxidizing environment of the endoplasmic environment and less prevalent in the reducing environment of the cytosol, but lysis upsets this equilibrium. Proteins involved in antioxidant defense feature prominently among the proteins identified. This is to be expected in the case of the various peroxiredoxins, which are probably the protein class in which sulfenation is best characterized. Peroxiredoxin isoforms 2, 3, 5, and 6 (PRX2, PRX3, PRX5, and PRX6) were all identified as sulfenated proteins in the SHR medulla. Peroxiredoxins share an ability to reduce hydroperoxides, but a specific antiperoxynitrite activity has been attributed to PRX573 and PRX6,66 which may be of importance in hypertensive kidney where peroxynitrite nitration products can accumulate.74 All of the peroxiredoxins, with the possible exception of the 1-Cys PRX6,75 appear to be exclusively reduced, in turn, by the thioredoxin system,39 so impairment of the thioredoxin system would hamper their ability to curtail ROS and RNS. Other antioxidant enzymes identified included 2684
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Tyther et al. Cu-Zn superoxide dismutase (Cu-Zn SOD), which eliminates superoxide, and aflatoxin B1 aldehyde reductase, which helps protect against lipoxidation products. Cu-Zn SOD has also been identified as a protein downregulated in SHR left ventricular hypertrophy, along with thiol-specific antioxidant protein.28 Certain isoforms of Cu-Zn SOD possess cysteine residues hyperoxidized to a sulfonic acid in Alzheimer’s and Parkinson’s Diseases.76 Sulfenation has not been implicated directly in alterations in aflatoxin B1 aldehyde reductase activity, but it is known to activate its fellow aldo-keto reductase family member aldose reductase in cardiac ischemia-reperfusion.77 Aflatoxin B1 aldehyde reductase and aldose reductase share significant sequence similarity and the latter has already been implicated in the renopathy arising from diabetic complications.78 An antioxidant role has also been suggested for translationally controlled tumor protein 1,79 and it has also recently been implicated in the development of hypertension.80 One quirk in the sulfenation study by Saurin et al.2 was the detection of ATP synthase β-subunit and troponin T, proteins that contain no cysteines, and whose presence they attributed to copurification with the redox active R-subunit of the ATP synthase and myofilament components. ATP synthase β-subunit was detected here as in the prior study, but the presence of the small heat shock protein alpha B-crystallin is similarly difficult to rationalize because it too possesses no cysteine residues.81 Alpha B-crystallin does, however, have a role to play in ischemic preconditioning82 and, because it is associated with PRX6,83 it may have copurified with this protein. Several proteins involved in energy production/metabolism were identified, which is consistent with similar studies of redox active thiols.84,85 Glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) has been detected in many oxidative stress studies scenarios and, in addition to its role in glycolysis, has also been implicated in apoptosis,86 transcription,87 and intracellular trafficking.88 Thus, alterations in activity mediated by sulfenation could have multiple consequences. Even “mild” oxidative stress likely to lead to sulfenic acid formation can impair GAPDH activity89 and, although S-nitrosylation of these residues may protect the enzyme,90 NO signaling is perturbed in hypertension.27 In addition to GAPDH, there are precedents for redox sensitivity in lactate dehydrogenase,2 creatine kinase,91 catechol-O-methyltransferase,92 and the 20S proteasome,93 which were among the proteins identified here. Dimethylarginine dimethylaminohydrolase 1 (DDAH1), which regulates nitric oxide synthase (NOS) activity through degrading NOS-inhibiting methylarginines, also has a critical cysteine group in its active site. This -SH can be S-nitrosylated,95 which may act as a regulatory feedback mechanism, or be oxidized by homocysteine,96 which also inhibits DDAH1 activity. Under conditions of enhanced oxidative stress, it is possible that this cysteine residue may be vulnerable to oxidation to sulfenic acid also. Inhibition of DDAH1 activity in turn increases the concentration of the NOS-inhibiting methylarginines, and contributes to hypertension by disrupting vascular homeostasis.97 Given that GSH biosynthesis has been shown to be perturbed in SHR kidney,98 it is interesting to consider the consequences of sulfenation of glutamate cysteine ligase and GSH synthetase, two of the key enzymes involved. Diminished GSH bioavailability may prevent S-glutathionylation of low pKa cysteine residues. This has been proposed as a means of protecting such residues from undesirable oxidation.99
Protein Sulfenation in Kidney of Spontaneously Hypertensive Rat In summary, we have observed enhanced sulfenation in the medulla of hypertensive rats in comparison to normotensive animals. Many of the identified sulfenated proteins have been reported as being redox sensitive in the past. However, it will be necessary to establish whether any functional changes accompany this PTM in these proteins. In doing so, our understanding of the role of ROS in renal function will be enhanced, and possible interventions to ameliorate hypertension may become apparent. Abbreviations: BIAM, biotin-maleimide; DTNB, 5,5′-Dithiobis (2-nitrobenzoic) acid; GSH/GSSG, reduced/oxidized glutathione; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NADPH, nicotinamide adenine dinucleotide; PTM, post-translational modification; ROS, reactive oxygen species; SHR, spontaneously hypertensive rat; TNB, 5-thio-2nitrobenzoic acid; TrxR, thioredoxin reductase.
Acknowledgment. The authors would like to acknowledge the contribution of the Proteomics Unit, University of Aberdeen, Scotland, in preparing this manuscript. We would also like to thank Ian Davidson and Dr. David Stead for their assistance. Our laboratory (R.T. and D.S.) is funded by the Higher Education Authority of Ireland Programme for Research in Third Level Institutions, Cycle 3. B.M. received an EMBARK fellowship from the Irish Research Council for Science Engineering and Technology.
Supporting Information Available: Sequence information for identified proteins. This material is available free of charge via the Internet at http://pubs.acs.org.
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(14) (15)
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References (1) Eaton, P. Protein thiol oxidation in health and disease: techniques for measuring disulfides and related modifications in complex protein mixtures. Free Radical Biol. Med. 2006, 40 (11), 1889–99. (2) Saurin, A. T.; Neubert, H.; Brennan, J. P.; Eaton, P. Widespread sulfenic acid formation in tissues in response to hydrogen peroxide. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (52), 17982–7. (3) Wilcox, C. S. Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2005, 289 (4), R913–35. (4) Vaziri, N. D.; Rodriguez-Iturbe, B. Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat. Clin. Pract. Nephrol. 2006, 2 (10), 582–93. (5) Claiborne, A.; Mallett, T. C.; Yeh, J. I.; Luba, J.; Parsonage, D. Structural, redox, and mechanistic parameters for cysteine-sulfenic acid function in catalysis and regulation. Adv. Protein Chem. 2001, 58, 215–76. (6) Claiborne, A.; Yeh, J. I.; Mallett, T. C.; Luba, J.; Crane, E. J., 3rd; Charrier, V.; Parsonage, D. Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation. Biochemistry 1999, 38 (47), 15407–16. (7) Kim, S. O.; Merchant, K.; Nudelman, R.; Beyer, W. F., Jr.; Keng, T.; DeAngelo, J.; Hausladen, A.; Stamler, J. S. OxyR: a molecular code for redox-related signaling. Cell 2002, 109 (3), 383–96. (8) Lee, J. W.; Soonsanga, S.; Helmann, J. D. A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (21), 8743–8. (9) Woo, H. A.; Chae, H. Z.; Hwang, S. C.; Yang, K. S.; Kang, S. W.; Kim, K.; Rhee, S. G. Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation. Science 2003, 300 (5619), 653–6. (10) Budanov, A. V.; Sablina, A. A.; Feinstein, E.; Koonin, E. V.; Chumakov, P. M. Regeneration of peroxiredoxins by p53-regulated sestrins, homologs of bacterial AhpD. Science 2004, 304 (5670), 596–600. (11) Zhou, W.; Zhu, M.; Wilson, M. A.; Petsko, G. A.; Fink, A. L. The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein. J. Mol. Biol. 2006, 356 (4), 1036–48. (12) Canet-Aviles, R. M.; Wilson, M. A.; Miller, D. W.; Ahmad, R.; McLendon, C.; Bandyopadhyay, S.; Baptista, M. J.; Ringe, D.; Petsko, G. A.; Cookson, M. R. The Parkinson’s disease protein DJ-1
(24)
(25) (26)
(27) (28)
(29)
(30) (31)
(32)
(33)
(34)
(35)
research articles
is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (24), 9103– 8. Irani, K.; Xia, Y.; Zweier, J. L.; Sollott, S. J.; Der, C. J.; Fearon, E. R.; Sundaresan, M.; Finkel, T.; Goldschmidt-Clermont, P. J. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 1997, 275 (5306), 1649–52. Rhee, S. G. Cell signaling. H2O2, a necessary evil for cell signaling. Science 2006, 312 (5782), 1882–3. Sundaresan, M.; Yu, Z. X.; Ferrans, V. J.; Irani, K.; Finkel, T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 1995, 270 (5234), 296–9. Waghray, M.; Cui, Z.; Horowitz, J. C.; Subramanian, I. M.; Martinez, F. J.; Toews, G. B.; Thannickal, V. J. Hydrogen peroxide is a diffusible paracrine signal for the induction of epithelial cell death by activated myofibroblasts. FASEB J. 2005, 19 (7), 854–6. Avshalumov, M. V.; Rice, M. E. Activation of ATP-sensitive K+ (K(ATP)) channels by H2O2 underlies glutamate-dependent inhibition of striatal dopamine release. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (20), 11729–34. Avshalumov, M. V.; Bao, L.; Patel, J. C.; Rice, M. E. H2O2 signaling in the nigrostriatal dopamine pathway via ATP-sensitive potassium channels: issues and answers. Antioxid. Redox Signaling 2007, 9 (2), 219–31. Touyz, R. M.; Schiffrin, E. L. Ang II-stimulated superoxide production is mediated via phospholipase D in human vascular smooth muscle cells. Hypertension 1999, 34 (4 Pt. 2), 976–82. Zafari, A. M.; Ushio-Fukai, M.; Akers, M.; Yin, Q.; Shah, A.; Harrison, D. G.; Taylor, W. R.; Griendling, K. K. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension 1998, 32 (3), 488–95. Epstein, F. H. Oxygen and renal metabolism. Kidney Int. 1997, 51 (2), 381–5. Zou, A. P.; Cowley, A. W., Jr. Nitric oxide in renal cortex and medulla. An in vivo microdialysis study. Hypertension 1997, 29 (1 Pt. 2), 194–8. Wu, F.; Park, F.; Cowley, A. W., Jr.; Mattson, D. L. Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney. Am. J. Physiol. 1999, 276 (6 Pt. 2), F874–81. Zou, A. P.; Cowley, A. W., Jr. Reactive oxygen species and molecular regulation of renal oxygenation. Acta Physiol. Scand. 2003, 179 (3), 233–41. Brezis, M.; Rosen, S. Hypoxia of the renal medulla--its implications for disease. N. Engl. J. Med. 1995, 332 (10), 647–55. Makino, A.; Skelton, M. M.; Zou, A. P.; Cowley, A. W., Jr. Increased renal medullary H2O2 leads to hypertension. Hypertension 2003, 42 (1), 25–30. Leclercq, B.; Jaimes, E. A.; Raij, L. Nitric oxide synthase and hypertension. Curr. Opin. Nephrol. Hypertens 2002, 11 (2), 185–9. Gallego-Delgado, J.; Lazaro, A.; Osende, J. I.; Esteban, V.; Barderas, M. G.; Gomez-Guerrero, C.; Vega, R.; Vivanco, F.; Egido, J. Proteomic analysis of early left ventricular hypertrophy secondary to hypertension: modulation by antihypertensive therapies. J. Am. Soc. Nephrol. 2006, 17 (12 Suppl. 3), S159–64. Meng, C.; Jin, X.; Xia, L.; Shen, S. M.; Wang, X. L.; Cai, J.; Chen, G. Q.; Wang, L. S.; Fang, N. Y. Alterations of mitochondrial enzymes contribute to cardiac hypertrophy before hypertension development in spontaneously hypertensive rats. J. Proteome Res. 2009, 8 (5), 2463–75. Laemmli, U. K. Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 1970, 277 (5259), 680–5. Rabilloud, T. A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 1992, 13 (7), 429–39. Cash, P.; Argo, E.; Bruce, K. D. Characterisation of Haemophilus influenzae proteins by two-dimensional gel electrophoresis. Electrophoresis 1995, 16 (1), 135–48. Allanore, Y.; Borderie, D.; Lemarechal, H.; Ekindjian, O. G.; Kahan, A. Acute and sustained effects of dihydropyridine-type calcium channel antagonists on oxidative stress in systemic sclerosis. Am. J. Med. 2004, 116 (9), 595–600. Raftery, M. J.; Yang, Z.; Valenzuela, S. M.; Geczy, C. L. Novel intraand inter-molecular sulfinamide bonds in S100A8 produced by hypochlorite oxidation. J. Biol. Chem. 2001, 276 (36), 33393–401. Balcerczyk, A.; Grzelak, A.; Janaszewska, A.; Jakubowski, W.; Koziol, S.; Marszalek, M.; Rychlik, B.; Soszynski, M.; Bilinski, T.; Bartosz, G. Thiols as major determinants of the total antioxidant capacity. BioFactors 2003, 17 (1-4), 75–82.
Journal of Proteome Research • Vol. 9, No. 5, 2010 2685
research articles (36) Schafer, F. Q.; Buettner, G. R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/ glutathione couple. Free Radical Biol. Med. 2001, 30 (11), 1191– 212. (37) Gilbert, H. F. Molecular and cellular aspects of thiol-disulfide exchange. In Advances in Enzymology,; A. Meister, E., Ed.; Wiley Interscience: New York, 1990; pp 69-173. (38) Torchinsky., Y. M. Sulphur in Proteins; Pergamon: Oxford, U.K., 1981. (39) Rhee, S. G.; Chae, H. Z.; Kim, K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radical Biol. Med. 2005, 38 (12), 1543–52. (40) Watson, W. H.; Yang, X.; Choi, Y. E.; Jones, D. P.; Kehrer, J. P. Thioredoxin and its role in toxicology. Toxicol. Sci. 2004, 78 (1), 3–14. (41) Haddad, J. J. Antioxidant and prooxidant mechanisms in the regulation of redox(y)-sensitive transcription factors. Cell. Signalling 2002, 14 (11), 879–97. (42) Lemarechal, H.; Anract, P.; Beaudeux, J. L.; Bonnefont-Rousselot, D.; Ekindjian, O. G.; Borderie, D. Impairment of thioredoxin reductase activity by oxidative stress in human rheumatoid synoviocytes. Free Radical Res. 2007, 41 (6), 688–98. (43) Tanito, M.; Nakamura, H.; Kwon, Y. W.; Teratani, A.; Masutani, H.; Shioji, K.; Kishimoto, C.; Ohira, A.; Horie, R.; Yodoi, J. Enhanced oxidative stress and impaired thioredoxin expression in spontaneously hypertensive rats. Antioxid. Redox Signaling 2004, 6 (1), 89– 97. (44) Kasuno, K.; Nakamura, H.; Ono, T.; Muso, E.; Yodoi, J. Protective roles of thioredoxin, a redox-regulating protein, in renal ischemia/ reperfusion injury. Kidney Int. 2003, 64 (4), 1273–82. (45) Poole, L. B.; Karplus, P. A.; Claiborne, A. Protein sulfenic acids in redox signaling. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 325–47. (46) Ellis, H. R.; Poole, L. B. Novel application of 7-chloro-4-nitrobenzo2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 1997, 36 (48), 15013–8. (47) Stamler, J. S.; Hausladen, A. Oxidative modifications in nitrosative stress. Nat. Struct. Biol. 1998, 5 (4), 247–9. (48) Gu, Z.; Kaul, M.; Yan, B.; Kridel, S. J.; Cui, J.; Strongin, A.; Smith, J. W.; Liddington, R. C.; Lipton, S. A. S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science 2002, 297 (5584), 1186–90. (49) Percival, M. D.; Ouellet, M.; Campagnolo, C.; Claveau, D.; Li, C. Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid. Biochemistry 1999, 38 (41), 13574–83. (50) Charles, R. L.; Schroder, E.; May, G.; Free, P.; Gaffney, P. R.; Begum, S.; Wait, R.; Heads, R. J.; Eaton, P. Protein sulfenation as a redox sensor: Proteomic studies using a novel biotinylated dimedone analogue. Mol. Cell. Proteomics 2007, 6 (9), 1473–84. (51) Hogg, P. J. Disulfide bonds as switches for protein function. Trends Biochem. Sci. 2003, 28 (4), 210–4. (52) Giles, G. I.; Tasker, K. M.; Jacob, C. Hypothesis: the role of reactive sulfur species in oxidative stress. Free Radical Biol. Med. 2001, 31 (10), 1279–83. (53) Bryk, R.; Griffin, P.; Nathan, C. Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature 2000, 407 (6801), 211–5. (54) Evans, R. G.; Fitzgerald, S. M. Nitric oxide and superoxide in the renal medulla: a delicate balancing act. Curr. Opin. Nephrol. Hypertens. 2005, 14 (1), 9–15. (55) Schnackenberg, C. G.; Welch, W. J.; Wilcox, C. S. Normalization of blood pressure and renal vascular resistance in SHR with a membrane-permeable superoxide dismutase mimetic: role of nitric oxide. Hypertension 1998, 32 (1), 59–64. (56) Lassegue, B.; Griendling, K. K. Reactive oxygen species in hypertension; An update. Am. J. Hypertens. 2004, 17 (9), 852–60. (57) Zhan, C. D.; Sindhu, R. K.; Pang, J.; Ehdaie, A.; Vaziri, N. D. Superoxide dismutase, catalase and glutathione peroxidase in the spontaneously hypertensive rat kidney: effect of antioxidant-rich diet. J. Hypertens. 2004, 22 (10), 2025–33. (58) Vaziri, N. D.; Lin, C. Y.; Farmand, F.; Sindhu, R. K. Superoxide dismutase, catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension. Kidney Int. 2003, 63 (1), 186–94. (59) Sindhu, R. K.; Ehdaie, A.; Farmand, F.; Dhaliwal, K. K.; Nguyen, T.; Zhan, C. D.; Roberts, C. K.; Vaziri, N. D. Expression of catalase and glutathione peroxidase in renal insufficiency. Biochim. Biophys. Acta 2005, 1743 (1-2), 86–92. (60) Gonzalez-Flecha, B.; Evelson, P.; Sterin-Speziale, N.; Boveris, A. Hydrogen peroxide metabolism and oxidative stress in cortical,
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(61)
(62)
(63)
(64)
(65)
(66)
(67) (68) (69)
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(72) (73)
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(75) (76)
(77) (78)
(79) (80)
(81)
medullary and papillary zones of rat kidney. Biochim. Biophys. Acta 1993, 1157 (2), 155–61. Chen, Y. F.; Cowley, A. W., Jr.; Zou, A. P. Increased H(2)O(2) counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2003, 285 (4), R827–33. Forman, H. J.; Fukuto, J. M.; Torres, M. Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. Am. J. Physiol.: Cell Physiol. 2004, 287 (2), C246–56. Salsbury, F. R., Jr.; Knutson, S. T.; Poole, L. B.; Fetrow, J. S. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid. Protein Sci. 2008, 17 (2), 299– 312. Lekgabe, E. D.; Kiriazis, H.; Zhao, C.; Xu, Q.; Moore, X. L.; Su, Y.; Bathgate, R. A.; Du, X. J.; Samuel, C. S. Relaxin reverses cardiac and renal fibrosis in spontaneously hypertensive rats. Hypertension 2005, 46 (2), 412–8. Nath, K. A.; Grande, J.; Croatt, A.; Haugen, J.; Kim, Y.; Rosenberg, M. E. Redox regulation of renal DNA synthesis, transforming growth factor-beta1 and collagen gene expression. Kidney Int. 1998, 53 (2), 367–81. Peshenko, I. V.; Shichi, H. Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite. Free Radical Biol. Med. 2001, 31 (3), 292–303. Turell, L.; Botti, H.; Carballal, S.; Radi, R.; Alvarez, B. Sulfenic acid--a key intermediate in albumin thiol oxidation. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2009, 877 (28), 3384–92. Parsonage, D.; Karplus, P. A.; Poole, L. B. Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (24), 8209–14. Peskin, A. V.; Low, F. M.; Paton, L. N.; Maghzal, G. J.; Hampton, M. B.; Winterbourn, C. C. The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents. J. Biol. Chem. 2007, 282 (16), 11885–92. Winterbourn, C. C.; Metodiewa, D. Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radical Biol. Med. 1999, 27 (3-4), 322–8. Denu, J. M.; Tanner, K. G. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry. 1998, 37 (16), 5633–42. Sohn, J.; Rudolph, J. Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction. Biochemistry. 2003, 42 (34), 10060–70. Dubuisson, M.; Vander Stricht, D.; Clippe, A.; Etienne, F.; Nauser, T.; Kissner, R.; Koppenol, W. H.; Rees, J. F.; Knoops, B. Human peroxiredoxin 5 is a peroxynitrite reductase. FEBS Lett. 2004, 571 (1-3), 161–5. Guo, W.; Adachi, T.; Matsui, R.; Xu, S.; Jiang, B.; Zou, M. H.; Kirber, M.; Lieberthal, W.; Cohen, R. A. Quantitative assessment of tyrosine nitration of manganese superoxide dismutase in angiotensin IIinfused rat kidney. Am. J. Physiol.: Heart Circ. Physiol. 2003, 285 (4), H1396–403. Fisher, A. B.; Dodia, C.; Manevich, Y.; Chen, J. W.; Feinstein, S. I. Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase. J. Biol. Chem. 1999, 274 (30), 21326–34. Choi, J.; Rees, H. D.; Weintraub, S. T.; Levey, A. I.; Chin, L. S.; Li, L. Oxidative modifications and aggregation of Cu, Zn-superoxide dismutase associated with Alzheimer and Parkinson diseases. J. Biol. Chem. 2005, 280 (12), 11648–55. Kaiserova, K.; Srivastava, S.; Hoetker, J. D.; Awe, S. O.; Tang, X. L.; Cai, J.; Bhatnagar, A. Redox activation of aldose reductase in the ischemic heart. J. Biol. Chem. 2006, 281 (22), 15110–20. Drel, V. R.; Pacher, P.; Stevens, M. J.; Obrosova, I. G. Aldose reductase inhibition counteracts nitrosative stress and poly(ADPribose) polymerase activation in diabetic rat kidney and highglucose-exposed human mesangial cells. Free Radical Biol. Med. 2006, 40 (8), 1454–65. Gnanasekar, M.; Ramaswamy, K. Translationally controlled tumor protein of Brugia malayi functions as an antioxidant protein. Parasitol. Res. 2007, 101 (6), 1533–40. Kim, M. J.; Kwon, J. S.; Suh, S. H.; Suh, J. K.; Jung, J.; Lee, S. N.; Kim, Y. H.; Cho, M. C.; Oh, G. T.; Lee, K. Transgenic overexpression of translationally controlled tumor protein induces systemic hypertension via repression of Na(+), K(+)-ATPase. J. Mol. Cell. Cardiol. 2007, 44 (1), 151–59. Eaton, P.; Fuller, W.; Shattock, M. J. S-thiolation of HSP27 regulates its multimeric aggregate size independently of phosphorylation. J. Biol. Chem. 2002, 277 (24), 21189–96.
research articles
Protein Sulfenation in Kidney of Spontaneously Hypertensive Rat (82) Eaton, P.; Fuller, W.; Bell, J. R.; Shattock, M. J. AlphaB Crystallin translocation and phosphorylation: signal transduction pathways and preconditioning in the isolated rat heart. J. Mol. Cell. Cardiol. 2001, 33 (9), 1659–71. (83) Fatma, N.; Kubo, E.; Sharma, P.; Beier, D. R.; Singh, D. P. Impaired homeostasis and phenotypic abnormalities in Prdx6-/-mice lens epithelial cells by reactive oxygen species: increased expression and activation of TGFbeta. Cell Death Differ. 2005, 12 (7), 734–50. (84) Hurd, T. R.; Prime, T. A.; Harbour, M. E.; Lilley, K. S.; Murphy, M. P. Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling. J. Biol. Chem. 2007, 282 (30), 22040– 51. (85) Leichert, L. I.; Jakob, U. Protein thiol modifications visualized in vivo. PLoS Biol. 2004, 2 (11), e333. (86) Hara, M. R.; Agrawal, N.; Kim, S. F.; Cascio, M. B.; Fujimuro, M.; Ozeki, Y.; Takahashi, M.; Cheah, J. H.; Tankou, S. K.; Hester, L. D.; Ferris, C. D.; Hayward, S. D.; Snyder, S. H.; Sawa, A. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 2005, 7 (7), 665–74. (87) Zheng, L.; Roeder, R. G.; Luo, Y. S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 2003, 114 (2), 255–66. (88) Tisdale, E. J.; Artalejo, C. R. A GAPDH mutant defective in Srcdependent tyrosine phosphorylation impedes Rab2-mediated events. Traffic 2007, 8 (6), 733–41. (89) Schmalhausen, E. V.; Pleten, A. P.; Muronetz, V. I. Ascorbateinduced oxidation of glyceraldehyde-3-phosphate dehydrogenase. Biochem. Biophys. Res. Commun. 2003, 308 (3), 492–6. (90) Ishii, T.; Sunami, O.; Nakajima, H.; Nishio, H.; Takeuchi, T.; Hata, F. Critical role of sulfenic acid formation of thiols in the inactivation of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide. Biochem. Pharmacol. 1999, 58 (1), 133–43. (91) Park, E. M.; Thomas, J. A. S-thiolation of creatine kinase and glycogen phosphorylase b initiated by partially reduced oxygen species. Biochim. Biophys. Acta 1988, 964 (2), 151–60.
(92) Cotton, N. J.; Stoddard, B.; Parson, W. W. Oxidative inhibition of human soluble catechol-O-methyltransferase. J. Biol. Chem. 2004, 279 (22), 23710–8. (93) Demasi, M.; Silva, G. M.; Netto, L. E. 20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated. J. Biol. Chem. 2003, 278 (1), 679–85. (94) Eaton, P.; Jones, M. E.; McGregor, E.; Dunn, M. J.; Leeds, N.; Byers, H. L.; Leung, K. Y.; Ward, M. A.; Pratt, J. R.; Shattock, M. J. Reversible cysteine-targeted oxidation of proteins during renal oxidative stress. J. Am. Soc. Nephrol. 2003, 14 (8 Suppl. 3), S290– 6. (95) Leiper, J.; Murray-Rust, J.; McDonald, N.; Vallance, P. S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (21), 13527–32. (96) Stuhlinger, M. C.; Tsao, P. S.; Her, J. H.; Kimoto, M.; Balint, R. F.; Cooke, J. P. Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine. Circulation 2001, 104 (21), 2569–75. (97) Leiper, J.; Nandi, M.; Torondel, B.; Murray-Rust, J.; Malaki, M.; O’Hara, B.; Rossiter, S.; Anthony, S.; Madhani, M.; Selwood, D.; Smith, C.; Wojciak-Stothard, B.; Rudiger, A.; Stidwill, R.; McDonald, N. Q.; Vallance, P. Disruption of methylarginine metabolism impairs vascular homeostasis. Nat. Med. 2007, 13 (2), 198–203. (98) Levonen, A. L.; Laakso, J.; Vaskonen, T.; Mervaala, E.; Karppanen, H.; Lapatto, R. Down-regulation of renal glutathione synthesis by systemic nitric oxide synthesis inhibition in spontaneously hypertensive rats. Biochem. Pharmacol. 2000, 59 (4), 441–3. (99) Tew, K. D. Redox in redux: Emergent roles for glutathione S-transferase P (GSTP) in regulation of cell signaling and Sglutathionylation. Biochem. Pharmacol. 2007, 73 (9), 1257–69.
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Journal of Proteome Research • Vol. 9, No. 5, 2010 2687
