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Chem. Res. Toxicol. 2008, 21, 2289–2299

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In Vitro and in Silico Characterization of Peroxiredoxin 6 Modified by 4-Hydroxynonenal and 4-Oxononenal James R. Roede,† David L. Carbone,‡ Jonathan A. Doorn,§ Oleg V. Kirichenko,† Philip Reigan,† and Dennis R. Petersen*,† Department of Pharmaceutical Sciences, School of Pharmacy, The UniVersity of Colorado Health Sciences Center, 4200 East Ninth AVenue, Campus Box C238, DenVer, Colorado 80262, Department of EnVironmental and Radiological Health Sciences, Colorado State UniVersity, Fort Collins, Colorado 80523, and DiVision of Medicinal and Natural Products, School of Pharmacy, UniVersity of Iowa, Iowa City, Iowa 52242 ReceiVed July 2, 2008

Peroxiredoxin 6 (PRX6) belongs to the 1-Cys class of peroxiredoxins and is recognized as an important antioxidant protein in tissues such as cardiac muscle, skin, and lung. Preliminary in vivo proteomic data have revealed that PRX6 is adducted by 4-hydroxynonenal (4HNE) in the livers of rats chronically fed an ethanol-containing diet. The goals of this study were to evaluate the in vitro effect of aldehyde adduction on PRX6 peroxidase activity, identify specific sites of aldehyde modification using mass spectrometry, and predict conformational changes due to adduction using molecular modeling. PRX6 was found to be resistant to inactivation via aldehyde modification; however, Western blots of adducted protein revealed that both 4HNE and 4-oxononenal (4ONE) caused extensive cross-linking, resulting in high molecular mass species. Tandem mass spectrometry (ESI-LC-MS/MS) analysis demonstrated multiple sites of modification, but adduction of the active site Cys47 was not observed. Molecular modeling simulations indicated that adduction at Cys91 results in a change in protein active site conformation, which potentially restricts access of 4-HNE to Cys47. The Cys91-Lys209 cross-linked adducts could provide the conformational changes required to inactivate the protein by either restricting access to electrophiles or preventing important amino acid interactions within the catalytic triad. Introduction Oxidative stress is a recognized feature of alcoholic liver disease (ALD)1 and is defined as an unbalanced increase of reactive oxygen species (ROS) with an overall pro-oxidant activity exerted on different molecules, which potentially affects molecular structure and function (1, 2). Cellular oxidative stress is prevented or attenuated by the aid of the cell’s antioxidant defenses, which include enzymatic systems, such as superoxide dismutase (SOD), catalase, glutathione peroxidases (GPx), and peroxiredoxins. Nonenzymatic antioxidants include glutathione (GSH) and vitamin E. Peroxiredoxins are a newly identified and expanding class of thiol-specific antioxidant proteins. These proteins are proposed to exert their protective role through peroxidase activity against hydrogen peroxide, peroxynitrite, organic hydroperoxides, and phospholipid hydroperoxides. Presently, peroxiredoxins are divided into two categories, 1-Cys or 2-Cys, based on the number of redox-active Cys residues that they contain. Peroxiredoxins 1-5 belong to the 2-Cys class, while peroxiredoxin 6 (PRX6) is the sole member of the 1-Cys class (3). PRX6 exists as a homodimer with a conserved, redox-active Cys47, which is involved in detoxifying peroxides as illustrated in Figure 1. During catalysis, the peroxide oxidizes the reactive * To whom correspondence should be addressed. Tel: 303-315-6159. Fax: 303-315-6281. E-mail: [email protected]. † The University of Colorado Health Sciences Center. ‡ Colorado State University. § University of Iowa. 1 Abbreviations: PRX6, peroxiredoxin 6; 4HNE, 4-hydroxynonenal; 4ONE, 4-oxononenal; NEM, N-ethylmaleimide; DTT, 1,4-dithiothreitol; ALD, alcoholic liver disease; GPx, glutathione peroxidase.

Figure 1. Reaction mechanism for PRX6. This scheme was adapted from ref 3.

cysteine to yield a cysteine sulfenic acid; however, this sulfenic acid is reduced back to the thiolate form with the aid of a thiolcontaining reductant, such as 1,4-dithiothreitol (DTT) or GSH (3). The identity of the physiological electron donor remains controversial because the proposed GPx-like activity cannot be demonstrated consistently (4-6). PRX6 is a cytosolic protein ubiquitously expressed throughout the body, with high expression in epithelial cells, such as hepatocytes and type II pneumocytes (4, 7). Both free radical generation and lipid peroxidation appear to play an important role in the pathomechanisms implicated in ALD (8). During the peroxidation of polyunsaturated fatty acids (PUFA), reactive aldehydes are generated endogenously. Unlike free radicals, such as hydroxyl radical, these aldehydes are rather long-lived and have the ability to diffuse from their site of origin

10.1021/tx800244u CCC: $40.75  2008 American Chemical Society Published on Web 10/23/2008

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to attack both intracellular and extracellular targets. Major aldehydic end products include 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE). These bifunctional aldehydes have the ability to covalently modify cellular macromolecules such as proteins and DNA (9). 4HNE and 4ONE are believed to react with Cys, His, and Lys residues via Michael addition. The 4HNE Michael adduct has the ability to cyclize to a hemiacetal, potentially limiting 4HNE’s ability to cross-link proteins; however, 4ONE does not cyclize, thereby increasing the likelihood of protein cross-links via a reaction between the carbonyl and a primary amine, forming a Schiff base (9, 10). Our laboratory and other investigators have demonstrated that these reactive aldehydes modify essential protein nucleophilic residues and inactivate or alter the normal function of many proteins such as HSP72, HSP90, ALDH2, and GAPDH (11-14). Recently, our laboratory has found that PRX6 is modified by 4HNE in the livers of ethanol-fed rats; therefore, this study was designed to determine whether aldehyde modification will affect the peroxidase activity of PRX6 and to locate and characterize specific sites of adduction. Recombinant human PRX6 was modified by either 4HNE or 4ONE, and peroxidase activity was assessed. Aldehyde-modified PRX6 was also subjected to a trypsin digest, and adducts were analyzed via tandem mass spectrometry. Also, computational-based molecular modeling simulations were conducted to assess residue accessibility and conformation changes due to aldehyde modification. Our results demonstrate that the peroxidase activity of PRX6 is very resistant to inactivation by 4HNE and 4ONE and that both of these biogenic aldehydes have the ability to cross-link PRX6 via Cys-Lys and Lys-Lys cross-links, resulting in inactivation.

Materials and Methods Reagents. Unless otherwise stated, all reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Purified human recombinant PRX6 protein was purchased from Laboratory Frontier (Seoul, South Korea). Sulfo-NHS-acetate (NHS) was purchased from Pierce (Rockford, IL). 4HNE and 4ONE were synthesized in our laboratory according to procedures described elsewhere (10). Animals. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of Colorado and were performed in accordance with published National Institutes of Health Guidelines. Male Sprague-Dawley rats were fed a modified Lieber-DeCarli diet (Bio-Serv, Frenchtown, NJ) for 60 days that consisted of 45% fat-derived calories, 16% protein-derived calories, and 35% ethanol- or sucrose-derived calories in treated or pair-fed controls, respectively. Upon completion of the feeding regimen, the animals were anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. Blood samples were collected from the inferior vena cava for determination of plasma alanine aminotransferase (ALT) activity using an assay kit from Diagnostic Chemicals Limited (Oxford, CT). Livers were removed and homogenized, and subcellular fractions were prepared as described elsewhere (11). Two-Dimensional Electrophoresis and in Vivo Adduct Detection. Cytosolic protein (300 µg) from ethanol-fed and pairfed control rat livers was loaded onto a first dimension tube gel. The 4.5% acrylamide tube gels containing 9.3 M urea were cast in glass tubes (i.d., 3 mm; o.d., 7 mm; L, 182 mm). The tube gels were focused with a 3-10 pH gradient using the following running conditions: 200 V for 2 h, 500 V for 2 h, and 800 V for 16 h. A 6-15% linear gradient slab gel was used to separate the proteins on the second dimension. Duplicate gels were run simultaneously, and one gel was stained overnight using Coomassie Brilliant Blue (R-250) solution consisting of 0.8% phosphoric acid and 605 mM ammonium sulfate, and the other gel was transferred to a PVDF membrane. The membrane was blocked in 5% nonfat milk (w/v) in tris-buffered saline containing 0.2% tween 20 (TBST). The

Roede et al. membranes were probed with custom primary antibodies (Synpep, Dublin, CA) against 4HNE-modified proteins. The blot was then probed with a horseradish peroxidase-conjugated secondary antibody and developed using enhanced chemiluminescence from GE Healthcare (Piscataway, NJ). The blot was then visualized using a STORM 860 scanner from Molecular Dynamics (Sunnyvale, CA). The Coomassie-stained gel was overlaid over the developed Western blot, and immunopositive spots were carefully matched, excised, and digested, and peptides were analyzed via mass spectrometry as previously described (11). Covalent Modification of PRX6. Modification of PRX6 protein was performed by incubating 7.7 µM PRX6 and concentrations of reactive aldehyde (4HNE or 4ONE) ranging from 0 to 770 µM for 30 min in 50 mM sodium phosphate buffer, pH 7.4, at 37 °C. Following the incubation period, free aldehyde was removed from the sample using Pierce Protein Desalting Columns (Rockford, IL). The removal of residual aldehyde was performed to prevent carryover that might interfere with subsequent assays. For amino acid blocking experiments, PRX6 was preincubated with Nethylmaleimide (NEM) (100-fold molar excess per Cys residue) and NHS (25-fold molar excess per Lys residue) for 1 h at 37 °C. The samples were then desalted and treated with aldehyde as described above. PRX6 Activity Assessment. A GPx activity kit from Cayman Chemical (Ann Arbor, MI) was used to assess the GPx-like activity of PRX6. A rhodanese protection assay, adapted from Kim et al. and Sorbo (15, 16), was also used to examine the peroxidase activity of PRX6. Briefly, PRX6 protein was modified with reactive aldehydes as previously described. Modified PRX6 (0.1 mg/mL) was incubated for 10 min at 37 °C in a solution containing 0.25 mM DTT, 7.5 µM iron(III) chloride, 0.1 mg/mL rhodanese, and 50 mM sodium phosphate, pH 7.4. Following this incubation, an aliquot of this solution was added to the reaction buffer containing 50 mM potassium cyanide, 20 mM Tris-HCl (pH 8.0), and 50 mM sodium thiosulfate and incubated for 10 min at room temperature. The reaction was stopped by the addition of 15% formaldehyde (v/v). A color reagent containing 165 mM iron(III) nitrate and 8.7% nitric acid (v/v) was then added to each stopped reaction, and a color change was observed. The absorbance of each reaction was then measured at a wavelength of 460 nm. Gel Staining and Western Blotting. Aldehyde-modified PRX6 protein samples were separated on a polyacrylamide gel via standard SDS-PAGE procedures. The protein was then transferred to a Hybond-P transfer membrane (GE Healthcare, Piscataway, NJ). The membrane was then blocked in a solution of 5% nonfat milk in TBST. The membrane was then probed with custom primary antibodies against 4-HNE, 4ONE-modified keyhole limpet hemocyanin, or PRX6 (Abcam, Cambridge MA). A horseradish peroxidaseconjugated secondary antibody was then applied. The membrane was developed using ECL-Plus reagent from GE Health Sciences. The chemiluminescence was visualized using a STORM 860 scanner from Molecular Dynamics (Sunnyvale, CA). For the 4HNE and 4ONE blots, the membrane was stripped and reprobed with a primary antibody against PRX6. Duplicate SDS-PAGE gels were run for each Western blot, and these were stained overnight with Coomassie brilliant blue to visualize the protein bands and potential protein cross-linking. LC-MS Analysis of Intact Protein. To analyze intact protein modified by 4-HNE, 25 µL of a 1 mg/mL PRX6 solution was diluted with 25 µL of 50 mM sodium phosphate buffer (pH 7.4), and the resultant solution was incubated with 1 µL of a 50 mM 4-HNE solution in acetonitrile (50× molar excess of HNE) or with 1 µL of 10 mM 4-HNE solution in acetonitrile (10× molar excess of HNE), at 37 °C for 30 min, followed by addition of 1 µL of a 1 M sodium borohydride solution in 0.1 M sodium hydroxide. The resultant solution was incubated at room temperature for 2 h and diluted with 0.01% TFA in water to a final PRX6 concentration of 100 ng/µL. Three microliter aliquots were used for injections on a 15 cm × 0.03 cm Jupiter C4 300 Å column (Phenomenex, Torrance, CA), installed on an Agilent 1100 capillary HPLC (Agilent Technologies, Wilmington, DE), which was interfaced with an

Aldehyde Modification of Peroxiredoxin 6 Agilent 1100 Series LC-MSD Trap mass spectrometer. The column was operated at 5 µL/min flow rate with a mobile phase temperature of 45 °C. The LC gradient used was the following: solvent A, 0.01% TFA in water; solvent B, 0.01% TFA in acetonitrile; isocratic elution at 35% A for 5 min followed by a linear ramp to 95% A in 20 min. MS conditions used comprised the following: spray needle at ground potential, capillary voltage at -4 kV, nebulizer gas pressure at 16 psi, drying gas flow at 6 L/min, drying gas temperature at 350 °C, trap drive at 110, ion current control at 30000, m/z range from 300 to 2200Th, and 10 averages per spectrum. The total ion chromatograms and mass spectra acquired were processed using Bruker Daltonik Data Analysis for LC-MSD Trap software (version 3.2). Total ion chromatograms were smoothed by means of Gauss algorithm (2.935s, 2 cycles), and the final mass spectra, used as an input for deconvolution routine, were obtained by averaging mass scans over the entire chromatographic peak. Deconvolution was performed by means of the related-ion deconvolution algorithm on the mass range from 20 to 30 kDa with the proton as the adduct ion. Algorithm parameters were employed as follows: abundance cutoff, 5%; maximum charge, 50; minimum number of peaks in a compound, 8; maximum number of compounds, 8; envelope cutoff, 75%; and molecular weight agreement, 0.02%. Trypsin Digestion and Tandem Mass Spectrometry. To prepare samples for mass spectrometry, PRX6 protein was treated as described above. Then, 0.1 M sodium borohydride in 0.1 N sodium hydroxide solution was added to the aldehyde reaction to yield a final concentration of 9 mM sodium borohydride and incubated for an additional 60 min at 37 °C. Following the sodium borohydride reduction, the samples were desalted using a Pierce Protein Desalting Column. Desalting was performed to remove the residual reduced aldehyde and sodium borohydride from the samples and prevent carryover to the trypsin digestion. The samples were heat denatured at 90 °C for 5 min and digested using trypsin (Promega, Madison WI). The digestion buffer contained 50 mM ammonium bicarbonate buffer (pH 7.8), 10% acetonitrile, 2 mM β-mercaptoethanol, and trypsin at a ratio of 1:20 (trypsin:PRX6). An Agilent (Palo Alto, CA) 1100 series LC-MSD Trap equipped with a Phenomenex (Torrence, CA) Jupiter 4 µm Proteo column (1 mm × 150 mm, 90 Å) was used to separate and identify peptides. The mobile phase consisted of 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (B) with a flow rate of 50 µL/min and gradient conditions as follows: 5% B at 0 min, 5% B at 5 min, 50% B at 50 min, 90% B at 65 min and held for 5 min, and 5% B at 85 min and held for 5 min. Mass spectrometric detection and analyses were performed using the positive ion mode with a capillary voltage of 4 kV. The nebulizer pressure was set at 20 psi, and the dry gas flow was set at 8 L/min with the dry gas temperature set at 350 °C. The scanning range for all analyses was 400-1800 m/z. MS/MS analysis was accomplished using the Auto MSn feature with fragmentation amplitude set at 1.5 V. The data were exported in the form of Mascot Generic Format (mgf) files, and peptides modified by 4-HNE or 4ONE were identified with the Mascot search program (http://www.matrixscience.com) (17) using the NCBI database and manually verified. A mass shift of +158 Da corresponded to a modification by either 4-HNE or 4ONE after sodium borohydride reduction. Molecular Modeling. Molecular modeling studies were performed on an SGI Octane 2 workstation using the Insight II software (Version 2005; Accelrys, Inc., San Diego, CA). The crystallographic coordinates for the 2.0 Å human peroxiredoxin protein, PDB code 1PRX (18), were obtained from the RCSB Protein Data Bank. The Ser91 residue was replaced with Cys, in both monomers, using the Homology Module. The monoadducted 4HNE and 4ONE species were constructed by attaching the C3 to the thiol of Cys91, and the cross-linked species were formed by attaching C3 to Cys91 and C1 to amino group of Lys209 using the Builder Module. The Cys47 residues in each monomer were modified to represent the anionic form at the beginning of the catalytic cycle. The potentials and charges were assigned using consistent valence forcefield (CVFF) parameters (19), and the ionizable residues were corrected for

Chem. Res. Toxicol., Vol. 21, No. 12, 2008 2291 physiologic pH. The modified structures were then subjected to a constrained minimization using the conjugate gradient method (1000 iterations). Each structure was solvated by a 10 Å layer of water molecules using TIP3P (20). Molecular dynamics simulations consisted of an initial equilibration for 40 ps, followed by 100 ps dynamics at 300 K. Trajectories were collected every 0.1 ps. The lowest potential energy structure was selected and then minimized using the conjugate gradient method (3000 iterations). The final minimized structures were then used for analysis and visualized using DS Visualizer 2.0 (Accelrys, Inc., San Diego, CA). Statistics. Statistics were performed using the software package GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA). Data from representative experiments were pooled, and means were compared using one-way ANOVA with a Tukey post-test. Means were considered to be significantly different if p < 0.05.

Results Ethanol Feeding Results in Liver Injury and an Increase in 4HNE-Protein Adducts. Rats fed a control or ethanolcontaining diet for 60 days displayed hepatic changes, consistent with early stage ALD characterized by significant elevations of plasma ALT activity, increased liver to body weight ratio, and hepatic steatosis, all of which have been previously described in our publications (8, 12). Cytosolic fractions prepared from the livers of control and ethanol-treated rats were subjected to 2D gel electrophoresis and probed with anti-4HNE antibodies as previously described (11, 12). Interestingly, a number of proteins displaying immunopositive staining were observed in samples prepared from control rats (Figure 2A). The in-gel digestion of selected spots and subsequent analysis by LC-MS/MS resulted in identification of the six proteins listed in Table 4, suggesting that certain of these proteins are highaffinity targets for modification by 4HNE generated during the steady-state levels of oxidative stress produced in many tissues characterized by high levels of metabolic and catabolic activity. It is apparent from Figure 2B that ethanol treatment increased the number of proteins modified by 4HNE as well as the degree to which certain proteins also present in control rats were modified. Of special interest are the very densely immunopositive spots designated as 1 and 5 in Figure 2B, which were subsequently identified in Table 1 as PRX6. Covalent Modification of PRX6 by 4HNE and 4ONE and Enzyme Activity. PRX6 is classified as a thiol-specific antioxidant protein proposed to function as a peroxidase in detoxifying a wide range of peroxides. It is hypothesized that PRX6 possesses GPx-like activity; however, after numerous optimization attempts, we were unable to demonstrate any sort of peroxidase activity using the GPx model described elsewhere (6) (data not shown). Therefore, a rhodanese protection assay was used to assess the effect of aldehyde modification on peroxidase activity (15). Briefly, rhodanese is a thiocyanate: cyanide sulfurtransferase that is known to be inactivated due to hydrogen peroxide-mediated oxidation of essential active site cysteine residues (15, 21). A thiol/Fe3+/O2 mixed function oxidase system was used to generate the hydrogen peroxide needed to inactivate rhodanese. In the presence of PRX6, rhodanese is protected from inactivation due to the peroxidase activity of PRX6. Thus, the effect of 4HNE or 4ONE modification on the peroxidase activity was assessed using this protection assay. As presented in Figure 3A, the modification of PRX6 by concentrations of 4HNE up to 100 M excess resulted in a concentration-dependent decrease in peroxidase activity. The IC50 for this inhibitory effect of 4HNE was calculated to be approximately 346 µM, a concentration that rarely occurs in vivo, suggesting that PRX6 is relatively insensitive to inactiva-

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Figure 2. PRX6 is adducted by 4HNE in the ethanol-treated rat. (A) Control and (B) ethanol. Note: PRX6 was identified in spots 2 and 5.

Table 1. Proteins Identified from 4HNE Immunopositive Spots from the Control Group spot

proteins

scorea

peptidesb

1

fumarylacetoacetase 4-hydroxyphenylpyruvate alloantigen F fumarylacetoacetase phosphoglucomutase liver regeneration related protein PRX6 dihydrofolate reductase GST Yb-2 actin-β actin-R1

454 235 251 127 260 254 505 162 471 526 261

13 7 8 3 5 8 17 6 16 18 9

3 4 5 7 11

a Highest MOWSE score calculated by Mascot. peptides identified.

b

Highest number of

Table 2. Proteins Identified from 4HNE Immunopositive Spots from the Ethanol-Fed Group spot

proteins

scorea

peptidesb

1

fumarylacetoacetase 4-hydroxyphenylpyruvate apolipoprotein A-I precursor PRX6 alloantigen F fumarylacetoacetase phosphoglucomutase liver regeneration related protein PRX6 catechol O-methyltransferase dihydrofolate reductase GST Yb-2 GST Yb-2 R-enolase actin-β actin-R1

454 235 561 326 251 127 260 254 505 61 162 471 493 536 526 261

13 7 16 11 8 3 5 8 17 4 6 16 16 15 18 9

2 3 4 5 6 7 8 9 11

a Highest MOWSE score calculated by Mascot. peptides identified.

b

Highest number of

tion by this aldehydic product of lipid peroxidation. Likewise, on the basis of the data presented in Figure 3B, the inhibitory effect of 4ONE was also found to be quite meager (Figure 3B), corresponding to a calculated IC50 value 339 µM. Whereas no reports of steady-state concentrations of 4ONE have been reported, it is unlikely that this level of 4ONE would occur in vivo. Cys47 is the conserved Cys residue in PRX6 that is essential for peroxidase activity (4); therefore, NEM, a selective Cys modifier, was used in an effort to assess the reactivity and accessibility of this residue to electrophiles. In addition to the

NEM experiments, lysine residues were also blocked using NHS. It is apparent from Figure 3C,D that preincubations with either Cys or Lys modifiers did not decrease the peroxidase activity of PRX6 below that present in control incubations, suggesting that Cys47 is protected against modification by NEM and that lysine modification alone does not alter the peroxidase activity of PRX6. 4HNE- and 4ONE-Mediated Cross-Linking. 4HNE and 4ONE are highly reactive, bifunctional aldehydes, which readily form Michael addition products with nucleophilic amino acids but can also undergo Schiff base formation with primary amines (22). To assess the ability of 4HNE and 4ONE to cross-link PRX6, the recombinant protein was incubated with increasing concentrations of aldehyde. Western blots using antisera directed against either 4HNE or 4ONE epitopes were performed to demonstrate adduction and to visualize cross-links. It is apparent from Figure 4 that pretreatment with 1-100-fold molar excess of 4HNE (Figure 4A) or 4ONE (Figure 4B) resulted in detectable, concentration-dependent cross-linking of PRX6 (indicated by the arrows). With respect to 4HNE pretreatment, high molecular mass bands, indicative of the PRX6 dimer, begin to appear at 38.5 µM 4HNE, or 5×, which intensified as the concentration of 4HNE approaches 50-fold molar excess equivalent to 385 µM and which increased further in samples exposed to 100× 4HNE. Higher molecular mass bands greater than 85 kDa became apparent following preincubations with 4HNE at concentrations greater than 385 µM. It is noteworthy that the extent of cross-linking resulting from 4HNE pretreatment is consistent with the loss of peroxidase activity presented in Figure 3, suggesting that formation of the dimers or higher molecular mass complexes is associated with a loss of PRX6 activity. The data presented in Figure 4B indicate that 4ONE is a more potent cross-linker as compared to 4HNE. High molecular mass bands (bands greater than 85 kDa) appeared at concentrations as low as 38.5 µM when 4ONE was added to the preincubations. As 4ONE concentrations were increased to 770 µM (100× molar excess), the presence of high molecular mass bands further increased in intensity along with the “dimer” bands. Comparison of the cross-linking data in Figure 4B with the loss of peroxidase activity presented in Figure 3B indicates that the progressive cross-linking of PRX6 correlates with a corresponding loss in peroxidase activity.

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Figure 3. Effect of (A) 4HNE, (B) 4ONE, (C) NEM, and (D) NHS on the peroxidase activity of PRX6 using a rhodanese protection assay (N ) 6, (SEM; **p < 0.001, *p < 0.05).

Protein cross-links can be attributed to Cys-Lys and/or LysLys intra- or interpeptide cross-links. To confirm the participation of Cys and Lys residues in the cross-links, PRX6 protein was pretreated with both a cysteine blocker, NEM, and an agent to block lysine residues, NHS. After this pretreatment, the samples were incubated in various concentrations of 4HNE or 4ONE. It is apparent from Figure 4C,D that pretreatments with these side chain-modifying agents markedly inhibited formation of PRX6 cross-links, even in the presence of 100× molar excess of 4HNE or 4ONE, an effect that was confirmed by immunoblots using anti-4HNE, anti-4ONE, or anti-PRX6 antibodies. LC-MS Analysis of Intact PRX6 Modified by 4HNE. On the basis of the data presented in Figure 4, there appear to be multiple sites of adduction for 4HNE and 4ONE. To further evaluate this, we performed LC-MS analysis to observe the addition of multiple aldehyde molecules to intact PRX6 protein. Only 4HNE was analyzed due to the fact that both 4HNE and 4ONE have similar reactivity and modify the same nucleophilic amino acids (10). LC-MS analysis of unadducted PRX6 and two 4HNE treated samples (10× and 50× molar excess of 4HNE) generated the total ion chromatograms displayed in Figure 5 (see the Materials and Methods). Superimposition of these chromatograms shows clearly that there is no peak retention time shift due to the presence of 4HNE-adducted protein species in the reaction mixtures under the conditions of the LC gradient employed. The unadducted PRX6 trace and both treated sample traces have similar peak shapes with no additional peaks detected in the adduction reactions. Neither change of the gradient slope nor variation of the mobile phase temperature resulted in separation of adducted and unadducted protein components. Denaturation of the protein species present in the reaction mixtures likewise failed to produce any change in the chromatogram appearance (data not shown). Mass spectra displayed in Figure 6A,C,E represent averaging of the scans taken across the chromatographic peak detected in the case of the unadducted, 10× 4HNE, and 50× 4HNE samples accordingly. These spectra were deconvoluted to produce the corresponding shifted spectra (Figure 6B,D,F). From comparison

of the latter, it is evident that the alkylation reaction generates adducted protein species with a mass shift equal to the mass of one 4HNE moiety with the aldehyde group being reduced to alcohol (158 Da). Unmodified PRX6 protein yielded only one major peak at an average mass of 24937 Da. In the 10× sample, we observed a major peak at 25097 Da, corresponding to a single 4HNE molecule adduction and a minor peak at 24940 Da that corresponded to the unadducted protein. The 50× treatment also yielded a single adduct peak at 25097 Da and several smaller unknown peaks. It is clear that the adduction reaction did not proceed to completion when 10× molar excess of 4HNE was employed, whereas 50× molar excess of 4HNE resulted in complete consumption of nonadducted PRX6. A reduction step during the sample preparation procedure appears to be crucial for the stabilization of the 4HNE-adducted protein species formed in the alkylation reaction. When this step was omitted, LC-MS analysis of 50× molar excess of HNE reaction sample showed the presence of a significant amount of nonadducted PRX6 (data not shown). This observation coincides with the notion that nonreduced 4HNE-protein adducts are sensitive to decomposition in the mass spectrometer interface (22). ESI-LC-MS/MS Identification of 4HNE and 4ONE Adducts. Tandem mass spectrometry analysis of PRX6 tryptic digests was employed to identify specific amino acid residues that were covalently modified by 4HNE and 4ONE in concentrations of 10× to 100× molar excess. Chemical reduction with sodium borohydride was employed to reduce and stabilize the adducts for subsequent detection with LC-MS/MS, resulting in a mass shift for both reduced 4HNE and reduced 4ONE of 158 Da. A summary of the 4HNE adducted peptides identified is presented in Table 2. Independent of the concentration of 4HNE used in the preincubations, the only target residue for modification was Cys91. As noted in Table 3, Cys91 was also a target for 4ONE modification as was Lys209 present in residues 205-215. The Cys91 and Lys209 modifications were observed when PRX6 was preincubated with 1× to 50× molar excess of 4ONE. Reactivity of Cys91 to Michael addition was confirmed

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Figure 4. Aldehyde-mediated cross-linking. PRX6 was treated with increasing concentrations of aldehyde, and aldehyde-mediated cross-linking was visualized using antibodies against 4HNE (A) or 4ONE (B). Aldehyde-mediated cross-links were blocked by pretreating PRX6 with NEM and NHS and then incubating with 4HNE or 4ONE. Adduct blocking was visualized with antibodies against 4HNE (C) or 4ONE (D). Cross-linking was confirmed by reprobing the blots with an antibody for PRX6 (C and D, bottom panels). High molecular mass bands are indicated by the arrows.

Figure 5. Total ion chromatogram of intact untreated PRX6 protein (green) or PRX6 treated with 10× (red) or 50× (blue) 4HNE.

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Figure 6. Total charge envelope for unmodified PRX6 protein (A), 10× 4HNE (C), and 50× 4HNE (E). Also included are the deconvoluted spectra for unmodified (B) PRX6 protein, 10× 4HNE (D), and 50× 4HNE (F). Note that the mass of the unmodified protein was observed to be approximately 24937 Da, and the addition of a single molecule of 4HNE would result in a mass shift of 158 Da (25097).

Table 3. 4HNE-Adducted Peptides Found Using LC-MS/MS position

peptide

scorec

% coveraged

RT (min)

[MH]+ calcd

[MH]+ obsd

85-97 85-97 85-97 85-97 85-97 85-97 85-97 85-97 85-97

DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK

697 466 626 724 565 731 806 532 518

80 61 65 81 67 73 79 67 59

22.4 23.7 22.5 22.5 23.5 22.7 22.3 23.6 23.7

1682.7709 1682.7709 1682.7709 1682.7709 1682.7709 1682.7709 1682.7709 1682.7709 1682.7709

1683.1654 1682.6654 1683.1054 1683.1854 1683.6454 1683.1654 1683.1454 1682.6454 1682.5654

a

d

b

adduct Cys Cys Cys Cys Cys Cys Cys Cys Cys

91 91 91 91 91 91 91 91 91

molar excess

[4HNE] (µM)e

10× 10× 10× 25× 25× 25× 50× 50× 100×

77 77 77 192.5 192.5 192.5 385 385 770

a Position of the residue in protein. b Peptide sequence. Note the modified residue in bold font. c Probability-based Mowse score generated by Mascot. % of protein covered and identified. e Treatement with 4HNE that yielded the specific adduct.

Table 4. 4ONE-Adducted Peptides Found Using LC-MS/MS

d

positiona

peptideb

scorec

% coveraged

RT (min)

[MH]+ calcd

[MH]+ obsd

adduct

molar excess

[4ONE] (µM)e

85-97 205-215 205-215 85-97 85-97 205-215 85-97 85-97 85-97 205.215 205-215

DINAYNCEEPTEK GVFTKELPSGK GVFTKELPSGK DINAYNCEEPTEK DINAYNCEEPTEK GVFTKELPSGK DINAYNCEEPTEK DINAYNCEEPTEK DINAYNCEEPTEK GVFTKELPSGK GVFTKELPSGK

487 581 554 539 629 629 424 591 829 829 591

62 68 66 60 68 68 59 76 84 84 76

23.7 28.2 28.3 24.5 24.6 28.5 24.6 24.4 24.6 28.6 28.3

1682.7709 1315.7387 1315.7387 1682.7709 1682.7709 1315.7387 1682.7709 1682.7709 1682.7709 1315.7387 1315.7387

1682.9454 1315.6454 1315.8454 1682.7454 1682.1454 1315.2054 1682.8054 1682.9054 1682.0854 1315.1454 1315.5654

Cys 91 Lys 209 Lys 209 Cys 91 Cys 91 Lys 209 Cys 91 Cys 91 Cys 91 Lys 209 Lys 209

1× 10× 25× 25× 25× 25× 50× 50× 50× 50× 50×

7.7 77 192.5 192.5 192.5 192.5 385 385 385 385 385

a Position of the residue in protein. b Peptide sequence. Note the modified residue in bold font. c Probability-based Mowse score generated by Mascot. % of protein covered and identified. e Treatement with 4ONE that yielded the specific adduct.

by modifying PRX6 with NEM and analyzing the adducts by LC-MS/MS (data not shown). Modification of Cys47 was not detected in any of the samples treated with 4HNE, 4ONE, or NEM. Histidine adducts were also not detected in either 4HNEor 4ONE-treated samples, and lysine adducts were only detected in the 4ONE-treated samples. In Silico Investigation of Aldehyde Modification. The examination of the PRX6 crystal structure revealed that many of the Lys residues are solvent accessible, whereas the His residues are buried within the globular protein structure (Figure

7). Furthermore, the Cys91 residues in both monomers of PRX6 appear to be excellent targets for 4HNE or 4ONE adduction, due to their location on the periphery of the protein. The results from our mass spectrometry analyses were applied to molecular modeling simulations, to investigate any conformational changes in the protein structure. In the first set of simulations 4HNE or 4ONE were covalently bound to Cys91, and distortions in the tertiary structure were observed postminimization (Figure 8). The distance between the R-carbons of the four amino acids, Val127, Met129, and Ala151 from one monomer

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Figure 7. Ribbon representation of the human PRX6 homodimer. The colors red and yellow are used to designate separate monomer subunits. The nucleophilic amino acid targets of 4HNE, Lys (colored blue), His (colored purple), and Cys (carbon atoms colored green) are shown in stick display style. 4HNE (colored by atom type) is shown in stick display style cross-linked between Cys91 and Lys209. The figures were constructed using Discovery Studio Visualizer 2.0 (Accelrys, Inc., San Diego, CA).

and Pro191 from the other monomer, positioned at the entrance of the PRX6 active site, were measured (Figure 8). From these modeling simulations, it is apparent that mono adduct formation has a significant impact on the distance between these four amino acid residues and, therefore, the conformation of the active site. The Michael addition of 4HNE and 4ONE to Cys91 resulted in shorter distances between the R-carbon atoms of Ala151 and Pro191 of 1.48 and 1.44 Å, respectively, while the distances between Met127 and Val129, Met127 and Pro191, and Val129 and Ala151 increased (Figure 8B,D,H,J), as compared with the corresponding distances in the unadducted protein (Figure 8A,G). In the 4HNE and 4ONE monoadducted species, the side chain residues of both Ala151 and Pro191 appear to move to restrict access to Cys47, and the side chain Met127 is orientated away from Cys47, altering the conformation of the active site entrance. The cyclic hemiacetal adduct of 4HNE was also examined (Figure 8E,K) and resulted in similar changes in protein conformation as the open chain monoadduct. The effect of the intermolecular cross-link between Cys91 of one monomer and Lys209 of the other monomer of PRX6 on the conformation of the active site was investigated. The crosslink was formed by Michael addition between the C3 of 4HNE or 4ONE and Cys91 and a Schiff base between the C1 aldehyde of 4HNE and 4ONE and Lys209 (Figure 7). The intermolecular cross-links formed by these R,β-unsaturated aldehydes resulted in significant distortions in the active site conformation of PRX6. The distance between Ala157 and Pro191 decreased by 1.76 and 1.54 Å, and Met127 and Pro191 decreased by 1.03 and 0.72 Å in the 4HNE and 4ONE cross-linked adducts, respectively (Figure 8C,E,I,L). As in the simulations with the monoadducts, the side chain residues of Ala157 and Pro191 moved to restrict access to Cys47; however, the movement of the Met127 side chain, away from Cys47, was more pronounced in the cross-linked adducts. In summary, the results from these modeling simulations suggest that the binding of 4HNE or 4ONE to Cys91 appreciably alters the conformation of the active site of PRX6. These

differences in conformation due to adduction also resulted in changes in the solvent accessible surface in both the mono- and the cross-linked adducted species. Therefore, these data support that adduction of PRX at Cys91 would result in a substantial change in the active site conformation, which may impact substrate accessibility.

Discussion Oxidative stress is implicated in the initiation and/or progression of many disease states, including ALD (1, 2). During the process of hepatic ethanol metabolism, ROS can be produced by CYP2E1 as a consequence of the oxidation of ethanol to acetaldehyde, mitochondrial dysfunction, and Kupffer cell activation (23-25). Free radical generation, specifically ROS, leads to lipid peroxidation and subsequent production of R,βunsaturated aldehydes, such as 4HNE and 4ONE. These aldehydes differ from ROS in that they have the potential to diffuse from their site of origin and modify intracellular and extracellular targets (9), potentially contributing to some of the cytotoxicity associated with oxidative stress. PRX6 has been shown to be an important antioxidant protein in other models of oxidative stress, such as lung damage due to hyperoxia (26) and cardiac ischemia reperfusion injury (27), and 4HNE has been shown to alter the normal function of many proteins (11-14). In this context, 4HNE-protein adducts have been detected in the livers of alcoholics and rats chronically treated with ethanol (28, 29). Using proteomic techniques, we report here that PRX6 is a target for 4HNE modification in a rat model of oxidative stress induced by chronic alcohol ingestion. The rhodanese protection assay was used to assess the peroxidase activity of recombinant human PRX6 indirectly. This specific assay takes advantage of the rhodanese enzyme, which is extremely sensitive to oxidation of a critical cysteine residue by hydrogen peroxide. PRX6 peroxide scavenging activity efficiently removes peroxides, indirectly protecting rhodanese

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Figure 8. Changes in the PRX6 active site conformation due to aldehyde modification. Solvent accessible surface representations of (A) PRX6, (B) 4ONE Cys91 Michael adduct, (C) 4ONE Cys91-Lys209 cross-link, (D) 4HNE Cys91 Michael adduct (open chain), (E) 4HNE Cys91 Michael adduct (cyclic hemiacetal), and (F) 4HNE Cys91-Lys209 cross-link. The residues Ala151, Val129, Met127, and Pro191 (colored by atom type) and Cys47 (carbon atoms colored green) are shown in stick display style. The distance (Å) between the R-carbons of Ala151, Val129, Met127, and Pro191 are shown for (G) PRX6, (H) 4ONE Cys91 Michael adduct, (I) 4ONE Cys91-Lys209 cross-link, (J) 4HNE Cys91 Michael adduct (open chain), (K) 4HNE Cys91 Michael adduct (cyclic hemiacetal), and (L) 4HNE Cys91-Lys209 cross-link. The figures were constructed using Discovery Studio Visualizer 2.0 (Accelrys, Inc., San Diego, CA).

against inactivation due to overoxidation of the critical cysteine residue. The results presented here, employing a range of concentrations of 4HNE or 4ONE in this protection assay, revealed that the peroxidase activity of this antioxidant protein is rather resistant to inactivation by these two aldehydes, which are documented to readily target cysteine residues through Michael addition reactions. The experimental results presented in Figure 3 demonstrate that PRX6 was equally resistant to the thiol-reactive reagent, NEM, and lysine-specific reagent, NHS. These results suggest that Cys47 is not readily modified by these Michael acceptors, and the inactivation observed at higher concentrations of 4HNE and 4ONE occurs through other mechanisms. Mean liver concentrations of 4HNE during oxidative stress have been reported to range from 8 to 12 µM (30, 31), and local concentrations could reach as high as 100 mM in peroxidizing membranes (32). Also, the IC50 for both of these aldehydes (346 and 339 µM for 4HNE and 4ONE, respectively) was well out of the range of physiological possibility. However, data presented in Figure 4 demonstrate that when present at relatively low concentrations, 4HNE and 4ONE have the ability to cross-link PRX6. These cross-links resulted in the generation of high molecular mass species; however, 4ONE appears to be a more potent cross-linker as compared to 4HNE. This difference

in potency of cross-linking can be attributed to the additional carbonyl group of 4ONE and the fact that 4ONE does not rearrange after Michael addition like 4HNE to form a cyclic hemiacetal; therefore, the ketone or aldehyde is available to participate in formation of a Schiff base once the Michael addition reaction has occurred (10, 22). Mass spectral data presented in Tables 2 and 3, along with immunoblot data in Figure 4, demonstrated that PRX6 is a target protein for both 4HNE and 4ONE. This 224 amino acid protein contains many nucleophilic residues that can be modified. Specifically, there are 18 Lys, three His, and two Cys residues contained on each monomer. LC-MS analysis of 4HNE modified PRX6 has confirmed that PRX6 can be adducted; however, only a single 4HNE molecule adduct was observed, not multiple adducts as one would predict due to the high number of nucleophilic amino acids present. Tandem mass spectrometry results did reveal multiple sites of modification, but analysis of the aldehyde modification did not reveal adduct formation at Cys47. Our results are consistent with a previous report that HEK293 cells exposed to thiol-reactive electrophiles, one of which reacts in the same manner as 4HNE and 4ONE, did not result in modification of Cys47 but did observe a modification of the peripheral cysteine, Cys91, of PRX6 (33). As mentioned previously, Cys47 is essential for peroxidase activity. X-ray

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crystallography assessments of PRX6 suggest that several positively charged residues in the active site, including His39, Arg132, and Arg155, participate in lowering the pKa of Cys47 by stabilizing its ionized state. The ionization increases the nucleophilicity of this cysteine (18). However, the increased nucleophilicity did not result in alkylation by 4HNE, 4ONE, NEM, or other thiol-reactive electrophiles (33). One possible explanation for this phenomenon is that the microenvironment of the active site pocket does not allow for aldehyde or NEM modification and/or local unfolding might be required. Another observed protein adduct was located on Lys209, a modification that was observed only in the 4ONE-treated samples. Lysine adducts tend to be transient and difficult to detect via LC-MS/MS (22). As presented in Figure 8, modification of this site potentially plays a key role in PRX6 crosslinking. Given the close proximity between Cys91 and Lys209, formation of a Michael addition product with Cys91 and subsequent formation of a Schiff base product through reaction of the carbonyl group with Lys209 would be expected to form an intermolecular cross-link. The formation of this cross-link would be consistent with data presented in Figure 4, which demonstrates that modification of Cys91 with NEM or the modification of Lys ε- amino groups with NHS efficiently blocks PRX6 cross-linking by 4HNE or 4ONE. Although a mechanism explaining how cross-linking would impair enzymatic activity of PRX6 remains to be established, the results of our molecular modeling studies, presented in Figure 8, suggest that the crosslinks formed by 4HNE and 4ONE, respectively, would have a significant impact on the active site geometry and impair substrate accessibility to the active site of PRX6. These particular cross-links could also impair the interactions of PRX6 with other proteins. In this context, it has been reported that PRX6 can heterodimerize with glutathione S-transferase pi (GST-π). This association is proposed to allow GST-π active involvement in the GSH-mediated reduction of the cysteine sulfenic acid after peroxide detoxification (34, 35). If intermolecular cross-links were present, this interaction would be prevented and render PRX6 inactive. Western blot data demonstrated significant aldehyde modification of PRX6; however, only Cys91 and Lys209 were consistently detected via LC-MS/MS, suggesting that additional modifications were present but not detected. Adducts were detected only when the samples were subjected to sodium borohydride reduction, and this leads us to believe that the aldehyde modifications of PRX6 are not completely stable. Of the 23 nucleophilic residues contained within the amino acid sequence of PRX6 with which both 4HNE and 4ONE will react, 18 of these are Lys residues. 4HNE and 4ONE can form stable adducts with both Cys and His residues without the aid of chemical reduction; however, Lys adducts are transient (10, 22). Michael addition of 4HNE or 4ONE to lysine is a reversible reaction, and it is widely accepted that Schiff bases are also reversible. Taken together, these observations suggest that PRX6 might be modified at many Lys residues, and these transient adducts allow this abundant hepatic protein to act as an aldehyde sink within the cytosol, protecting other more sensitive proteins from inactivation. Because of the fact that oxidative stress is one of the many factors contributing to ALD and that PRX6 has been shown to be a target for reactive aldehydes in vitro and in vivo, our laboratory will further investigate the actual role of PRX6 in the liver with regard to the mechanism of initiation and progression of ALD.

Roede et al.

Acknowledgment. This work was supported in part by Grants NIH/NIAAA RO1AA09300 and NIH/NIEHS RO1ES09410 (D.R.P.), NIH/NIEHS F32 ES11937 (J.A.D.), NIH/NIAAA F31 AA014308 (D.L.C.), and NIH/NIAAA F31 AA016710 (J.R.R.).

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