Departments of 1Pathology, 2Environmental Health Sciences, and 3Biostatistics, University of Alabama at Birmingham, and 4Medical Research Council-Dunn Human Nutrition Unit, Cambridge, UK
Submitted 11 September 2003 ; accepted in final form 9 November 2003
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ABSTRACT |
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mitochondrial thiol proteins; cysteine; reactive oxygen species; reactive nitrogen species
Recently, a method using a novel lipophilic compound (4-iodobutyl) triphenyl phosphonium (IBTP) was established, which selectively labels reduced thiol groups within mitochondrial proteins (15, 25). This lipophilic cation is concentrated in the organelle due to the large membrane potential across the inner mitochondrial membrane. IBTP was shown to colocalize with mitochondrial enzymes in human fibroblasts with essentially uniform labeling of all mitochondria (15). Once IBTP is taken up into the mitochondrion, the thiolate groups present within proteins displace the iodo group of IBTP to form a stable phosphonium thioether. Selective labeling of mitochondrial proteins is dependent on the membrane potential, because uncouplers essentially remove all IBTP adducts in both cells and isolated mitochondria. These IBTP-labeled proteins can easily be identified by immunoblotting with an antibody raised against the triphenylphosphonium group. Thus thiols present within proteins that have been oxidized or modified as a consequence of oxidants can be identified by decreased labeling with IBTP using gel electrophoresis and immunoblotting with the anti-triphenylphosphonium antibody (anti-TPP) (15).
It was predicted that use of this technique in combination with a mitochondrial proteomics approach would enable the identification of specific mitochondrial proteins that are modified in an in vivo model of oxidative or nitrosative stress. For these studies, we chose a model of chronic alcohol consumption that is known to induce mitochondrial production of ROS/RNS, oxidation of mitochondrial proteins, and mitochondrial dysfunction (3, 4, 8, 30). Thus a protocol was developed in which we were able to identify specific mitochondrial proteins that had altered thiol redox status as a consequence of chronic alcohol exposure. Detection of these posttranslational modifications demonstrates at the molecular level a mechanism by which chronic alcohol consumption might negatively affect mitochondrial energy metabolism and contribute to alcohol-induced liver toxicity. Furthermore, the results of these studies demonstrate that this approach can be extended to a wide variety of other pathophysiological conditions where oxidative and nitrosative stress has been implicated in the etiology.
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MATERIALS AND METHODS |
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Animals and diets. Male Sprague-Dawley rats (200 g) were individually housed in suspended cages and maintained under a 12:12-h light-dark cycle for the entire duration of the feeding protocol. Nutritionally adequate ethanol and control liquid diets were formulated and prepared according to Lieber and DeCarli (14). The ethanol diet provides 36% of the total daily caloric intake as ethanol, 11% as carbohydrate, 18% as protein, and 35% as fat. Weight-matched control rats were pair fed and received identical diets except that ethanol calories were substituted isocalorically by dextrin maltose. Animals were maintained on the diets for at least 31 days (2, 4).
Isolation of rat liver mitochondria and labeling with IBTP. Coupled liver mitochondria were prepared by differential centrifugation of liver homogenates (23, 26) using ice-cold mitochondria isolation medium containing 0.25 M sucrose, 1 mM EDTA, and 5 mM Tris·HCl (pH 7.5). Protease inhibitors were added to the isolation buffer to prevent protein degradation (2, 4). The total mitochondria protein from control animals was 224 ± 20 mg and from ethanol-treated animals was 257 ± 10 mg (means ± SE, n = 8; P = 0.19). Respiratory control ratios for mitochondria isolated from control animals was 4.9 ± 0.3 and for ethanol-fed animals was 3.5 ± 0.2 (means ± SE, n = 8; P = 0.0004).
For IBTP labeling of reduced mitochondrial protein thiols, mitochondria (1.0 mg/ml) were incubated at 37°C in respiration buffer containing (in mM) 120 KCl, 5 KH2PO4, 1 EGTA, 25 sucrose, 5 MgCl2, and 3 HEPES (pH 7.4), and respiration was initiated by the addition of succinate and ADP. After 3 min, 5 µM IBTP was added to mitochondria and the reaction was allowed to proceed for 10 min. The reaction was stopped by the addition of iodoacetic acid. Control experiments were also performed with labeling in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Incubation of mitochondria with this concentration of IBTP has no effect on mitochondrial respiration and does not deplete mitochondrial glutathione (15). Mitochondrial suspensions were centrifuged at 12,500 rpm, the supernatant was discarded, and pellets were stored at -80°C until used for gel electrophoresis.
Electrophoresis and immunoblotting. For one-dimensional (1D) SDS-PAGE, mitochondrial samples from control and ethanol-fed rats were separated on 1018% gradient gels, transferred to nitrocellulose membranes, and probed with a 1:10,000 dilution of anti-TPP followed by incubation with 1:3,000 dilution of donkey anti-rabbit HRP-conjugated secondary antibody. For two-dimensional (2D) gel electrophoresis, mitochondria were suspended in tube gel sample buffer [9.5 M urea, 2.0% CHAPS, 1.0% DTT, and 0.8% (vol/vol) of each of pH 310, 58, and 46 ampholines]. After 1-h incubation to fully solubilize mitochondria membranes, 50 µg of protein were loaded onto isoelectric focusing gels (pH gradient 48.5) and focused overnight for 16 h at 400 V, followed by 1 h at 800 V to sharpen bands. Isoelectric focusing gels were equilibrated in 62.5 mM Tris·HCl, 3.0% SDS, 10% glycerol, 0.005% bromophenol blue, and 1.0% DTT before being loaded onto 820% gradient gels for SDS-PAGE. For each control and ethanol pair of samples, gels were run in duplicate with one gel Coomassie blue stained and the other to be used for immunoblotting with the anti-TPP antiserum. The anti-TPP membranes were incubated with a 1:30,000 dilution of the anti-TPP antiserum for 1 h followed by incubation with a 1:5,000 dilution of secondary antibody for 1 h. IBTP-labeled protein thiols for both 1D and 2D gels were visualized by enhanced chemiluminescence (ECL) detection.
Image analysis of two-dimensional gels and anti-TPP blots. For Coomassie blue-stained gels, each control and its matching ethanol pair were stained and destained in an identical manner. Gels were scanned and were used for analysis with PDQuest software (BioRad, Hercules, CA). Protein spots were identified using the spot wizard tool, and a match set was created containing all gels so that matched protein spots would be given the same identification number. The control gel from one pair was used as the master image for protein spot matching. For each pair of gels, the total protein density in the entire gel was nearly identical (control: 971,715 ± 277,153 vs. ethanol: 1,045,240 ± 348,049 relative density units, means ± SE; P = 0.43), thus normalization of individual protein spot densities did not change the outcome of the analysis. We did not observe any significant differences among the matched spots on the Coomassie blue-stained gel.
For anti-TTP Western blots, three pairs of control and ethanol blots were processed and developed together to facilitate comparison. Blot images were obtained using FluorChem software on an AlphaInnotech camera imager (San Leandro, CA), and the resulting TIFF files were analyzed using PDQuest software. Protein spots were identified as described above. To correct for possible intergel protein loading differences, the density for each protein spot on the immunoblot was normalized to the total protein density in the corresponding Coomassie blue-stained gel. Normalized spot densities from control blots were compared with normalized spot densities from ethanol blots using a one-tailed paired Student's t-test.
Identification of IBTP-labeled mitochondrial proteins. Protein spots immunoreactive for IBTP labeling were cut from gels and processed for identification using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry essentially as described by Brookes and colleagues (5). Films and images of IBTP-immunoreactive protein spots were aligned with the duplicate stained gels so that the corresponding immunoreactive protein spot could be identified. MALDI-TOF analysis was accomplished using a PE-Biosystems Voyager Elite instrument (Framingham, MA) equipped with a nitrogen laser (337 nm) and operated using a delayed extraction mode. The peptide masses were entered into "Mascot," and the National Center for Biotechnology Information database was searched to identify the proteins.
Aldehyde dehydrogenase activity and immunoblotting. Mitochondrial aldehyde dehydrogenase (ALDH) activity was measured by the procedure described previously (16). Mitochondria were pretreated with pyrazole (0.2 mM), rotenone (2 µM), and allopurinol (20 µM) to inhibit alcohol dehydrogenase, NADH dehydrogenase, and xanthine oxidase activities, respectively. The specific activity of ALDH is expressed as nanomoles of NADH produced per minute per milligram of protein and was determined over the linear portion of the reaction. ALDH protein was quantified by immunoblotting with an anti-rat liver ALDH antibody (32).
Statistical analysis. Data are reported as means ± SE for sample sizes of three to eight, and statistical evaluations between samples means were made by paired Student's t-test. The minimum level of significance was set at P 0.05.
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RESULTS |
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In the first series of experiments, the extent of IBTP labeling was assessed using a low-resolution separation of mitochondrial proteins by 1D SDS-PAGE (Fig. 1A) and immunoblotting against IBTP (Fig. 1B). These experiments are important to determine whether the extent of IBTP labeling in mitochondria from control and ethanol-fed rats was similar. It is clear that there is no significant difference in the density of the five major bands resolved on the 1D gel with respect to either the protein or IBTP immunoreactive labeling (Fig. 1, AC). As expected, labeling with IBTP was prevented by abolishing the mitochondrial membrane potential with the uncoupler FCCP or by pretreating mitochondria with the thiol-alkylating agent iodoacetic acid before the addition of IBTP (Fig. 1B). Taken together, these data demonstrate that the labeling of mitochondrial proteins by IBTP is dependent on the presence of the mitochondrial membrane potential and that the reaction of IBTP is specific to thiols.
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In the low-resolution 1D SDS-PAGE gels, the labeling of individual proteins is likely to be masked by the comigration of proteins with very similar molecular weights. In the next series of experiments, the high-resolution separation of matrix mitochondrial proteins was performed using separation in the 1D by isoelectric focusing (IEF) followed by 2D SDS-PAGE. The inner membrane proteins are underrepresented by this technique, because they precipitate in the first dimension of the isoelectric focusing gel. However, we chose to concentrate on the matrix proteins, because only a few proteins in complex I are labeled by IBTP (15). The proteins separated by this approach show a very similar pattern for mitochondria isolated from both control and ethanol-treated animals (Fig. 2A). A master map for this profile generated from six individual separations identified 50 proteins in common between mitochondria isolated from control and ethanol-fed animals (Fig. 2B). There was no significant difference in the total protein density between the separations for mitochondria from control and ethanol-fed animals. This subproteome of the mitochondria is dominated by matrix proteins and indicates that chronic ethanol consumption has little effect on the levels of these proteins (15).
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The increased resolution of proteins using 2D gels resulted in the five major IBTP-labeled bands resolved on the 1D gels (Fig. 1B) being separated into 40 discrete spots (Fig. 3A). Moreover, the 2D gel analysis revealed that there was differential IBTP labeling in specific mitochondrial proteins from ethanol-treated animals compared with controls. Several protein spots immunoreactive for IBTP (Fig. 3B) were matched to the corresponding spots within the gel and identified using MALDI-TOF mass spectrometry. The protein spot numbers in Fig. 3B correspond to the identified proteins listed in Table 1. The groups of proteins identified as numbers 4 and 7 were found to be isoforms of the same protein. A total of seven distinct proteins were identified, of which two proteins (circled in Fig. 3A), mitochondrial aldehyde dehydrogenase and glucose-regulated protein 78 (GRP78), showed a consistent and reproducible decrease in IBTP labeling in mitochondria isolated from ethanol-treated animals compared with controls (Table 1). The majority of proteins showed no significant change, whereas two proteins showed higher levels in the mitochondria from ethanol-treated animals, although this failed to reach significance.
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The low-Km ALDH is responsible for >90% of acetaldehyde oxidation in liver (31) and contains numerous cysteine residues including three in the active site (9). Because the oxidation or modification of these critical thiol groups may have a negative impact on protein function, the activity of low-Km ALDH in mitochondria was measured. There was a significant decrease in the specific activity of ALDH in mitochondria from ethanol-treated animals (6.51 ± 0.6 nmol NADH produced·min-1·mg protein-1) compared with control (9.64 ± 0.61 nmol NADH produced·min-1·mg protein-1; P = 0.006, n = 5) when low concentrations (5 µM) of acetaldehyde were used in the assay. Addition of the reductant -mercaptoethanol (0.3 mM) did not restore activity of ALDH in the mitochondria from ethanol-treated animals to those measured in controls. The inability of a strong thiol reductant to restore ALDH activity indicates that an irreversible oxidative modification to the active site cysteine may have occurred as a consequence of chronic alcohol consumption. Thus the decrease in activity and IBTP labeling of the low-Km ALDH was due to the oxidation of critical thiols and was not due to a decrease in the amount of the ALDH protein. This was confirmed by measuring the levels of the enzyme by Western blot analysis with an antibody directed against the enzyme, which showed no significant difference between the amount of ALDH immunoreactive protein in mitochondria from ethanol or control animals (Fig. 3C).
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DISCUSSION |
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Mitochondria from ethanol-treated animals are known to have a decreased rate of respiration (8); thus it is possible that the levels of IBTP that accumulated in mitochondria might be different between control and ethanol groups. However, this is probably not the case, because Lin et al. (15) demonstrated that the uptake of IBTP into mitochondria was dependent on the mitochondrial membrane potential, which is not altered by chronic alcohol consumption alone (20). It was not possible to label mitochondria with IBTP over a wide range of membrane potentials due to limited viability of the mitochondrial preparations. To minimize the impact of the membrane potential, IBTP labeling was performed under conditions of State 3 respiration. Under these circumstances, control of respiration is less dependent on the activity of the electron transport chain. Initial studies using standard 1D SDS-PAGE and immunoblotting for the IBTP protein adduct indicated this was indeed the case and was not significantly altered as a consequence of chronic alcohol consumption (Fig. 1). However, the use of 2D electrophoresis revealed that there was differential labeling of several mitochondrial proteins in response to chronic ethanol treatment (Fig. 3). It is important to note that of the subset of proteins labeled by IBTP, only a limited number showed changes again consistent with a minimal effect of membrane potential on the IBTP available for reaction with thiols. Furthermore, 2D gel electrophoresis allowed the identification of several IBTP-labeled proteins by MALDI-TOF mass spectrometry (Table 1). The low-Km mitochondrial ALDH and the GRP78 consistently showed a significant decrease in IBTP labeling in mitochondria isolated from ethanol-fed rats. GRP78 is a member of the heat-shock protein 70 family and is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER) (10). GRP78 has been shown to be an alcohol-response gene in a model of alcohol-induced hepatotoxicity in mice (12). Although GRP78 is predominantly localized in the ER, it has been demonstrated in mitochondria (5, 21). However, the functional significance of an alteration in the thiol redox status of GRP78 in the context of chronic alcohol consumption and mitochondrial dysfunction is currently not known.
One of the major proteins showing decreased labeling with IBTP was the low-Km ALDH, which resides in the mitochondrial matrix and contributes to the metabolism of acetaldehyde, the oxidation product of ethanol (31), and, as such, is an important component of cellular defenses against toxic aldehydes (16). In addition, there is considerable evidence that chronic ethanol consumption induces lipid peroxidation in the liver, which results in the generation of reactive aldehydes, which are capable of forming adducts with proteins (19, 27). Recent studies (19) have determined that these aldehyde-protein adducts can be immunogenic and, as such, could potentially contribute to the pathophysiology of alcoholic liver injury. Analysis of low-Km ALDH in the present study showed a significant decrease in the specific activity of this enzyme in mitochondria isolated from ethanol-fed rats compared with controls, which was not due to a chronic alcohol-related decrease in ALDH protein. It is likely that some of the cysteine residues in ALDH may be particularly susceptible to modification by ethanol-dependent formation of electrophilic reactants including aldehydes or RNS. We postulate that the decrease in low-Km ALDH activity was due to modification of the cysteine residue present in the active site of the enzyme by chronic alcohol-related increases in ROS/RNS. Oxidative modification and inactivation of low-Km ALDH in response to chronic ethanol consumption represents a mechanism that could potentially amplify further production of reactive lipidderived aldehyde species leading to irreversible injury to mitochondria.
In conclusion, these studies have established a sensitive protocol for the identification of mitochondrial proteins with altered redox thiol status, which occurs following chronic alcohol consumption. Whether these modifications contribute to mitochondrial dysfunction following chronic alcohol exposure is not known. However, emerging evidence strongly suggests that alterations in protein thiol status might have biological relevance and contribute to the pathologies associated with several disease states. This area of research is at an early stage of development, but our demonstration that it is possible to focus on redox changes of an important subpopulation of proteins is of great significance for the identification of proteins modified and inactivated by ROS/RNS.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants AA-13682 (to S. M. Bailey) and AA-13395 (to V. Darley-Usmar). The MALDI-TOF mass spectrometer in the Univ. of Alabama at Birmingham (UAB) mass spectrometry shared facility was purchased with funds provided by NCRR Grant S10 RR-11329. Operation of the mass spectrometry shared facility was supported, in part, by the UAB Comprehensive Cancer Center core support Grant P30 CA-13148.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* A. Venkatraman and A. Landar contributed equally to the work.
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REFERENCES |
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