Chem. Res. Toxicol. 2002, 15, 699-706
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Identification of Seven Proteins in the Endoplasmic Reticulum as Targets for Reactive Metabolites of Bromobenzene Yakov M. Koen and Robert P. Hanzlik* Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-7582 Received December 31, 2001
The hepatotoxicity of bromobenzene is strongly correlated with the covalent binding of chemically reactive metabolites to cellular proteins, but up to now relatively few hepatic protein targets of these reactive metabolites have been identified. To identify additional hepatic protein targets we injected an hepatotoxic dose of [14C]bromobenzene to phenobarbital-pretreated male Sprague-Dawley rats ip. After 4 h, their livers were removed and homogenized, and the homogenates fractionated by differential ultracentrifugation. The highest specific radiolabeling (6.1 nmol equiv 14C/mg of protein) was observed in a particulate fraction (P25) sedimented at 25000g from a 6000g supernatant fraction. Proteins in this fraction were separated by twodimensional electrophoresis and, after transblotting, analyzed for radioactivity by phosphorimaging. More than 20 radiolabeled protein spots were observed in the blots. For 17 of these spots, peptide mass maps were obtained using in-gel digestion with trypsin, followed by MALDITOF mass spectrometric analysis of the resulting peptide mixtures. By searching genomic databases, the 17 sets of MS-derived peptide masses were found to match predicted tryptic fragments of just 7 proteins. Spots 1-4 matched with 78 kDa glucose regulated protein (GRP78), protein disulfide isomerase isozyme A1 (PDIA1), endoplasmic reticulum protein ERp29, and PDIA6, respectively. Spots 5 and 6, 7-11, and 12-17 presented as apparent “charge trains” of spots, each of which gave peptide mixtures closely similar to those of other spots within the train. The proteins present in these sets of spots were identified as transthyretin, serum albumin precursor and PDIA3, respectively. The possible relationship of the adduction of these proteins to the toxicological outcome is discussed.
Introduction The cytotoxicity of many small, relatively unreactive organic compounds has been associated with their biotransformation to chemically reactive metabolites which then covalently bind to cellular protein nucleophiles (1). For many well-studied organic pro-toxicants (e.g., acetaminophen, halothane, diclofenac, and bromobenzene), the association between the protein covalent binding of their metabolites and the ensuing toxicity is so strong that the former is generally assumed to cause the latter. Over the past several decades much has been learned about the enzymatic mechanisms of pro-toxin activation and the chemical mechanisms of macromolecule adduction (2, 3). More recently interest has focused on the identification of the individual proteins that are targeted by reactive metabolites of a given pro-toxin (46). Early efforts to identify cellular proteins targeted by chemically reactive metabolites utilized classical methods for protein isolation and purification, combined with the use of either radioactivity or adduct-specific antibodies for detecting the presence of adduct moieties, and either N-terminal or internal peptide microsequencing for protein identification. Our laboratory has been interested in elucidating mechanisms underlying the hepatotoxicity of bromobenzene. We have previously reported on the identification * To whom correspondence should be addressed. Fax: (785) 8645326. E-mail:
[email protected].
of the structures of metabolite-protein adducts formed in the livers of rats treated with hepatotoxic doses of bromobenzene (7, 8). Using classical techniques of protein separation we subsequently identified six different hepatocellular proteins targeted by bromobenzene metabolites including two microsomal esterase enzymes (9), microsomal epoxide hydrolase1 and the cytosolic proteins fatty acid binding protein, carbonic anhydrase III and glutathione transferase Ya (10). More recently, the techniques of two-dimensional gel electrophoresis, in-gel digestion and peptide mass mapping have significantly accelerated the process of identifying proteins in complex mixtures (11-13). In this manuscript we report on the use of this latter approach to identify 17 radiolabeled protein spots in a two-dimensional gel separation of liver proteins from rats treated with an hepatotoxic dose of [14C]bromobenzene.
Experimental Procedures Materials. [14C]Bromobenzene (5.17 Ci/mol) was prepared in our laboratory, stored, and handled as described previously (10, 14). Trypsin, sequencing grade, from bovine pancreas was obtained from Boehringer Mannheim (Indianapolis, IN). 4-Vinylpyridine was obtained from Sigma and was distilled and stored under nitrogen at -20 °C. Dithiothreitol and phenylmethanesulfonyl fluoride were obtained from Sigma. CHAPS2 was obtained from Pierce. Tris, SDS, glycine, Sequi-blot PVDF 1
E. M. Rombach and R. P. Hanzlik, unpublished results.
10.1021/tx0101898 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/19/2002
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membranes (0.2 µm), prestained pI markers, and broad-range molecular mass markers were obtained from Bio-Rad. All other electrophoresis supplies were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). HPLC-grade solvents and analytical grade inorganic salts were obtained from Fisher (Fair Lawn, NJ). Deionized water (resistivity > 17 MΩ/cm) was used for preparation of all buffers. Treatment of Animals and Preparation of Subcellular Fractions. Male Sprague-Dawley rats (150-180 g, Charles River Laboratories, Wilmington, MA) were housed in a temperature and humidity controlled room with a 12 h light/dark cycle and ad libitum access to food and water. After acclimating for at least 3 days, animals were given 3 daily ip injections of sodium phenobarbital (80 mg/kg) in 0.9% saline (1.0 mL/kg). After the third dose, food was withheld overnight and the next morning the rats were injected with [14C]bromobenzene (2 mmol/ kg, ip) in corn oil (1 mL/kg) and killed 4 h later by decapitation under CO2 narcosis. Livers were removed and homogenized in ice-cold 50 mM potassium phosphate buffer, pH 7.4, containing 0.15 M KCl, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride (buffer A; 4 mL/g tissue). The homogenate was successively centrifuged at 6500gavg (10 min), 25000gavg (20 min) and 100000gmax (60 min). The 6500g pellet was discarded; the 25000g pellet (25P fraction), 100000g pellet (microsomes), and 100000g supernatant (cytosol) fractions were collected and processed further as follows. The 25P fraction was resuspended in buffer A and resedimented at 25000g. The final 25P pellet was homogenized in 100 mM potassium phosphate (pH 7.4), 0.5 mM EDTA, 20% glycerol (buffer B). The microsomal fraction was washed by homogenization in 0.1 M sodium pyrophosphate buffer, pH 8.2 (1.3 mL/g tissue), followed by centrifugation at 100000g. The final microsomal pellet was resuspended in buffer B. Cytosol was clarified by recentrifugation at 100000g and dialyzed against 20 mM potassium phosphate, pH 7.4, containing 0.5 mM dithiothreitol (4 × 60 volumes), to remove reversibly bound radioactivity. The washed subcellular fractions were aliquoted and stored at -70 °C. Determination of Covalently Bound Radiolabel. Aliquots of the subcellular fractions were precipitated with 10% trichloroacetic acid, and protein precipitates were successively washed with acetone, methanol/water (80:20 v/v, three times), acetone and diethyl ether, dried by rotary evaporation, dissolved in 1 M NaOH and neutralized with 1 M HCl, after which the radioactivity was measured by scintillation counting and protein determined by Bradford assay using a standard kit (Bio-Rad). Two-Dimensional Electrophoresis. Electrophoretic separations were performed using a Multiphor II unit equipped with programmable Power Supply 3501 EPS and IEF2 Kit. Immobiline DryStrips (pH 3-10, nonlinear gradient, 18 cm) were used for the first dimension and ExcelGel (XL SDS 12-14, 24.5 × 18 cm) for the second dimension. Electrophoresis was performed according to the manufacturer’s instructions with the following modifications. Aliquots of the 25P fraction (100 µL, 1 mg of protein) were mixed with 300 µL of rehydration solution (9 M urea, 4% CHAPS, 0.5% IPG buffer (pH 3-10 NL), 0.065 M dithiothreitol, and a trace of bromophenol blue), and incubated at ambient temperature for 60 min. The resulting solubilisate was applied to the entire IPG strip by placing the sample with the strip in a rehydration tray for 16 h. Isoelectrofocusing was conducted at 20 °C using the following voltage gradient: 0-300 V, 1 min; 300 V, 3 h; 300-1000 V, 2 h; 1000-3500 V, 1 h; 3500 V, 15 h (total 57 kV h). After focusing, the strips were incubated with standard SDS-PAGE equilibration solution [50 mM TrisHCl, (pH 6.8), 6 M urea, 30% glycerol, 2% SDS] supplemented with 130 mM dithiothreitol, for 15 min, followed by incubation 2 Abbreviations: 2-D, two-dimensional; CHAPS, 3[(cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; ER, endoplasmic reticulum; IEF, isoelectric focusing; IPG, immobilized pH gradient; MALDI-TOF, matrix-assisted laser desorption ionizationtime-of-flight; MS, mass spectrometry; PDI, protein disulfide isomerase; PVDF, poly(vinylidene difluoride); SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; TTR, transthyretin.
Koen and Hanzlik
Figure 1. SDS-PAGE and phosphorimaging analysis of liver homogenate fractions 4 h after ip injection of [14C]bromobenzene. Aliquots of the P25, microsomal and cytosolic fractions containing equivalent amounts of protein (25 µg) were separated by a 4 to 20% gradient SDS-PAGE followed by electroblotting to a PVDF membrane. The membrane was stained with Coomassie R250 and exposed to a Molecular Dynamics phosphorimaging plate. Panel A shows protein patterns on the PVDF membrane; panel B shows results of phosphorimaging of the same membrane. Lane 1, P25 mitochondrial fraction; lane 2, microsomal fraction; lane 3, cytosolic fraction. with the same solution except that DTT was replaced by 200 mM 4-vinylpyridine, for 15 min. The strips were then applied on the flat-bed SDS-gel, and the proteins were further separated by SDS-PAGE carried out at the following conditions: 15 °C; 20 mA, 45 min; 40 mA, 5 min; 50 mA, 2 h. Proteins were visualized by staining with 0.025% Coomassie R250, 30% methanol, 0.5% acetic acid for 2 h, followed by destaining with 30% methanol. In all separation experiments, prestained pI markers and broad-range molecular mass markers were used for calibration of the first- and second dimension gels, respectively. To locate radiolabeled proteins, they were transferred from nonstained SDS-gel to a Sequi-blot PVDF membrane (0.2 µm) by electroblotting in semi-dry conditions using Multiphor II with NovaBlot Kit according to the Manufacturer’s instructions. The blots were stained with 0.1% Coomassie R250, 50% methanol, 7% acetic acid, then destained with 50% methanol, 7% acetic acid and subjected to phosphorimaging analysis for 14C using a Molecular Dynamics storage phosphor screen and Bio-Rad Molecular Imager FX scanning unit and software. The integrated image densities of the radioactive spots on the phosphorimage (Figure 2B) and of the corresponding spots on the Coomassie-stained PVDF blot (Figure 2A) were measured (in arbitrary but internally consistent units). For each spot the numeric ratio of the two image densities was computed as an index of the relative degree of adduction of the protein(s) in that spot by [14C]bromobenzene metabolites (See Table 1). In-Gel Digestion. Aliquots of the 25P fraction (1 mg protein) were separated by two-dimensional electrophoresis as described above, and individual protein spots were excised and in-gel digested with trypsin as described previously (10). The resulting peptide mixtures were concentrated by vacuum-evaporation and immediately submitted for mass spectrometric analysis or stored at -20 °C until analyzed. As a control, protein-free gel fragments of approximately the same size were processed identically. Mass Spectrometry of Tryptic Digests. Aliquots of peptide mixtures were mixed with saturated solution of R-cyanohydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid and applied to a sample plate. The samples were then analyzed on a Voyager-DE STR MALDI-TOF mass spectrometer (Perseptive Biosystems) operated in positive reflector mode [accelerating voltage, 20 kV; mirror voltage ratio, 1.12; extraction delay, 180 ns]. Data acquisition was performed over the m/z range 700-3000. Mass spectra were externally calibrated using a standard mixture of known peptides covering the entire mass range, and where possible the calibration was verified using internal m/z peaks arising from trypsin autolysis. Partial
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Table 1. Densities of Individual Spots in Two-Dimensional Gel Separation of Proteins Shown in Figure 1a spot no.
protein (panel A)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 + 16 17
18 104 2.3 6.3 0.91 2.0 10 13 30 22 11.5 91 59 44 18 7.0
14C
content (panel B)
relative normalized adduct densityb
217 740 43 69 106 267 27 62 65 124 86 80 438 374 223 102
13 7.9 21 12 130 148 3.0 5.3 2.4 6.3 8.3 1.0 8.2 9.4 14 16
a Data are in arbitrary units representing the integrated density of each of the numbered spots in the two panels of Figure 1. b Ratio of integrated values for panel B/panel A normalized to the ratio for spot 12.
sequence determination of selected peptides was performed by analysis of post-source decay mass spectra. Database Searching. The observed monoisotopic peptide masses were compared to the theoretical peptide masses of all proteins available from SWISS-PROT and TrEMBL databases (http://ca.expasy.org) and from the NCBInr database (http:// www.ncbi.nlm.nih.gov), using MS-Fit (http://prospector.ucsf.edu/) and ProFound (http://129.85.19.192/prowl-cgi/ProFound.exe) peptide mass fingerprinting programs. The parameters used in the search were: mass tolerance
