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Protein N-Acylation: H2O2-Mediated Covalent Modification of Protein by Lipid Peroxidation-Derived Saturated Aldehydes Kousuke Ishino,† Takahiro Shibata,† Takeshi Ishii,‡ Yu-Ting Liu,§ Shinya Toyokuni,§ Xiaochun Zhu,| Lawrence M. Sayre,*,| and Koji Uchida*,† Graduate School of Bioagricultural Sciences, Nagoya UniVersity, Nagoya 464-8601, Japan, School of Food and Nutritional Sciences, UniVersity of Shizuoka, Shizuoka 422-8526, Japan, Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto UniVersity, Kyoto 606-8501, Japan, and Department of Chemistry, Case Western ReserVe UniVersity, CleVeland, Ohio 44106 ReceiVed February 28, 2008
Various lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the degenerated proteins have been found in cells and tissues during aging, oxidative stress, and in a variety of pathological states. The critical agents that give rise to this protein degeneration may be represented by aldehydes. Although the covalent modification of proteins by aldehydes alone has been well-studied, the effect of reactive oxygen species, such as H2O2, upon aldehyde modification of the protein has received little attention. We have now established a unique protein modification in which H2O2 and, to a lesser extent, alkyl hydroperoxides mediate the binding of alkanals to the lysine residues of protein to generate structurally unusual N-acylation products. Upon the reaction of a lysinecontaining peptide, NR-benzoylglycyl-lysine, with hexanal in the presence of H2O2, a product containing one molecule of hexanal per peptide was detected. On the basis of the chemical and spectroscopic evidence, the product was identified to be the acylation product, N-hexanoyllysine. H2O2 mediated the N-acylation of the lysine derivative by the saturated aldehydes of 1-6 carbons in length. The H2O2-mediated acylation of the protein was immunochemically confirmed by reaction of the proteins with hexanal in the presence of H2O2. Furthermore, the enhanced N-acylations (N-acetylation and N-hexanoylation) were also observed in the kidney of rats exposed to ferric nitrilotriacetate, a well-characterized inducer of oxidative stress. Mechanistic studies using a phosphonium lysine derivative suggest a Baeyer-Villiger-like reaction proceeding through peroxide addition to the aldehyde Schiff base. These data suggest that the hydroperoxides, including H2O2, might be involved not only in the oxidative modification of protein but also in the covalent binding of the saturated aldehydes to proteins under oxidative stress. Introduction It is estimated that most of the proteins in the human body are post-translationally modified. Such modifications include phosphorylation, methylation, glucosylation, etc. They are enzyme-mediated and homeostatically important, either to carry out a particular structural or functional role or to allow the efficient recycling of the amino acid constituents. Several lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the modified proteins have been found in cells during aging, oxidative stress, and in various pathological states including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis (1–4). It has also been suggested that many of the effects of cellular dysfunction under oxidative stress are mediated by the products of the nonenzymatic reactions, such as the peroxidative degradation of polyunsaturated fatty acids (5, 6). Lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them are a variety of different aldehydes. The primary products of lipid peroxidation, lipid hydroperoxides, can undergo * To whom correspondence should be addressed. E-mail: uchidak@ agr.nagoya-u.ac.jp or
[email protected]. † Nagoya University. ‡ University of Shizuoka. § Kyoto University. | Case Western Reserve University.
carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals giving rise to the formation of short-chain, unesterified aldehydes of 3-9 carbons in length and a second class of aldehydes still esterified to the parent lipid (7). The important agents that give rise to the modification of a protein may be represented by reactive aldehydic intermediates, such as alkanals, ketoaldehydes, 2-alkenals, and 4-hydroxy2-alkenals (3, 5, 7). These reactive aldehydes are considered important mediators of cell damage due to their ability to covalently modify biomolecules, which can disrupt important cellular functions and can cause mutations (7). Furthermore, the adduction of aldehydes to apolipoprotein B in low-density lipoproteins (LDL) has been strongly implicated in the mechanism by which LDL is converted to an atherogenic form that is taken up by macrophages, leading to the formation of foam cells (8, 9). Among the variety of lipid peroxidation-derived aldehydes, the saturated aldehydes, such as hexanal, are most abundantly formed (10, 11). Upon reaction with proteins, these aldehydes react with lysine residues to form an imine or Schiff base adduct (12). However, because of the reversible nature of such unconjugated Schiff bases, these aldehydes have received relatively little attention as the causative agent for modification of nucleophilic biomolecules. In the present study, we established a novel mechanism of irreversible covalent protein
10.1021/tx800080x CCC: $40.75 2008 American Chemical Society Published on Web 05/31/2008
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Figure 1. Reaction of a lysine-containing peptide with hexanal in the presence of H2O2. (A) HPLC profiles of NR-benzoylglycyl-lysine incubated with hexanal in the presence of H2O2. The reaction mixture, containing50 mM NR-benzoylglycyl-lysine, 50 mM hexanal, and 50 mM H2O2 in 50 mM sodium phosphate buffer (pH 7.2), was incubated for 24 h at 37 °C. HPLC was performed using a Develosil ODS-HG-5 column (4.6 mm × 250 mm) equilibrated in a solution of 100% aqueous to 100% acetonitrile in 0.01% trifluoroacetic acid at a flow rate of 0.8 mL/min. The elution profiles were monitored by absorbance at 235 nm. (B) Mass spectrum of the main product a generated from the reaction of NR-benzoylglycyl-lysine with H2O2/hexanal.
modification by aldehydes, in which hydrogen peroxide (H2O2)1 and alkyl hydroperoxides mediate the binding of saturated aldehydes to the lysine residues of protein to generate structurally unusual acylation products.
Materials and Methods Meterials. Human serum albumin (HSA) (fatty acid-free), NRbenzoylglycyl-lysine, tert-butyl hydroperoxide (70% aqueous solution), cumene hydroperoxide (80%), and 3-chloroperoxybenzoic acid were obtained from Sigma-Aldrich (St. Louis, MO). Hexanoic anhydride was purchased from Acros. HEPES and L-ascorbic acid were obtained from Fisher Scientific. Sequence grade modified trypsin was purchased from Promega Co., Ltd. Dithiothreitol (DTT) 1 Abbreviations: BSA, bovine serum albumin; Fe3+-NTA, ferric nitrilotriacetate; H2O2, hydrogen peroxide; mAb, monoclonal antibody; MALDITOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Ishino et al. was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). H2O2 (31%, W/V) was obtained from Mitsubishi Gas Co., Ltd. R-Cyano-4-hydroxycinnamic acid was purchased from Bruker Daltonics Japan (Tokyo, Japan). The anti-N-acetyllysine monoclonal antibody (mAb) 4G12 was obtained from Upstate Co., Ltd. The anti-N-hexanoyllysine mAb 5H4 was kindly provided by NOF Co., Ltd. The synthesis of [4-(5-amino-1-carboxypentylcarbamoyl)benzyl]triphenylphosphonium bromide hydrobromide (PLys) will be described elsewhere. General Procedures. The nuclear magnetic resonance (NMR) spectra were recorded using Bruker AMX400 or Varian Inova (400 MHz) instruments. The ultraviolet absorption spectra were measured by a Hitachi U-Best-50 spectrophotometer. Liquid chromatographymass spectrometry (LC-MS) was conducted using a Jasco PlatformII-LC instrument. The LC-MS analysis was performed using a Develosil ODS-HG-5 column (4.6 mm × 250 mm) eluted with linear gradient from 100% water containing 0.1% acetic acid to 100% acetonitrile containing 0.1% acetic acid for 30 min at a flow rate of 0.8 mL/min. The elution profiles were monitored by absorbance at 235 nm. Reaction of Nr-Benzoylglycyl-lysine with Aldehydes in the Presence and Absence of H2O2. NR-Benzoylglycyl-lysine (50 mM) was incubated with 50 mM aldehydes in the presence and absence of H2O2 (50 mM) in 50 mM sodium phosphate buffer (pH 7.2) at 37 °C. The reaction mixtures were analyzed with a reverse-phase HPLC on Develosil ODS-HG-5 column (4.6 mm × 250 mm) (Nomura Chemicals, Aichi, Japan) eluted with a linear gradient from 100% water containing 0.01% TFA to 100% acetonitrile containing 0.1% acetic acid for 30 min. The elution profiles were monitored by absorbance at 235 nm. The NMR data of NRbenzoylglycyl-lysine and the major product (peak a) are provided as Supporting Information (Figures S1-S5). Synthesis of Nr-Benzoylglycyl-NE-hexanoyllysine. The authentic NR-benzoylglycyl-N-hexanoyllysine was prepared as previously reported (13). Synthesis of NE-Hexanoyl-PLys. A solution of PLys (39 mg, 57 µmol) in 8 mL of THF-H2O (1:1,v/v) was basified to pH 10 with 0.2 N aqueous NaOH. A solution of hexanoic anhydride (124 mg, 580 µmol) in 1 mL of THF was added dropwise, keeping the pH 9-10 with 0.2 N aqueous NaOH. At the end, the pH was allowed to come to pH 7.5 over 30 min, and the reaction mixture was acidified to pH 2-3 with 1 N aqueous HBr and then extracted with CH2Cl2 (3 × 20 mL). The CH2Cl2 layer was evaporated, and the residue was dissolved in 3 mL of H2O-CH3CN (2:1, v/v) and purified by a semipreparative HPLC, yielding the product as a white solid (30 mg, 75%). 1H NMR (400 MHz, CDCl3): δ 8.02 (br, 1H), δ 7.77 (br, 3H), δ 7.44-7.70 (12H), δ 7.43 (br, 2H), δ 7.13 (br, 1H), δ 6.80 (br, 2H), δ 4.77 (m, 2H), δ 3.12 (br, 2H), 2.14 (br, 2H), δ 1.78 (br, 2H), δ 1.15-1.60 (10H), δ 0.80 (t, 3H, J ) 6.6 Hz). HRMS (FAB) calcd for C38H44N2O4P+ (M+), 623.3033; found, 623.3036. In Vitro Modification of HSA by H2O2/Hexanal. Modification of the protein by H2O2/hexanal was performed by incubating HSA (1.0 mg/ml) with 10 mM hexanal in the presence and absence of H2O2 (10 mM) in 0.1 M sodium phosphate buffer (pH 7.2) at 37 °C for 24 h. Reaction of PLys with Hexanal in the Presence and Absence of (i) Fe(II) and L-Ascorbic Acid, (ii) H2O2, (iii) tert-Butylhydroperoxide or Cumene Hydroperoxide, or (iv) 3-Chloroperoxybenzoic Acid. A solution of PLys (50 mM, 8 µL) and n-hexanal (20 mM, 20 µL) was added to 100 mM, pH 7.4, HEPES buffer (total volume, 200 µL) either alone or containing (i) L-ascorbic acid (100 mM, 2 µL) and FeSO4·(NH4)2SO4 (50 mM, 2 µL), (ii) H2O2 (4.5 µL of 0.3, 0.015, 0.0015, or 0.00015% aqueous solutions), (iii) tert-butyl hydroperoxide (5.2 µL of 0.7% aqueous solution or cumene hydroperoxide (20 µL of 0.38% in EtOH), or (iv) 3-chloroperoxybenzoic acid (20 µL of 20 mM in EtOH). The solutions were incubated for 2 days at 37 °C for LC-MS analysis. HPLC-ESI-MS/MS. Reversed-phase HPLC was performed on two different systems, in both cases using a 5 µm 4.6 mm × 250 mm Agilent Zorbax SB-C18 column with a binary eluent at a flow
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Figure 2. Proposed structure and 1H-detected HMBC spectrum of the main product generated from the reaction of NR-benzoylglycyl-lysine with H2O2/hexanal. Cross-peaks permit the assignment of the resonances of the partial structure, -CH2-NH-CO-CH2-.
rate of 400 µL/min. Eluent A was 95% H2O, 5% MeOH, and 0.1% formic acid. Eluent B was 95% MeOH, 5% H2O, and 0.1% formic acid. System I utilized a Surveyor MS Pump with the gradient program (70% B for 5 min, 70-100% B over 40 min, 100 to 70% B over 5 min, and 70% B for 5 min), and ESI-MS analysis was performed with a Thermo Finnigan LCQ Advantage mass spectrometer. Two scan events were used as follows: (i) m/z 400-1000 full scan MS and (ii) data-dependent scan MS/MS on the most intense ion from i or from the parent mass list. System II utilized a Surveyor MS Pump Plus with a gradient program (50% B for 10 min, 50-80% B over 35 min, 80 to 50% B over 5 min, and 50% B for 5 min), and ESI-MS analysis was performed with a Thermo Finnigan LCQ DecaXP Max mass spectrometer. One scan event was used as follows: m/z 618.4-628.4 zoom scan MS or m/z 623.4 MS/MS. In both cases, the instruments were set in the positive mode using nitrogen as the sheath and auxiliary gas; the capillary temperature was 300 °C, the capillary voltage was 35.00 V, and the source voltage was 4.50 kV. The MS/MS normalized collision energy was set to 38%. All data were processed with the Qual browser of the Xcalibur. Enzyme-Linked Immunosorbent Assay (ELISA). The noncompetitive ELISA was performed as previously described (14). SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDSPAGE was performed according to Laemmli (15). Immunoblot Analysis. A gel was transblotted onto a nitrocellulose membrane, incubated with Block Ace (40 mg/mL) for blocking, washed, and treated with anti-N-hexanoyllysine mAb
5H4. This procedure was followed by the addition of horseradish peroxidase conjugated to a goat antimouse IgG F(ab′)2 fragment and ECL reagents (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). The bands were visualized using a Cool Saver AE-6955 (ATTO, Tokyo, Japan). Animal Experiments. The ferric nitrilotriacetate (Fe3+-NTA) solution was prepared immediately before use by the method described by Toyokuni et al. (16) with a slight modification. Briefly, ferric nitrate nonahydrate and the nitrilotriacetic acid disodium salt were each dissolved in deionized water to form 80 and 160 mM solutions, respectively. They were mixed at the volume ratio of 1:2 (molar ratio, 1:4), and the pH was adjusted with sodium hydrogen carbonate to 7.4. Male SPF slc: Wistar rats (Shizuoka Laboratory Animal Center, Shizuoka), weighing 130-150 g (6 weeks of age), were used. They were kept in a stainless steel cage and given commercial rat chow (Funabashi F-2, Chiba) as well as deionized water (Millipore Japan, Osaka) ad libitum. The animals were divided into acute and subacute toxicity groups. In the acute toxicity group, the animals received a single intraperitoneal injection of Fe3+-NTA (15 mg Fe/kg body weight). In the subacute toxicity group, the animals received a 3 week treatment with a 5 mg Fe3+NTA/kg body weight daily dose for 3 days followed by a 10 mg Fe/kg body weight daily dose for the next 2 days and 5 times a week for the next 2 weeks as previously described (17). The animals were sacrificed by decapitation, and both kidneys of each animal were immediately removed. In the case of the subacute toxicity experiments, the animals were sacrificed 48 h after the final
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Results
Figure 3. LC-MS analysis of the main product and authentic NRbenzoylglycyl-N-hexanoyllysine. (A) Mass spectra: upper, authentic NR-benzoylglycyl-N-hexanoyllysine; lower, the main product a generated from the reaction of NR-benzoylglycyl-lysine with H2O2/hexanal. (B) Selected ion-current chromatograms obtained from the LC-MS analysis monitored with m/z 406: upper, authentic NR-benzoylglycylN-hexanoyllysine; lower, the main product a generated from the reaction of NR-benzoylglycyl-lysine with H2O2/hexanal.
injection. Both kidneys of each animal were immediately removed. One of them was fixed in Bouin’s solution, embedded in paraffin, cut into 3 µm thick slices, and used for immunohistochemical analyses by an avidin-biotin complex method with alkaline phosphatase. Briefly, after deparaffinization with xylene and ethanol, normal rabbit serum (Dako Japan Co., Ltd., Kyoto; diluted to 1:75) for the inhibition of the nonspecific binding of the secondary antibody, a primary antibody (anti-N-acetyllysine mAb 4G12 or anti-N-hexanoyllysine mAb 5H4), biotin-labeled rabbit antimouse IgG serum (Vector Laboratories; diluted 1:300), and avidin-biotin complex (Vector; diluted 1:100) were sequentially used. Procedures using PBS or the IgG fraction (0.5 mg/ml) of the normal mouse serum instead of a primary antibody showed no or negligible positivity.
Reaction of a Lysine-Containing Peptide with Hexanal in the Presence of H2O2. To characterize the effect of H2O2 on the aldehyde-lysine adduction chemistry, a lysine-containing dipeptide, NR-benzoylglycyl-lysine, was incubated with an equimolar quantity of hexanal, a dominant oxidation product of n-6 polyunsaturated fatty acids, in the presence and absence of H2O2, and the products were characterized by reverse-phase HPLC at various times over 24 h. In the absence of H2O2, the hexanal-lysine Schiff base adduct and/or its precursor carbinolamine were expected to be present at equilibrium, although no product was detected by HPLC. This may be due to the solvent conditions used to perform the separation. In the presence of H2O2, however, the reaction of the lysine derivative with hexanal gave a single product (product a) concomitant with the consumption of hexanal (Figure 1A). The LC-MS analysis showed a 98 Da increase in the mass value of the unmodified lysine derivative and gave the [M + H]+ peak at m/z 406 (Figure 1B), suggesting the formation of a product containing one molecule of hexanal per lysine. To characterize the chemical structure of the product, isolation by HPLC on the reversed-phase column was carried out. After purification, the structure of the product was characterized by NMR analysis (Supporting Information, Figures S1-S5). As compared with the 13C NMR spectra between the NR-benzoylglycyl-lysine and the product, one signal (δC 176.32, S), corresponding to the carbonyl group, newly appeared in the 13C NMR spectrum of the product. The 1H-detected multiple-bond heteronuclear multiple quantum coherence (HMBC) experiment showed the correlations of the -CH2 protons (δH 3.16) and methylene protons (δH 2.15) to the carbonyl carbon (Figure 2), suggesting the binding of hexanal to the -amino group via an amide bond. Thus, the most likely structure was the hexanoyl derivative of the peptide, NR-benzoylglycyl-N-hexanoyllysine (Figure 2). To confirm this structure, we prepared the authentic N-hexanoyllysine by carbodiimide conjugation of NR-benzoylglycyl-lysine with hexanoic acid. The LC-MS analysis revealed that the product was inseparable from the authentic hexanoylated adduct (Figure 3). When 50 mM NR-benzoylglycyl-lysine was incubated with an equimolar quantity of hexanal and H2O2, the yield of the product increased as a function of time and approximately 20% of the lysine derivative was converted to the hexanoyl derivative after 24 h of incubation (Figure 4A). It was also observed that the yield of N-hexanoyllysine was dependent upon the concentration of H2O2 (Figure 4B,C). The data showed that the adduct was detected even when 50 mM NR-benzoylglycyl-lysine was incubated with 50 mM hexanal and 0.1 mM H2O2 (Figure 4B). N-Acylation of Lysine by H2O2/Saturated Aldehydes. The identification of N-hexanoyllysine led us to examine the possibility that aldehydes ubiquitously generate acylation products upon the reaction of primary amines in the presence of H2O2. To evaluate the H2O2-catalyzed acylation of the lysine derivative by other aldehydes, we incubated NR-benzoylglycyllysine with a variety of aldehydes in the presence and absence of H2O2 and monitored the products by LC-MS. Figure 5A shows the chromatograms for the reaction mixture of the lysine derivative with alkanals with 1-6 carbon length in the presence of H2O2 monitored at 235 nm, in which we observed that these aldehydes also reacted with the lysine derivative in the presence of H2O2. The identity of the products was confirmed by the selected ion current chromatograms obtained from the LC-MS analysis (Figure 5B). The data showed that these
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Figure 4. Formation of N-hexanoyllysine upon reaction of the lysine derivative with hexanal in the presence of H2O2. (A) Time-dependent formation of N-hexanoyllysine. The reaction mixture, containing 50 mM NR-benzoylglycyl-lysine, 50 mM hexanal, and 50 mM H2O2 in 50 mM sodium phosphate buffer (pH 7.2), was incubated at 37 °C. (B and C) H2O2-dependent formation of N-hexanoyllysine. (B) Selected ion-current chromatograms obtained from the LC-MS analysis monitored with m/z 406 and (C) the yields of the product (N-hexanoyllysine). In panel B, the arrow represents the peak corresponding to the N-hexanoyllysine adduct. The reaction mixture, containing 50 mM NR-benzoylglycyl-lysine and 50 mM hexanal in 50 mM sodium phosphate buffer (pH 7.2), was incubated with H2O2 (0-10 mM) for 24 h at 37 °C.
alkanals gave pseudomolecular ion peaks, which corresponded to the expected acylation products. Interestingly, the alkyl ketones, such as 2-hexanone and 3-hexanone, did not serve as the substrates for the acylation (data not shown), suggesting that the aldehyde group might be essential for the reaction. In addition, the unsaturated aldehydes, such as ketoaldehydes and glycolaldehyde, failed to generate the acylation products (data not shown). N-Acylation of Protein by H2O2/Hexanal. To examine if the N-acylation by H2O2/aldehydes could occur in a protein, bovine serum albumin (BSA) was incubated with hexanal in the presence and absence of H2O2 and the formation of Nhexanoyllysine in the protein was immunochemically characterized using the mAb 5H4 specific to N-hexanoyllysine (Supporting Information, Figures S6). As shown in Figure 6A (left panel), the ELISA analysis showed that the anti-N-hexanoyllysine antibody had no immunoreactivity to the protein that had been incubated with H2O2 or hexanal alone, whereas the addition of the aldehyde and H2O2 to the reaction mixture resulted in a significant increase in the immunoreactivity with the antibody. The immunoblot analysis also showed the formation of immunoreactive materials in the protein treated with hexanal and H2O2 (Figure 6B). Moreover, we have also observed that the reaction of BSA with H2O2/acetaldehyde resulted in the formation of immunoreactive materials with an anti-N-acetyllysine mAb (data not shown). These data suggested that the lysine residues of the protein could be nonenzymatically acylated by alkanals in the presence of H2O2. Protein N-Acylation in the Kidney of Rats Exposed to Fe3+-NTA. It has been shown that iron overload using Fe3+-
NTA induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage that eventually leads to a high incidence of renal adenocarcinoma in rodents (18, 19). Toyokuni et al. (20) previously determined the production of C2-C12 saturated and unsaturated aldehydes and C7-C12 acetaldehyde-derived acyloins in the kidney of rats exposed to this renal carcinogen and demonstrated that large amounts of saturated aldehydes, including hexanal, were produced. Using the anti-N-acetyllysine mAb 4G12 and anti-N-hexanoyllysine mAb 5H4, the occurrence of the N-acylation in this oxidative stress-related carcinogenesis model was assessed. The kidneys were excised at the time of sacrifice and then fixed with Bouin’s fixative. The hematoxylin and eosin-stained sections of the paraffin-embedded tissues were analyzed for histological damage. The morphological changes in the kidneys of the rats treated with Fe3+-NTA vs time are very similar to previous reports on ddY mice (16, 21). In the control rat kidney, an almost negligible level of immunoreactivity was observed (Figure 6C, panels a and b). The intense immunoreactivities were found in some of the renal proximal tubular cells 3 h after the administration of 15 mg Fe/kg body weight of Fe3+-NTA (Figure 6C, panels b and e). Modest immunoreactivities were still observed at 3 weeks in some of the regenerating cells (Figure 6C, panels c and f). The immunoreactivities were significantly found not only in the cytoplasm but also in some of the nuclei. This pattern of distribution in the rat kidney is consistent with that of the distribution of the membrane lipid peroxidation products and their conjugates with cytosolic proteins (22). Preadsorption of mAb 5H4 by free N-hexanoyllysine abolished the immunostaining (Supporting Information, Figure S7), indicating the
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Figure 5. N-Acylation of lysine by H2O2/saturated aldehydes. The reaction mixture, containing 50 mM NR-benzoylglycyl-lysine, 50 mM saturated aldehydes, and 50 mM H2O2 in 50 mM sodium phosphate buffer (pH 7.2), was incubated for 24 h at 37 °C. (A) HPLC profiles of NR-benzoylglycyl-lysine incubated with C1-C6 saturated aldehydes in the presence of H2O2. (B) Selected ion-current chromatograms obtained from the LC-MS analysis.
specific reactivity of the antibody with the epitope. Moreover, no significant immunoreaction product deposits were seen in sections processed with the omission of the primary antibody (data not shown). Mechanism of N-Acylation. To illuminate mechanistic aspects of the aldehyde-dependent lysine N-acylation, a number of potential oxidant systems were comparatively evaluated using 2 mM reactants and oxidants, and a phosphonium NR-acylated lysine derivative (PLys) that gives enhanced signal sensitivity in MS studies. Reactions were analyzed by HPLC-ESI-MS/MS using the authentic N-hexanoyl derivative of PLys as reference. As shown in Figure 7A, incubation of PLys and hexanal alone or in the presence of Fe(II) and ascorbate (in air) as a typical metal-catalyzed oxidant system did not result in amide formation (traces b and c), whereas amide formation was observed using H2O2 (trace d), tert-butyl hydroperoxide (trace e), cumene hydroperoxide (trace f), and 3-chloroperoxybenzoic acid (not shown). Confirmation of the identity of the product was by MS/ MS (Figure 7B). Estimation of the relative yield of amide product by LC-MS (data not shown) revealed that the both alkyl hydroperoxides effected 15-20% of the amount of hexanoyl derivative as did H2O2. 3-Chloroperoxybenzoic acid was about twice as efficacious as H2O2 but had little physiological relevance. In addition, a separate study was performed to investigate the dependence of H2O2 concentration on the formation of hexanal-derived amide. As shown in Figure 7C, dropping the H2O2 concentration down to as low as 1 µm still resulted in detectable amide formation. This result supports the likely generation of aldehyde-derived amide adducts at physiological concentrations of H2O2.
Discussion A growing body of evidence suggests that many of the effects of cellular dysfunction under oxidative stress are mediated by the products of nonenzymatic reactions, such as the peroxidative degradation of polyunsaturated fatty acids. Lipid peroxidation proceeds by a free radical chain reaction mechanism and yields lipid hydroperoxides as the major initial reaction products. Subsequently, the decomposition of lipid hydroperoxides generates a number of breakdown products that display a wide variety of damaging actions. A number of reactive aldehydes derived from the lipid peroxidation have been implicated as causative agents in cytotoxic processes initiated by the exposure of biological systems to oxidizing agents (7). In the present study, we examined the effect of the oxidizing agent, H2O2, upon the aldehyde modification of lysine residues and detected the Nacylated lysine as the major products. Furthermore, the formation of N-acylation products was immunochemically confirmed in the protein treated with H2O2/hexanal in vitro and in the Fe3+NTA-induced carcinogenesis model in vivo. To the best of our knowledge, this is the first report that demonstrates the involvement of H2O2 in the covalent binding of relatively inert saturated aldehydes to primary amines. Hexanal, an aldehyde produced in high quantity during lipid peroxidation (10, 11, 20, 23–25), shows metabolic, genotoxic, and mutagenic effects, as well as inhibitory effects on cell proliferation (7). It has also been shown that hexanal is by far the major aldehyde and that its production correlates well with the oxidation of polyunsaturated fatty acid in LDL and reflects the degree of LDL oxidation in vitro (26–29). Moreover, a remarkable overproduction of this aldehyde was shown in skin fibroblasts from a patient with cardiomyopathy and cataracts
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Figure 6. Immunochemical detection of N-acylated lysines in vitro and in vivo. BSA (1 mg/mL) was incubated with hexanal (0-50 mM) in the presence and absence of H2O2 (0-50 mM), and the formation of N-hexanoyllysine in the protein was immunochemically characterized using the anti-N-hexanoyllysine mAb 5H4. (A) ELISA, (B) immunoblot analysis, and (C) immunochemical detection of Nacetyllysine and N-hexanoyllysine in the kidney of rats exposed to Fe3+-NTA. The formation of N-acetyllysine (panels Ca-Cc) and Nhexanoyllysine (panels Cd-Cf), immunoreactive with the anti-Nacetyllysine mAb 4G12 and anti-N-hexanoyllysine mAb 5H4, respectively, was immunohistochemically assessed in a rat renal carcinogenesis model with Fe3+-NTA (×200). Panels in part C: a and d (serial sections), control; b and e (serial sections), 3 h after Fe3+-NTA treatment; and c and f (serial sections), 3 weeks after Fe3+-NTA treatment.
both under basal conditions and after menadione or doxorubicin treatment in vivo (30). Other short-chain aldehydes, such as acetaldehyde, have also been detected in micromolar amounts in the effluent perfusates of hearts perfused with a free radicalgenerating system and are proposed to be useful markers for monitoring oxidative stress during reperfusion of ischemic myocardium (31). On the other hand, the formation of reactive oxygen species during lipid peroxidation has also been implicated. Park and Floyd (32) demonstrated that the incubation of DNA with autooxidized methyl linolenate generated the H2O2related oxidized DNA base, 8-hydroxy-2′-deoxyguanosine, and that the generation of this oxidized product was dependent on the presence of the transition metal ions and was inhibited by various scavengers, including catalase. More recently, Shirai et al. (33) reported that the 13-hydroperoxyoctadecadienoic acid treatment of pheochromocytoma PC-12 cells generated reactive oxygen species detectable by the fluorescent probe, 2′,7′dichlorofluorescin. However, these data, claiming that H2O2 can be produced as a result of lipid peroxidation, are indirect and
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Figure 7. LC-MS analysis of the reaction of PLys and hexanal in the presence of various oxidant conditions. (A) LC-MS selected ion chromatograms (system I, see Materials and Methods) of m/z 623.2 (corresponding to the mass of N-hexanoyl-Plys) from total ion chromatograms for authentic N-hexanoyl-PLys (trace a) and for the reaction mixture of PLys and hexanal (2 mM each) alone (trace b) or in the presence of Fe(II)ascorbate (trace c), 2 mM H2O2 (trace d), 2 mM tert-butyl hydroperoxide (trace e), or 2 mM cumene hydroperoxide (trace f). (B) Tandem mass spectra of the m/z 623.2 peak for authentic N-hexanoyl-PLys (trace a) and for the reaction mixture of PLys and hexanal in the presence of 2 mM H2O2 (trace b), 2 mM tert-butyl hydroperoxide (trace c), or 2 mM cumene hydroperoxide (trace d). A 100% relative abundance for the base peak (m/z 397) corresponds to ion intensities of 3.17 × 105 (trace a), 5.09 × 105 (trace b), 4.22 × 104 (trace c), and 1.92 × 104 (trace d). (C) LC-MS selected ion chromatograms (system II, see Materials and Methods) of m/z 623.2 (corresponding to the mass of N-hexanoyl-Plys) from total ion chromatograms for authentic N-hexanoyl-PLys (trace A) and for the reaction mixture of PLys and hexanal (2 mM each) in the presence of 1-100 µm H2O2.
lack direct measurement using the analytical methodology. Thus, it is still uncertain if H2O2 would be formed during lipid peroxidation. Meanwhile, lipid peroxidation is a chain process that involves both alkoxy and hydroperoxy radicals; therefore, ROOH is certainly formed in the process, which can affect the amide-forming reaction. Thus, it may not be unlikely that H2O2
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Figure 8. Proposed mechanism for N-acylation by saturated aldehydes in the presence of H2O2.
would be formed possibly through 1,2-elimination of some of the alkyl hydroperoxides where the new CdC can go into conjugation. It is well-recognized that saturated aldehydes mainly form Schiff bases through the formation of unstable carbinolamine intermediates. However, because of the reversibility of this reaction, the simple aldehydes have received relatively little attention as protein-modifying reagents. We found in this study that H2O2, and to a lesser extent alkyl hydroperoxides, are capable of mediating covalent modification of proteins by saturated aldehydes. This finding suggests the possibility that saturated aldehydes, in combination with H2O2 or ROOH, may contribute to the modification of nucleophilic biomolecules and the development of tissue damage under oxidative stress. A probable mechanism for the reaction is the imine analogue of the Baeyer-Villiger reaction of ketones with peroxides to give esters, which also pertains to the mechanism of oxidation of aldehydes to carboxylic acids by ROOH. The reaction would proceed by addition of ROOH (R ) H, alkyl) to the Schiff base, followed by 1,2-migration of hydride and expulsion of H2O or alkyl-OH, respectively (Figure 8). The reaction may be acidcatalyzed (to create a better leaving group) and would be more efficient for the latter reason using a peracid rather than ROOH. At the same time, however, it is well-known that potent oxene donors like peracids react with imines (usually in organic solvent) directly to generate oxaziridines, semistable species that decompose to amides only upon heating or in the presence of transition metal catalysts (34). Although the Schiff base-derived peroxycarbinolamine could decompose to oxaziridine in competition with 1,2-hydride migration, the distinction of the Baeyer-Villiger as opposed to oxaziridine pathway has been pointed out in the literature (35). In our reaction using 3-chloroperoxybenzoic acid at low concentration, no oxaziridine product was observed by MS, although it is unclear whether such species would survive the ionization conditions. In any event, because the generation of circulating peracids in physiological oxidative stress is unlikely, it seems unnecessary at this time to invoke a competing oxaziridine pathway for amide formation in this study. The occurrence of protein N-acylation under oxidative stress in vivo was confirmed in the kidney of rats exposed to Fe3+NTA, a well-characterized inducer of oxidative stress. The iron chelate was originally used as an experimental model of iron
overload (36). Later, repeated intraperitoneal injections of Fe3+NTA were reported to induce acute and subacute renal proximal tubular necrosis and a subsequent high incidence (60-92%) of renal adenocarcinoma in male rats and mice (18, 19). A single injection of Fe3+-NTA causes a number of time-dependent morphological alterations in the structure and the function of the renal proximal tubular cells and their mitochondria. During the early stage of injury, typical cellular changes are the loss of the brush border, cytoplasmic vesicles, mitochondrial disorganization, and dense cytoplasmic deposits in the proximal tubular cells. Most of the damaged epithelia shows the typical appearance of necrotic cells, and more than half of the proximal tubular cells are removed. This study showed that the N-acylation products, such as N-acetyllysine and N-hexanoyllysine, could be generated in the renal proximal tubules of rats exposed to oxidative stress 3 h after the administration with Fe3+-NTA (Figure 7, panels b and e). The immunoreactivities were detected even 3 weeks after the administration of Fe3+-NTA (Figure 7, panels c and f), suggesting that the long retention of protein N-acylation may play a role in the Fe3+-NTA-induced carcinogenesis. It has been suggested that lipid peroxidation is one of the basic mechanisms of Fe3+-NTA-induced acute renal injury and is closely associated with renal carcinogenesis (37). This hypothesis can be supported by a number of studies showing the accumulation of protein-bound lipid peroxidation products, such as acrolein (25), crotonaldehyde (38), malondialdehyde (39), and 4-hydroxy-2-nonenal (16). These and the findings that the H2O2-related oxidized products, such as 8-oxo-2′-deoxyguanosine, are accumulated in this experimental carcinogenesis model (40) suggested that the lipid peroxidation-derived saturated aldehydes and H2O2, simultaneously generated in the kidney of rats exposed to Fe3+-NTA, might be involved in the formation of the N-acylation products. On the other hand, it is well-established that protein N-acylation of lysine residues can occur in vivo via enzymatic means on both nuclear and cytoplasmic proteins (41–43). The increased staining seen in the Fe3+-NTA-treated animals could, therefore, be an enzymatic response to the oxidative stress rather than direct adduction of the aldehydes generated by oxidative stress to proteins involving the H2O2-mediated reactions observed in vitro. In any event, given the regulatory functions of every known lysine modification, N-acylation is likely to play an important role in protein folding and functions under oxidative stress.
Protein N-Acylation
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N-Hexanoyllysine was previously identified as a product of the reaction of the lysine residue with the oxidized linoleate, 13-hydroperoxyoctadecadienoic acid (13). The authors speculated the direct interaction between the lipid hydroperoxide and the lysine residues of protein as an underlying mechanism. Mets et al. (44) also isolated a similar N-hexanoylated derivative of pyridoxamine and proposed a mechanism in which the conjugated diene hydroperoxides oxidatively decompose to ketoaldehydes, which then react with the primary amine to form the N-acylated product through the formation of a hemiacetal derivative. However, until this study, bona fide reactive species responsible for these N-acylations had remained unidentified. On the basis of the fact that lipid peroxidation generates a great number of oxidized products, including aldehydes and reactive oxygen species, the combined action of saturated aldehydes and either H2O2 or ROOH is likely to occur. Thus, N-hexanoyllysine, which has been considered to be one of the earlier and stable markers for lipid peroxidation-derived protein modification as compared to the aldehyde-derived protein adducts (45), may actually be the product of the lysine modification by hexanal, originating from the lipid hydroperoxide, and either H2O2 or the circulating lipid hydroperoxide directly as the oxidant. Acknowledgment. This work was supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology and by the Center of Excellence (COE) Program in the 21st Century in Japan (K.U.) and from the NIH (HL 53315, L.M.S.). This paper is respectfully dedicated to the memory of Dr. Earl R. Stadtman (NIH). Supporting Information Available: 1H NMR and 13C NMR analysis spectra, HMBC spectrum, specificity of anti-hexanoyllysine mAb, and absorption control experiment. This material is available free of charge via the Internet at http://pubs.acs.org.
References (1) Stadtman, E. R. (1986) Oxidation of proteins by mixed-function oxidation systems: Implication in protein turnover, ageing and neutrophil function. Trends Biochem. Sci. 11, 11–12. (2) Stadtman, E. R. (1990) Metal ion-catalyzed oxidation of proteins: Biochemical mechanism and biological consequences. Free Radical Biol. Med. 9, 315–325. (3) Stadtman, E. R. (1992) Protein oxidation and aging. Science 257, 1220– 1224. (4) Stadtman, E. R., and Oliver, C. N. (1991) Metal-catalyzed oxidation of proteins. Physiological consequences. J. Biol. Chem. 266, 2005– 2008. (5) Uchida, K. (2000) Role of reactive aldehyde in cardiovascular diseases. Free Radical Biol. Med. 28, 1685–1696. (6) Uchida, K. (2003) 4-Hydroxy-2-nonenal: A product and mediator of oxidative stress. Prog. Lipid Res. 42, 318–343. (7) Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biol. Med. 11, 81–128. (8) Steinberg, D. (1995) Role of oxidized LDL and antioxidants in atherosclerosis. AdV. Exp. Med. Biol. 369, 39–48. (9) Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C., and Witztum, J. L. (1989) Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320, 915– 924. (10) Esterbauer, H., and Zollner, H. (1989) Methods for determination of aldehydic lipid peroxidation products. Free Radical Biol. Med. 7, 197– 203. (11) Spiteller, P., Kern, W., Reiner, J., and Spiteller, G. (2001) Measurement of n-alkanals and hydroxyalkenals in biological samples. Biochim. Biophys. Acta 1531, 188–208. (12) Fenaille, F., Guy, P. A., and Tabet, J. C. (2003) Study of protein modification by 4-hydroxy-2-nonenal and other short chain aldehydes analyzed by electrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 14, 215–226.
(13) Kato, Y., Mori, Y., Makino, Y., Morimitsu, Y., Hiroi, S., Ishikawa, T., and Osawa, T. (1999) Formation of N-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification. J. Biol. Chem. 274, 20406– 20414. (14) Toyoda, K., Nagae, R., Akagawa, M., Ishino, K., Shibata, T., Ito, S., Shibata, N., Yamamoto, T., Kobayashi, M., Takasaki, Y., Matsuda, M., and Uchida, K. (2007) Protein-bound 4-hydroxy-2-nonenal: An endogenous triggering antigen of anti-DNA response. J. Biol. Chem. 282, 25769–25778. (15) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. (16) Toyokuni, S., Uchida, K., Okamoto, K., Hattori-Nakakuki, Y., Hiai, H., and Stadtman, E. R. (1994) Formation of 4-hydroxy-2-nonenalmodified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate. Proc. Natl. Acad. Sci. U.S.A. 91, 2616–2620. (17) Nishiyama, Y., Suwa, H., Okamoto, K., Fukumoto, M., Hiai, H., and Toyokuni, S. (1995) Low incidence of point mutations in H-, K- and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate. Jpn. J. Cancer Res. 86, 1150–1158. (18) Ebina, Y., Okada, S., Hamazaki, S., Ogino, F., Li, J.-L., and Midorikawa, O. (1986) Nephrotoxicity and renal cell carcinoma after use of iron- and aluminum-nitrilotriacetate complexes in rats. J. Natl. Cancer Inst. 76, 107–113. (19) Li, J.-L., Okada, S., Hamazaki, S., Ebina, Y., and Midorikawa, O. (1987) Subacute nephrotoxicity and induction of renal cell carcinoma in mice treated with ferric nitrilotriacetate. Cancer Res. 47, 1867– 1869. (20) Toyokuni, S., Luo, X. P., Tanaka, T., Uchida, K., Hiai, H., and Lehotay, D. C. (1997) Induction of a wide range of C(2-12) aldehydes and C(7-12) acyloins in the kidney of Wistar rats after treatment with a renal carcinogen, ferric nitrilotriacetate. Free Radical Biol. Med. 22, 1019–1027. (21) Toyokuni, S., Okada, S., Hamazaki, S., Minamiyama, Y., Yamada, Y., Liang, P., Fukunaga, Y., and Midorikawa, O. (1990) Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-induced renal lipid peroxidation in ddY mice. Cancer Res. 50, 5574–5580. (22) Uchida, K., Fukuda, A., Kawakishi, S., Hiai, H., and Toyokuni, S. (1995) A renal carcinogen ferric nitrilotriacetate mediates a temporary accumulation of aldehyde-modified proteins within cytosolic compartment of rat-kidney. Arch. Biochem. Biophys. 317, 405–411. (23) Des Rosiers, C., Rivest, M.-J., Boily, M.-J., Jette´, M., Carrobe´-Cohen, A., and Kumar, A. (1993) Gas chromatographic-mass spectrometry of tissue malondialdehyde, 4-hydroxynenal, and other aldehydes after reduction to stable alcohols. Anal. Biochem. 208, 161–170. (24) Esterbauer, H., Dieber-Rotheneder, M., Waeg, G., Striegl, G., and Jurgens, G. (1990) Biochemical, structural, and functional properties of oxidized low-density lipoprotein. Chem. Res. Toxicol. 3, 77–92. (25) Kawai, Y., Furuhata, A., Toyokuni, S., Aratani, Y., and Uchida, K. (2003) Formation of acrolein-derived 2′-deoxyadenosine adduct in an iron-induced carcinogenesis model. J. Biol. Chem. 278, 50346–50354. (26) Esterbauer, H., Jurgens, G., Quehenberger, O., and Koller, E. (1987) Autoxidation of human low density lipoprotein: Loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J. Lipid Res. 28, 495–509. (27) Frankel, E. N., German, J. B., and Davis, P. A. (1992) Headspace gas chromatography to determine human low-density lipoprotein oxidation. Lipids 27, 1047–1051. (28) Frankel, E. N., Kanner, J., German, J. B., Parks, E., and Kinsella, J. E. (1993) Inhibition of oxidation of human low-density-lipoprotein by phenolic substances in red wine. Lancet 341, 454–457. (29) Frankel, E. N., Hu, M. L., and Tappel, A. L. (1989) Rapid headspace gas-chromatography of hexanal as a measure of lipid peroxidation in biological samples. Lipids 24, 976–981. (30) Luo, X., Pitkanen, S., Kassovska-Bratinova, S., Robinson, B. H., and Lehotay, D. C. (1997) Excessive formation of hydroxyl radicals and aldehydic lipid peroxidation products in cultured skin fibroblasts from patients with complex I deficiency. J. Clin. InVest. 99, 2877–2882. (31) Cordis, G., Bagchi, D., Maulik, N., and Das, D. K. (1994) Highperformance liquid chromatographic method for the simultaneous detection of malondialdehyde, acetaldehyde, formaldehyde, acetone and propionaldehyde to monitor the oxidative stress in heart. J. Chromatogr. 661, 181–191. (32) Park, J. W., and Floyd, R. A. (1992) Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free Radical Biol. Med. 12, 245–250. (33) Shirai, M., Kawai, Y., Yamanishi, R., Kinoshita, T., Chuman, H., and Terao, J. (2006) Effect of a conjugated quercetin metabolite, quercetin
1270
(34)
(35)
(36)
(37) (38) (39)
(40)
Chem. Res. Toxicol., Vol. 21, No. 6, 2008
3-glucuronide, on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells. Free Radical Res. 40, 1047–1053. Leung, C. H., Voutchkova, A. M., Crabtree, R. H., Balcells, D., and Eisenstein, O. (2007) Atom economic synthesis of amides via transition metal catalyzed rearrangement of oxaziridines. Green Chem. 9, 976– 979. Maurizio Ceruti, M., Viola, F., Balliano, G., Grosa, G., Caputo, O., Gerst, N., Schuber, F., and Cattell, L. (1988) Synthesis of a squalenoid oxaziridine and other new classes of squalene derivatives, as inhibitors of sterol biosynthesis. Eur. J. Med. Chem. 23, 533–537. Awai, M., Narasaki, M., Yamanoi, Y., and Seno, S. (1979) Induction of diabetes in animals by parenteral administration of ferric nitrilotriacetate. A model of experimental hemochromatosis. Am. J. Pathol. 95, 663–674. Toyokuni, S. (1996) Iron-induced carcinogenesis: The role of redox regulation. Free Radical Biol. Med. 20, 553–566. Ichihashi, K., Osawa, T., Toyokuni, S., and Uchida, K. (2001) Endogenous formation of protein adducts with carcinogenic aldehydes: Implications for oxidative stress. J. Biol. Chem. 276, 23903–23913. Uchida, K., Sakai, K., Itakura, K., Osawa, T., and Toyokuni, S. (1997) Protein modification by lipid peroxidation products: formation of malondialdehyde-derived N-(2-propenol)lysine in proteins. Arch. Biochem. Biophys. 346, 45–52. Akatsuka, S., Aung, T. T., Dutta, K. K., Jiang, L., Lee, W. H., Liu, Y. T., Onuki, J., Shirase, T., Yamasaki, K., Ochi, H., Naito, Y., Yoshikawa, T., Kasai, H., Tominaga, Y., Sakumi, K., Naka-
Ishino et al.
(41)
(42)
(43) (44)
(45)
beppu, Y., Kawai, Y., Uchida, K., Yamasaki, A., Tsuruyama, T., Yamada, Y., and Toyokuni, S. (2006) Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis. Am. J. Pathol. 169, 1328–1342. Sadoul, K., Boyault, C., Pabion, M., and Khochbin, S. (2008) Regulation of protein turnover by acetyltransferases and deacetylases. Biochimie 90, 306–312. Chen, Y., Sprung, R., Tang, Y., Ball, H., Sangras, B., Kim, S. C., Falck, J. R., Peng, J., Gu, W., and Zhao, Y. (2007) Lysine propionylation and butyrylation are novel post-translational modifications in histones. Mol. Cell. Proteomics 6, 812–819. Ito, K. (2007) Impact of post-translational modifications of proteins on the inflammatory process. Biochem. Soc. Trans. 35, 281–283. Metz, T. O., Alderson, N. L., Chachich, M. E., Thorpe, S. R., and Baynes, J. W. (2003) Pyridoxamine traps intermediates in lipid peroxidation reactions in vivo: evidence on the role of lipids in chemical modification of protein and development of diabetic complications. J. Biol. Chem. 278, 42012–42019. Kato, Y., Yoshida, A., Naito, M., Kawai, Y., Tsuji, K., Kitamura, M., Kitamoto, N., and Osawa, T. (2004) Identification and quantification of N-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry. Free Radical Biol. Med. 37, 1864–1874.
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