The Responses of Mitochondrial Proteome in Rat Liver to the

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The Responses of Mitochondrial Proteome in Rat Liver to the Consumption of Moderate Ethanol: The Possible Roles of Aldo-Keto Reductases Liang Shi,†,‡ Yuan Wang,†,‡ Shuyang Tu,†,‡ Xiaolei Li,†,‡ Maomao Sun,†,‡ Sanjay Srivastava,§ Ningzhi Xu,†,‡ Aruni Bhatnagar,*,§ and Siqi Liu*,†,‡,§ Beijing Genomics Institute, Chinese Academy of Science, Beijing, China, Beijing Proteomics Institute, Beijing, China, and Departement of Medicine, University of Louisville, Louisville, Kentucky Received December 16, 2007

A large body of evidence supports the view that mitochondria are a primary target of alcohol stress. Changes in mitochondrial proteins due to moderate ethanol intake, however, have not been broadly and accurately estimated. For this study, rats were fed low doses of ethanol and the mitochondria were isolated from heart, kidney, and liver, using ultracentrifugation with Nycodenz density gradient. The mitochondrial proteins were well resolved upon two-dimensional electrophoresis (2DE), and the alcohol-responsive 2DE spots were identified by matrix-assisted laser desorption/ionization-time-offlight mass spectrometry (MALDI-TOF/TOF MS). Compared with the control group, the proteins extracted from liver mitochondria of ethanol-fed rats exhibited the significant changes on 2DE images, whereas the 2DE images obtained from the kidney and the heart mitochondria remained almost unchanged by ethanol feeding. Significantly, over 50% of the alcohol-responsive proteins in liver mitochondria were members of aldo-keto reductase family (AKR), which were usually present in cytoplasm. The organelle distributions of AKR proteins in liver mitochondria were further confirmed by Western blot analysis as well as by confocal microscopy. In addition, translocations of AKR were examined in the CHANG cell line, which was cultured with and without ethanol. The results of Western blot strongly suggested that the abundances of AKR proteins in the mitochondria were greatly reduced by the presence of ethanol in culture medium. The results of this study show that, even with moderate ethanol feeding, the mitochondrial proteome in rat liver was more sensitive to alcohol stress than that of either the kidney or the heart. The translocation of AKR proteins may be involved in the detoxification of liver cells. Keywords: liver • mitochondria • alcohol • AKR • 2DE • MALDI-TOF/TOF MS • confocal

Introduction Alcohol abuse is associated with a wide variety of negative health outcomes including morbidity, mortality, and disability. A large body of data demonstrates that chronic consumption of excessive alcohol eventually results in many disorders in human organs, particularly the liver.1 In contrast, it has been suggested that consumption of moderate levels of alcohol could have protective effects.2 For instance, young adults consuming only 10-20 g of alcohol per day were found to have lower blood pressure than the abstinent group.3 Moderate alcohol consumption was also found to be associated with an increase in plasma high density lipoprotein (HDL) cholesterol concentrations and a decrease in low density lipoprotein (LDL) cholesterol concentrations.4 The molecular mechanisms underlying the benefits or the risk related with alcohol use are complex and require systematic investigation at a molecular level. * To whom correspondence should be addressed. E-mail: aruni.bhatnagar@ louisville.edu; [email protected]. † Chinese Academy of Science. ‡ Beijing Proteomics Institute. § University of Louisville. 10.1021/pr700853j CCC: $40.75

 2008 American Chemical Society

Ethanol intake is associated with an increase in oxidative stress. Nevertheless, the mechanisms, how ethanol triggers an increase of reactive oxygen species (ROS), remain unclear. The recent findings suggest that the mitochondrion may significantly contribute to the overall increase of oxidant levels in hepatocytes exposed to ethanol, acutely or chronically. Bailey et al. proposed that the oxidation of ethanol directly enhanced mitochondrial ROS production.5 On the other hand, the major intracellular antioxidant, reduced glutathione (GSH), 10-15% of which is located in mitochondria, was depleted in the liver of alcohol-fed rats.6 Obviously, mitochondria are likely to be a primary target of ethanol-induced ROS. The detailed molecular mechanisms for these events are well studied. With a variety of proteomics approaches, a coordinated decrease in both mitochondrial and nuclear encoded subunits of the respiratory complexes was detected, particularly in those that comprise cytochrome c oxidase.7 Moreover, many mitochondrial proteins altered their post-translational status in response to chronic alcohol consumption.8 Alcohol-dependent inactivation of ALDH, complexes of respiration chain, I, III, IV and V, and several β-oxidation enzymes were modified via oxidation and nitroJournal of Proteome Research 2008, 7, 3137–3145 3137 Published on Web 07/03/2008

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sation of thiols. However, all the studies have been accomplished upon the alcoholism animal model, and these results still lack convincing evidence to address the pathological and molecular development in animal model with ethanol intake at low dose. A primarily related question is if the tissue tolerance under moderate ethanol stress is similar to heavy ethanol intake. Next question should be addressed whether mitochondrial proteins are sensitive to ethanol intake at low dose. Finally, if the abundance of mitochondrial proteins is affected, is this proteomic change comparable to one responding to high ethanol intake? Hence, investigation of mitochondrial proteomes under moderate ethanol-feeding is highly expected to answer the inquiries above. To overview changes in mitochondrial proteomes induced by moderate intake of ethanol, three rat tissues, heart, kidney, and liver, were collected from the animals administrated with and without moderate alcohol for 12 weeks. The mitochondrial proteins prepared from these tissues were well separated by 2DE. Although after alcohol administration few differential 2DE spots were observed in rat heart or kidney mitochondrial proteins, most significant alterations in 2DE images were found in the protein samples of liver mitochondria. Surprisingly, upon MALDI-TOF/TOF MS, almost 50% of the alcohol-responsive 2DE spots in rat liver mitochondria were identified to be members of the AKR superfamily. These proteomic observations were further confirmed by other biological analysis, such as Western blot and confocal microscopy. Taken together, these data demonstrated for the first time that some members of the AKR family were located at rat liver mitochondria and relocated responding to the rising stress established of moderate ethanol.

Materials and Methods Chemicals. Acrylamide, ammonium persulfate, IPG strips (5 × 180 mm or 5 × 70 mm, pH 3-10 linear), ampholyte pH 3-10, urea, 3-[(3-chloromidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and all ultrapure reagents for electrophoresis were obtained from Bio-Rad (Hercules, CA). Tetramethylethylendiamine (TEMED), dithiothreitol (DTT), iodoacetamide (IAM), ammonium bicarbonate, trifluoroacetic acid, and R-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma (St. Louis, MO). Modified porcine trypsin was from Promega (Madison, WI). Swine antirabbit immunoglobulins labeled with fluorescein isothiocyanate (FITC) were purchased from Dako (Carpinteria, CA). Rat Model. The procedures of animal treatment were in accordance with recommendations published in the Guide for the Care and Use of Laboratory Animals.10 Adult male Wistar rats weighting about 300 g were divided into ethanol intake group and water drinking control. Ethanol-fed rats received 6% (v/v) ethanol in their drinking water during whole experimental period. The rats were housed under a 12 h/12 h light-dark cycle with access to standard rodent chow. The percentage composition by weight of the diet was 19% protein, 9.6% fat, 4.3% fiber, and 61% carbohydrate. Rat bodies were weighed once a week, and blood alcohol concentrations were measured in all the rats three times during the study. After 12 weeks feeding, the rats were sacrificed, and the freshly isolated tissues were immediately prepared for mitochondria or stored at -80 °C until use for proteomic experiments. Mitochondrial Preparation. The rat tissues were minced and homogenized with a hand-held Dounce homogenizer in the precool homogenization buffer, 220 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl, pH 7.4, and protease and phospho3138

Journal of Proteome Research • Vol. 7, No. 8, 2008

Shi et al. tase inhibitors. Using gradient centrifuge, the crude mitochondria were pelleted at 8000g. The crude preparation was further centrifuged through a Nycodenz density gradient from 20 to 34% at 52 000 g for 1.5 h. The refined rat mitochondria were enriched upon density fractionation. The integrity and purity of the purified mitochondria were assessed by transmission electron microscopy (TEM) and Western blot using mitochondrial specific antibody, prohibitin (Santa Cruz, CA) and cytoplasm specific antibody, acrA1 generated from our laboratory specific antibody. The purified mitochondria were stored at -80 °C until use. Two-Dimensional Electrophoresis. The mitochondrial proteins were extracted by the lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 10 mM DTT, 1 mM PMSF, 2 mM EDTA and 40 mM Tris-HCl, pH7.4. Total of 100 µg proteins/ sample were loaded for 2DE. Isoelectric focusing, firstdimensional electrophoresis, was carried out in the 18 cm, pH 3-10 L IPG strips and proceeded to 70 kVh with 0.05 mA/strip at room temperature. After reduction of 1% DTT for 15 min and alkylation of 2.5% IAM for 15 min, second-dimension SDSPAGE was conducted in 24 cm 12% acrylamide gels at 15 W/gel until the dye front reached to the bottoms of gels. The 2DE gels were stained by silver nitrate with a modified protocol. All the silver-stained gels were scanned by a laser densitometer at 500 pixel resolution (Powerlook 2100XL, UMAX, Dallas, TX). The gel images were analyzed with Imagemaster Platinum version 5.0 (GE Healthcare, Fairfield, CT). The differential 2DE spots were achieved through a paired comparison of 2DE images for the control and the alcohol-fed samples. Of these paired gels, the 2DE spots with 3 fold changes in spot volume were defined as the significantly differential ones. These spots were finally determined by statistical estimation upon parallel samples (4 rats/group) as well as parallel runs (2 runs/ sample). Identification of Differential 2DE Spots by MALDI-TOF/ TOF MS. The differential 2DE spots were manually excised and diced into small pieces (