// Praveen K. Vayalil 1 , Aimee Landar 1 1 Department of Pathology, Division of Molecular and Cellular Pathology, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA Correspondence to: Praveen K. Vayalil, e-mail: praveen@uab.edu Keywords: mitochondria, prostate cancer, oncobioenergetics, bioenergetics, mitochondrial oncobioenergetic index Received: June 19, 2015 Accepted: September 04, 2015 Published: October 15, 2015 ABSTRACT Mitochondrial function is influenced by alterations in oncogenes and tumor suppressor genes and changes in the microenvironment occurring during tumorigenesis. Therefore, we hypothesized that mitochondrial function will be stably and dynamically altered at each stage of the prostate tumor development. We tested this hypothesis in RWPE-1 cells and its tumorigenic clones with progressive malignant characteristics (RWPE-1 < WPE-NA22 < WPE-NB14 < WPE-NB11 < WPE-NB26) using high-throughput respirometry. Our studies demonstrate that mitochondrial content do not change with increasing malignancy. In premalignant cells (WPE-NA22 and WPE-NB14), OXPHOS is elevated in presence of glucose or glutamine alone or in combination compared to RWPE-1 cells and decreases with increasing malignancy. Glutamine maintained higher OXPHOS than glucose and suggests that it may be an important substrate for the growth and proliferation of prostate epithelial cells. Glycolysis significantly increases with malignancy and follow a classical Warburg phenomenon. Fatty acid oxidation (FAO) is significantly lower in tumorigenic clones and invasive WPE-NB26 does not utilize FAO at all. In this paper, we introduce for the first time the mitochondrial oncobioenergetic index (MOBI), a mathematical representation of oncobioenergetic profile of a cancer cell, which increases significantly upon transformation into localized premalignant form and rapidly falls below the normal as they become aggressive in prostate tumorigenesis. We have validated this in five prostate cancer cell lines and MOBI appears to be not related to androgen dependence or mitochondrial content, but rather dependent on the stage of the cancer. Altogether, we propose that MOBI could be a potential biomarker to distinguish aggressive cancer from that of indolent disease.
Vascular endothelial cells (ECs) are important for maintaining vascular homeostasis. Dysfunction of ECs contributes to cardiovascular diseases, including atherosclerosis, and can impair the healing process during vascular injury. An important mediator of EC response to stress is the GTPase Rac1. Rac1 responds to extracellular signals and is involved in cytoskeletal rearrangement, reactive oxygen species generation and cell cycle progression. Rac1 interacts with effector proteins to elicit EC spreading and formation of cell-to-cell junctions. Rac1 activity has recently been shown to be modulated by glutathiolation or S-nitrosation via an active site cysteine residue. However, it is not known whether other redox signaling compounds can modulate Rac1 activity. An important redox signaling mediator is the electrophilic lipid, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). This compound is a downstream product of cyclooxygenase and forms covalent adducts with specific cysteine residues, and induces cellular signaling in a pleiotropic manner. In this study, we demonstrate that a biotin-tagged analog of 15d-PGJ2 (bt-15d-PGJ2) forms an adduct with Rac1 in vitro at the C157 residue, and an additional adduct was detected on the tryptic peptide associated with C178. Rac1 modification in addition to modulation of Rac1 activity by bt-15d-PGJ2 was observed in cultured ECs. In addition, decreased EC migration and cell spreading were observed in response to the electrophile. These results demonstrate for the first time that Rac1 is a target for 15d-PGJ2 in ECs, and suggest that Rac1 modification by electrophiles such as 15d-PGJ2 may alter redox signaling and EC function.
Cellular redox signalling is mediated by the post-translational modification of proteins in signal-transduction pathways by ROS/RNS (reactive oxygen species/reactive nitrogen species) or the products derived from their reactions. NO is perhaps the best understood in this regard with two important modifications of proteins known to induce conformational changes leading to modulation of function. The first is the addition of NO to haem groups as shown for soluble guanylate cyclase and the newly discovered NO/cytochrome c oxidase signalling pathway in mitochondria. The second mechanism is through the modification of thiols by NO to form an S-nitrosated species. Other ROS/RNS can also modify signalling proteins although the mechanisms are not as clearly defined. For example, electrophilic lipids, formed as the reaction products of oxidation reactions, orchestrate adaptive responses in the vasculature by reacting with nucleophilic cysteine residues. In modifying signalling proteins ROS/RNS appear to change the overall activity of signalling pathways in a process that we have termed 'redox tone'. In this review, we discuss these different mechanisms of redox cell signalling, and give specific examples of ROS/RNS participation in signal transduction.
Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology. Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology. Chronic ethanol consumption causes liver damage by a complex process thought to involve oxidative and nitrosative stress, hypoxia, up-regulation of proinflammatory cytokines, and defects in energy metabolism (1Arteel G.E. Gastroenterology. 2003; 124: 778-790Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 2Cunningham C.C. Bailey S.M. Biol. Signals Recept. 2001; 10: 271-282Crossref PubMed Scopus (107) Google Scholar, 3Hoek J.B. Pastorino J.G. Alcohol. 2002; 27: 63-68Crossref PubMed Scopus (402) Google Scholar). As both a source for the formation and target of modifications mediated by reactive oxygen and nitrogen species, the mitochondrion is recognized as a site critical in the cellular stress response induced by chronic ethanol exposure. Increased mitochondrial production of reactive oxygen species (4Kulielka E. Dicker E. Cederbaum A.I. Arch. Biochem. Biophys. 1994; 309: 377-386Crossref PubMed Scopus (146) Google Scholar, 5Bailey S.M. Cunningham C.C. Hepatology. 1998; 28: 1318-1326Crossref PubMed Scopus (167) Google Scholar, 6Bailey S.M. Pietsch E.C. Cunningham C.C. Free Radic. Biol. Med. 1999; 27: 891-900Crossref PubMed Scopus (181) Google Scholar), oxidation of mitochondrial proteins (7Venkatraman A. Landar A. Davis A.J. Ulasova E. Page G. Murphy M.P. Darley-Usmar V. Bailey S.M. Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 286: G521-G527Crossref PubMed Scopus (78) Google Scholar, 8Wieland P. Lauterberg B.H. Biochem. Biophys. Res. Commun. 1995; 213: 815-819Crossref PubMed Scopus (90) Google Scholar, 9Rouach H. Fataccioli V. Gentil M. French S.W. Morimoto M. Nordmann R. Hepatology. 1997; 25: 351-355Crossref PubMed Scopus (200) Google Scholar, 10Bailey S.M. Patel V.B. Young T.A. Asayama K. Cunningham C.C. Alcohol. Clin. Exp. Res. 2001; 25: 726-733Crossref PubMed Scopus (150) Google Scholar), depressed oxidative phosphorylation activity (11Bailey S.M. Free Radic. Res. 2003; 37: 585-596Crossref PubMed Scopus (83) Google Scholar, 12Cunningham C.C. Coleman W.B. Spach P.I. Alcohol Alcohol. 1990; 25: 127-136Crossref PubMed Scopus (147) Google Scholar, 13Hoek J.B. Curr. Top. Bioenerg. 1994; 17: 197-241Crossref Scopus (34) Google Scholar), and disrupted fatty acid metabolism (14Koteish A. Diehl A.M. Semin. Liver Dis. 2001; 21: 89-104Crossref PubMed Scopus (408) Google Scholar, 15Jaeschke H. Gores G.J. Cederbaum A.I. Hinson J.A. Pessayre D. Lemasters J.J. Toxicol. Sci. 2002; 65: 166-176Crossref PubMed Scopus (1046) Google Scholar, 16Baraona E. Lieber C.S. Recent Dev. Alcohol. 1998; 14: 97-134Crossref PubMed Scopus (79) Google Scholar) occur following consumption of alcohol, indicating that ethanol induces significant changes in mitochondria physiology. These responses are also accompanied by a profound increase in the sensitivity of the respiratory chain to inhibition by NO, which we propose plays a key role in contributing to the hypoxia associated with ethanol-dependent hepatotoxicity (17Venkatraman A. Shiva S. Davis A.J. Bailey S.M. Brookes P.S. Darley-Usmar V. Hepatology. 2003; 38: 141-147Crossref PubMed Scopus (51) Google Scholar). While mechanisms responsible for ethanol-induced mitochondrial dysfunction have been investigated, the impact of chronic ethanol consumption on the overall content of mitochondrial proteins, the "mitochondrial proteome," has not been studied. Earlier studies by Cunningham and colleagues (18Coleman W.B. Cunningham C.C. Biochim. Biophys. Acta. 1990; 1019: 142-150Crossref PubMed Scopus (73) Google Scholar, 19Coleman W.B. Cunningham C.C. Biochim. Biophys. Acta. 1991; 1058: 178-186Crossref PubMed Scopus (64) Google Scholar) have demonstrated that ethanol consumption decreases the synthesis of the 13 mitochondrially encoded proteins that comprise respiratory complexes I, III, and IV and the ATP synthase. It is proposed that the inhibition of mitochondrial protein synthesis following chronic ethanol exposure is due to defects in mtDNA (20Cahill A. Wang X. Hoek J.B. Biochem. Biophys. Res. Commun. 1997; 235: 286-290Crossref PubMed Scopus (98) Google Scholar, 21Cahill A. Stabley G.J. Wang X. Hoek J.B. Hepatology. 1999; 30: 881-888Crossref PubMed Scopus (67) Google Scholar) and a decline in the number of functional mitochondrial ribosomes resulting from chronic ethanol exposure (22Cahill A. Baio D.L. Ivester P.I. Cunningham C.C. Alcohol. Clin. Exp. Res. 1996; 20: 1362-1367Crossref PubMed Scopus (25) Google Scholar, 23Patel V.B. Cunningham C.C. Arch. Biochem. Biophys. 2002; 398: 41-50Crossref PubMed Scopus (31) Google Scholar). It is important to note that the mitochondrial proteome involves over 100 proteins as components of oxidative phosphorylation, most of which are encoded by the nuclear genome. Little or no information is available on the effects of ethanol on these nuclear encoded proteins, and because they are thought to play an important regulatory role in mitochondrial function this is potentially important in understanding the basis of mitochondrial pathologies. Indeed the mechanisms by which chronic ethanol alters the mitochondrial genome or levels of other mitochondrial proteins and the role that these changes may play in the development of liver injury from chronic ethanol exposure are not known. Ethanol-dependent hepatotoxicity, particularly in the early stages that precede cirrhosis, is then an ideal model to examine the response of the mitochondrial proteome in an organ exposed to a metabolic stress.Two-dimensional gel electrophoresis is the central tool for proteomic analysis. This technique combines isoelectric focusing (IEF) 1The abbreviations used are: IEF, isoelectric focusing; BN, blue native; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; QPCR, quantitative PCR; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Hsp, heat shock protein. 1The abbreviations used are: IEF, isoelectric focusing; BN, blue native; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; QPCR, quantitative PCR; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Hsp, heat shock protein. in the first dimension where proteins are separated according to differences in net charge followed by the separation of proteins based on molecular weight in the second dimension using standard SDS-PAGE. This technique is capable of resolving hundreds to thousands of proteins in a complex biological sample on a single two-dimensional gel that can then be identified by mass spectrometry. A number of proteomic approaches have been used with mitochondria including sucrose density gradient fractionation and immunocapture techniques, and these have identified ∼600 proteins as members of the mitochondrial proteome (24Murray J. Zhang B. Taylor S.W. Oglesbee D. Fahy E. Marusich M.F. Ghosh S.S. Capaldi R.A. J. Biol. Chem. 2003; 278: 13619-13622Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 25Taylor S.W. Fahy E. Zhang B. Glenn G.M. Warnock D.E. Wiley S. Murphy A.N. Gaucher S.P. Capaldi R.A. Gibson B.W. Ghosh S.S. Nat. Biotechnol. 2003; 21: 281-286Crossref PubMed Scopus (591) Google Scholar). While conventional two-dimensional IEF/SDS-PAGE is well suited to identify changes in the levels of the more hydrophilic proteins of the mitochondrion, e.g. matrix proteins, analysis of membrane proteins is hampered by the fact that many of these proteins precipitate at the basic end of the IEF gels and are thus poorly resolved on conventional two-dimensional gels (26Lopez M.F. Kristal B.S. Chernokalskaya E. Lazarev A. Shestopalov A.I. Bogdanova A. Robinson M. Electrophoresis. 2000; 21: 3427-3440Crossref PubMed Scopus (188) Google Scholar, 27Santoni V. Molloy M. Rabilloud T. Electrophoresis. 2000; 21: 1054-1070Crossref PubMed Scopus (827) Google Scholar, 28Hanson B.J. Schulenberg B. Patton W.F. Capaldi R.A. Electrophoresis. 2001; 22: 950-959Crossref PubMed Scopus (87) Google Scholar). Therefore, alternate protein separation techniques are required to define changes in the levels of membrane proteins. To address this issue we used the two-dimensional blue native (BN)-PAGE technique developed by Schagger and von Jagow (29Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1887) Google Scholar, 30Schagger H. Methods Enzymol. 1996; 264: 555-566Crossref PubMed Google Scholar) and modified by Brookes and colleagues (31Brookes P.S. Pinner A. Ramachandran A. Coward L. Barnes S. Kim H. Darley-Usmar V.M. Proteomics. 2002; 2: 969-977Crossref PubMed Scopus (141) Google Scholar) to complement our two-dimensional gel analyses of the mitochondrial proteome. Using this approach changes in the levels of both mitochondrial and nuclear encoded proteins that comprise the respiratory complexes following chronic ethanol consumption can be assessed. Moreover because the first dimension BN-PAGE is done under non-denaturing conditions information regarding protein-protein interactions and the assembly of oxidative phosphorylation complexes is retained. This is particularly important because it will enable probing the molecular basis of the observation that chronic ethanol consumption decreases the functioning of the oxidative phosphorylation system (12Cunningham C.C. Coleman W.B. Spach P.I. Alcohol Alcohol. 1990; 25: 127-136Crossref PubMed Scopus (147) Google Scholar, 13Hoek J.B. Curr. Top. Bioenerg. 1994; 17: 197-241Crossref Scopus (34) Google Scholar).In this investigation, global alterations in mitochondrial protein levels and mtDNA damage were studied in a well characterized rodent model of chronic ethanol feeding (32Lieber C.S. DeCarli L.M. Alcohol. Clin. Exp. Res. 1982; 6: 523-531Crossref PubMed Scopus (600) Google Scholar) that is known to cause hepatic mitochondrial dysfunction (12Cunningham C.C. Coleman W.B. Spach P.I. Alcohol Alcohol. 1990; 25: 127-136Crossref PubMed Scopus (147) Google Scholar, 13Hoek J.B. Curr. Top. Bioenerg. 1994; 17: 197-241Crossref Scopus (34) Google Scholar) and oxidative and nitrosative stress (5Bailey S.M. Cunningham C.C. Hepatology. 1998; 28: 1318-1326Crossref PubMed Scopus (167) Google Scholar, 33Baraona E. Zeballos G.A. Shoichet L. Mak K.M. Lieber C.S. Alcohol. Clin. Exp. Res. 2002; 26: 883-889Crossref PubMed Google Scholar) in the early stage of the disease process. Using both the proteomic approaches described above, alterations in the levels of proteins involved in β-oxidation of fatty acids and the oxidative phosphorylation system were found in mitochondria isolated from the livers of rats chronically exposed to ethanol. Moreover significant liver mtDNA damage was detected in response to ethanol consumption. This result is important because it demonstrates that ethanol exposure alters the mitochondrial genome during the very early stages of ethanol-induced liver injury, i.e. fatty liver stage, in young animals. These data are consistent with the hypothesis that chronic ethanol initiates reactions that induce mtDNA damage, which in turn negatively impacts mitochondrial protein synthesis and functioning of the oxidative phosphorylation system. Furthermore the results from these proteomic analyses reveal that although the response of liver to chronic ethanol exposure is a complex process the number of changes in proteins is limited to a specific group of metabolic pathways.EXPERIMENTAL PROCEDURESMaterials—Lieber-DeCarli ethanol and control liquid diets were purchased from Bio-Serv (Frenchtown, NJ). Immobiline pH gradient strips, ampholytes, and molecular weight standards were purchased from Amersham Biosciences. Monoclonal antibodies specific for complex I iron-sulfur protein 5 and 7, complex III subunit I, complex IV subunit I, and ATP synthase α and β subunits were purchased from Molecular Probes (Eugene, OR). All other biochemicals were obtained from Sigma or Bio-Rad.Animals and Diets—Male Sprague-Dawley rats (200 g) were individually housed in suspended cages and maintained under a 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 (32Lieber C.S. DeCarli L.M. Alcohol. Clin. Exp. Res. 1982; 6: 523-531Crossref PubMed Scopus (600) Google Scholar). 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 as described previously (5Bailey S.M. Cunningham C.C. Hepatology. 1998; 28: 1318-1326Crossref PubMed Scopus (167) Google Scholar). These studies were approved by the local institutional animal care and use committee.Isolation of Rat Liver Mitochondria—Coupled liver mitochondria were prepared by differential centrifugation of liver homogenates (34Spach P.I. Bottenus R.E. Cunningham C.C. Biochem. J. 1982; 202: 445-452Crossref PubMed Scopus (49) Google Scholar, 35Thayer W.S. Rubin E. J. Biol. Chem. 1979; 254: 7717-7723Abstract Full Text PDF PubMed Google Scholar) 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 (10Bailey S.M. Patel V.B. Young T.A. Asayama K. Cunningham C.C. Alcohol. Clin. Exp. Res. 2001; 25: 726-733Crossref PubMed Scopus (150) Google Scholar). The total mitochondria yield from control animals was 220 ± 20 mg of protein and from ethanol-treated animals was 260 ± 10 mg of protein (n = 8, p = 0.19).Two-dimensional Gel Electrophoresis—First dimension IEF was performed using 11-cm immobilized pH gradient strips with pH range 3–10 using an in-gel rehydration method. Briefly mitochondrial protein from control and ethanol-fed rats were diluted in a rehydration solution containing 40 mm Tris-HCl, pH 8.8, 7 m urea, 2 m thiourea, 4% CHAPS, 2% ampholytes (pH 3–10), and 5 mm tributylphosphine and incubated for 30 min at room temperature to fully solubilize samples. After incubation, 180 μg of protein was applied to IEF gels, and the gels were allowed to rehydrate overnight. IEF was performed for 1 min at 300 V, 1.5 h at a 300–3500-V gradient, and then held at 3500 V for 4.5 h using an Amersham Biosciences Multiphor II system. After IEF, the immobilized pH gradient strips were equilibrated for 20 min in 50 mm Tris-HCl, pH 8.8, 6 m urea, 20% glycerol, 2% SDS, 0.005% bromphenol blue, and 65 mm dithiothreitol followed by a 20-min incubation in the same buffer containing 2.5% iodoacetamide in place of dithiothreitol before SDS-PAGE. Two-dimensional SDS-PAGE was performed using 10% homogenous Tris-glycine acrylamide gels (Bio-Rad Criterion precast gels). After electrophoresis, gels were stained with Sypro Ruby Red (Molecular Probes), and gel images were captured with excitation and emission wavelengths of 480 and 620 nm, respectively, using the ProXpress imager (PerkinElmer Life Sciences).Blue Native Gel Electrophoresis—BN-PAGE was performed using the method developed by Schagger and von Jagow (29Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1887) Google Scholar, 30Schagger H. Methods Enzymol. 1996; 264: 555-566Crossref PubMed Google Scholar) and modified by Brookes et al. (31Brookes P.S. Pinner A. Ramachandran A. Coward L. Barnes S. Kim H. Darley-Usmar V.M. Proteomics. 2002; 2: 969-977Crossref PubMed Scopus (141) Google Scholar). All buffers and solutions were kept at 4 °C and pH 7.0. Mitochondrial pellets (1 mg of protein) were resuspended in 113 μl of extraction buffer containing 0.75 m aminocaproic acid, 50 mm BisTris, and 1% n-dodecyl-β-d-maltoside and allowed to incubate on ice for 60 min. After incubation, samples were centrifuged at 14,000 rpm for 5 min, and the supernatant containing the extracted mitochondrial complexes was collected and kept on ice. Protein content was measured by the Bradford method, and 2.5 μl of a 5% (w/v) suspension of Coomassie Brilliant Blue G-250 (Serva Blue G) in 0.5 m aminocaproic acid was added to 60 μg of protein (∼30 μl by volume). Samples were then stored on ice for no more than 30 min before being loaded onto a non-denaturing 5–16.5% gradient gel to separate the individual oxidative phosphorylation complexes intact. Electrophoresis buffers and conditions are as described previously (31Brookes P.S. Pinner A. Ramachandran A. Coward L. Barnes S. Kim H. Darley-Usmar V.M. Proteomics. 2002; 2: 969-977Crossref PubMed Scopus (141) Google Scholar).After native electrophoresis, the entire vertical lane containing all the mitochondrial protein complexes was cut from the gel, rotated 90°, and laid on top of a denaturing 5–15% Tris-Tricine SDS-PAGE gel to resolve the individual polypeptides of the complexes based on molecular weight (31Brookes P.S. Pinner A. Ramachandran A. Coward L. Barnes S. Kim H. Darley-Usmar V.M. Proteomics. 2002; 2: 969-977Crossref PubMed Scopus (141) Google Scholar). Samples from each pair of control and ethanol-treated mitochondria were resolved on one SDS-PAGE gel to control for intergel differences. In some experiments the individual oxidative phosphorylation complexes were excised from the first dimension BN-PAGE gel and applied to the top of a denaturing SDS-polyacrylamide gel to resolve individual proteins in that complex. For these experiments, each control and ethanol sample was run in duplicate so that one gel could be stained and the second gel could be immunoblotted to nitro-cellulose membranes. Gels were stained using a mixture of Coomassie Blue R-250 and G-250 (0.05% (w/v) each in 25% isopropanol, 10% glacial acetic acid). For immunoblot analysis, proteins were transferred to 0.2-μm nitrocellulose membranes and blocked with 5% nonfat milk. Levels of mitochondrial proteins were detected using a 1:10,000 dilution of primary antibodies (complex I iron-sulfur protein 5 and 7, complex III subunit I, complex IV subunit I, or ATP synthase α and β subunits) followed by incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-mouse IgG. Binding of the secondary antibody was visualized by enhanced chemiluminescence.Image Analysis of Two-dimensional Gels—Mitochondrial proteins from control and ethanol-fed rats were separated by the proteomic methods described above. Gels were scanned, saved as TIFF files, and analyzed for differences in protein expression using PDQuest Image Analysis software (Bio-Rad). Because the gels were stained with Sypro Ruby Red it was necessary to invert the data from the gels so that the fluorescent protein spots could be identified by the software as dark protein spot density against a light background. Protein spots were identified and a matched set containing five control gels with the corresponding ethanol gels was created. To compare protein spot density across different gels a reference gel (i.e. master image) was selected. To increase the likelihood that the highest possible number of protein spots in each gel could be matched to the corresponding protein spots in the master image, the selection of the master image was based on the highest number of detected proteins spots and the best protein spot resolution across the whole proteome. Thus, the control gel from pair 1 met all the requirements and was used as the master image for protein spot matching purposes. Automatic matching of protein spots in each gel to corresponding protein spots in the master image was performed, and the protein spots that were incorrectly matched to the reference gel were manually corrected. To correct for any possible intergel protein loading differences, the data for all protein spots in a given gel were normalized to the total density in valid protein spots for that gel. Normalized protein spot densities were transferred to Microsoft Excel, and -fold changes for each pair were determined. The -fold changes were then averaged resulting in a mean-fold change ± S.E. Normalized protein spot densities from control gels were compared with normalized protein spot densities from ethanol gels using a two-tailed paired Student's t test. Scanned TIFF images for one-dimensional and two-dimensional BN-PAGE were analyzed using Scion Image Beta 4.02 (Scion Corp.) after removing the background uniformly across all the pairs. The areas under the curve for the individual spots obtained from mitochondria isolated from ethanol-fed animals were normalized to their respective pair-fed controls and represented as -fold change. A two-tailed paired Student's t test was performed on the BN-PAGE raw data.Mass Spectrometry—Protein spots found by the image analysis to have altered levels of expression as a result of chronic ethanol feeding were excised from gels and subjected to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry essentially as described in Brookes et al. (31Brookes P.S. Pinner A. Ramachandran A. Coward L. Barnes S. Kim H. Darley-Usmar V.M. Proteomics. 2002; 2: 969-977Crossref PubMed Scopus (141) Google Scholar) to identify these proteins. Gel pieces were washed with 5% acetonitrile to remove Coomassie Blue, SDS, and salts. After destaining, the gel pieces were dried, rehydrated with a trypsin-containing solution, and allowed to digest for 16–20 h. The in-gel tryptic digest was extracted several times with 50% acetonitrile, 5% formic acid. An aliquot of the peptide extract was mixed with an equal volume of the matrix α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, and 1.0 μl of this mixture was spotted onto a gold target plate for MALDI-TOF analysis 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 the MASCOT search engine, 2See www.matrixscience.com. and the National Center for Biotechnology Information data base was searched to match the tryptic peptide fingerprint with a parent polypeptide.Mitochondrial DNA Damage Assessment by Quantitative PCR (QPCR)—Genomic DNA was extracted from liver homogenates, quantified using PicoGreen (Molecular Probes) on a Cytofluor 4000 Series fluorometer, and 15 ng was used for QPCR. QPCR assesses generalized DNA damage in a gene-specific manner in which DNA lesions block rTth polymerase and lead to a decrease in amplification (36Ballinger S.W. Patterson C. Yan C.N. Doan R. Burow D.L. Young C.G. Yakes F.M. Van Houten B. Ballinger C.A. Freeman B.A. Runge M.S. Circ. Res. 2000; 86: 960-966Crossref PubMed Scopus (365) Google Scholar, 37Ballinger S.W. Van Houten B. Jin G.F. Conklin C.A. Godley B.F. Exp. Eye Res. 1999; 68: 765-772Crossref PubMed Scopus (182) Google Scholar). Sensitivity of the assay is increased through amplification of large targets that increases the probability of encountering a DNA lesion. A 16,059-bp QPCR product, which encompasses all but 236 bp of NADH5/6 genes in the mitochondrial genome was amplified using primer set M13597 FOR (bp 13597–13620) and 13361 REV (bp 13361–13337). Copy number differences in mitochondrial DNA were normalized using a short QPCR, which yields products directly related to gene copy number using primer pair 13597F/13713R (5′-CCCAGCTACTACCATCTTCAAGT/GATGGTTTGGGAGATTGGTTGATGT-3′) for mitochondrial DNA.Mitochondrial DNA damage was quantified by comparing the relative efficiency of amplification of large (>15-kb) fragments of DNA and normalizing this to gene copy numbers by the amplification of smaller (<250-bp) fragments, which have a statistically negligible likelihood of containing damaged bases. Hence, to measure mtDNA damage, long (L) and short (S) QPCRs were performed to determine DNA damage and number of gene copies present, respectively. To calculate lesion frequencies, the long QPCR values (L) were divided by the corresponding short QPCR results (S) to account for potential copy number differences between samples (L/S). Normalized values from damaged (Ld/Sd = Ad) samples were compared with non-damaged controls (Lo/So = Ao) resulting in a relative amplification ratio (Ad/Ao). Assuming a random distribution of lesions and using the Poisson equation (f(x) = eλλx/x! where λ = the average lesion frequency) for the non-damaged templates (zero class; x = 0), the average lesion frequency per DNA strand was determined: λ = -ln Ad/Ao·A.RESULTSFor these experiments, animals were fed a diet containing 36% total calories as ethanol for 5–6 weeks. Under these conditions a number of changes in mitochondrial function occur including inhibition of mitochondrial protein synthesis, increased formation of reactive oxygen and nitrogen species, and increased sensitivity to the regulation of respiration by NO (5Bailey S.M. Cunningham C.C. Hepatology. 1998; 28: 1318-1326Crossref PubMed Scopus (167) Google Scholar, 17Venkatraman A. Shiva S. Davis A.J. Bailey S.M. Brookes P.S. Darley-Usmar V. Hepatology. 2003; 38: 141-147Crossref PubMed Scopus (51) Google Scholar, 18Coleman W.B. Cunningham C.C. Biochim. Biophys. Acta. 1990; 1019: 142-150Crossref PubMed Scopus (73) Google Scholar). This exposure of ethanol decreased the activity of mitochondrial complexes I, III, and IV (Table I) in agreement with previous studies (12Cunningham C.C. Coleman W.B. Spach P.I. Alcohol Alcohol. 1990; 25: 127-136Crossref PubMed Scopus (147) Google Scholar). Chronic ethanol consumption had no effect on citrate synthase activity (Table I) and total mitochondria protein yield, indicating that ethanol feeding had little effect on overall mitochondrial content or purity. This preparation does contain some minor contamination from other nonmitochondrial membrane sources but is functionally viable for the periods needed for respiratory measurement and proteomics.Table IChronic ethanol consumption decreases the activity of respiratory complexes I, III, and IV in liver Complex I activity was assessed by measuring the oxygen consumption of mitochondria isolated from control and ethanol-fed rats in the presence of glutamate/malate (5 mm) and ADP (0.5 mm). Antimycin-sensitive complex III activity was assessed indirectly by measuring the respiratory rate in the presence of succinate (15 mm), ADP (0.5 mm), and antimycin (2 nm). Complex IV (cytochrome c oxidase) and citrate synthase activities were measured using standard spectrophotometric methods (65Shepherd J.A. Garland G.P. Methods Enzymol. 1969; 13: 11-19Crossref Scopus (411) Google Scholar, 66Wharton D.C. Tzagoloff A. Methods Enzymol. 1967; 10: 245-250Crossref Scopus (1330) Google Scholar). Data are expressed as the mean ± S.E. for five pairs of control and ethanol-fed rats, and statistical analysis was performed using a paired
Supplementary Figure 3 from Homotypic Gap Junctional Communication Associated with Metastasis Suppression Increases with PKA Activity and Is Unaffected by PI3K Inhibition
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