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Mitochondrial dysfunction is recognized as a contributing factor to a number of diseases including chronic alcohol induced hepatotoxicity. While there is a detailed understanding of the metabolic pathways and proteins of the liver mitochondrion, little is known of how changes in the mitochondrial proteome contribute to the development of hepatic pathologies. In this short overview the insights gained from study of changes in the mitochondrial proteome in alcoholic liver disease will be described. Profiling the liver mitochondrial proteome has the potential to shed light on the alcohol-mediated molecular defects responsible for mitochondrial and cellular dysfunction. The methods presented herein demonstrate the power of using complementary proteomics approaches, i.e. 2-D IEF/SDS-PAGE and BN-PAGE, to identify changes in the abundance of mitochondrial proteins following chronic alcohol consumption. This proteomic data can then be integrated into a logical and mechanistic framework to further our understanding of the role of mitochondrial dysfunction in the pathogenesis of alcohol induced liver disease.
It is widely recognized that a key component in the development of alcohol induced liver disease is the diminished capacity of liver to generate and maintain sufficient levels of ATP. This resulting decrease in bioenergetic capacity is thought to contribute to decreased hepatocyte viability and ultimately the pathological changes that occur in alcohol-dependent hepatotoxicity. The importance of mitochondrial dysfunction in this disease process has long been appreciated due to chronic alcohol mediated defects on the oxidative phosphorylation system, which leads to decreased ATP synthesis (1,2). Moreover, evidence indicates that these chronic alcohol induced alterations in mitochondria structure and function also contribute to increased production of reactive oxygen and nitrogen species in mitochondria (3). This increase in mitochondrial oxidant production could result in post-translational oxidative modifications and inactivation of mitochondrial macromolecules, particularly proteins, thereby further compromising mitochondria function in the chronic alcohol consumer.
While mechanisms responsible for alcohol dependent mitochondrial dysfunction have been studied, the impact of chronic alcohol consumption on the overall content of mitochondrial proteins, i.e. the global mitochondrial proteome, has only recently been investigated (4,5). Early studies by Cunningham and colleagues demonstrated that chronic alcohol consumption decreases the synthesis of the 13 mitochondrial encoded proteins that are components of complexes I, III, IV, and V (6,7) due to defects in mtDNA (4,8,9) and a decrease in functional mitochondrial ribosomes (10,11). It is important to note however that there are well over 600 proteins that comprise the mitochondrial proteome (12,13), and that close to 100 of these are components of the oxidative phosphorylation system, most of which are encoded by the nuclear genome. As very little information is available on the effects of chronic alcohol consumption on the total mitochondrial proteome, especially effects on nuclear encoded proteins, we have used two complementary proteomics approaches, 2-D isoelectric focusing (IEF)/SDS-PAGE and blue native-PAGE (BN-PAGE) to improve our understanding of the impact these changes have on mitochondrial functionality and their contribution to the development of alcohol induced liver pathology.
It is hypothesized that chronic alcohol-induced alterations to the mitochondrial proteome negatively affects mitochondrial bioenergetics and signaling, which contribute, in part, to the development of liver pathology. To address this question our laboratories have begun to profile the mitochondrial proteome using both conventional 2-D IEF/SDS-PAGE and BN-PAGE. Recent advancements in the field have now made it possible to identify post-translational modifications to mitochondrial proteins by ROS/RNS, identify defects in the assembly of multi-protein complexes in mitochondria, and separate the more hydrophobic respiratory complex proteins of the inner membrane using newly refined gel electrophoresis methods (14).
A scheme depicting our strategy for detecting alterations to the mitochondrial proteome in models of chronic alcohol-induced liver injury is shown in Figure 1. Briefly, mitochondria isolated from the liver of control and alcohol-fed animals can be subjected to conventional 2-D IEF/SDS-PAGE and BN-PAGE. The high-resolution protein “maps” generated via these electrophoresis methods can then be analyzed for changes in protein abundance or a variety of well-defined ROS/RNS-mediated post-translational modifications using immunoblotting techniques. Finally, proteins of interest can then be identified using several mass spectrometry techniques. Proteomic studies from our laboratories are considered significant in that novel changes in several key energy metabolism pathways including fatty acid oxidation, TCA cycle, and oxidative phosphorylation system were found to be altered in mitochondria isolated from animals exposed to ethanol chronically (4,5,15). These alterations were in response to alcoholic mediated metabolic stress and correlated to those pathways with a direct impact on the etiology of disease. A detailed discussion of these techniques is provided in the sections below.
|Protein Amount||Quantity of Sample||Quantity of Rehydration Buffer|
|50 µg||5 µL||155 µL|
|100 µg||10 µL||150 µL|
|200 µg||20 µL||140 µL|
|Resolving gel solutions||Stacking gel solution|
|Protogel||0.66 µL||1.60 µL||1.91 µL||0.60 µL|
|Water||1.97 µL||0.56 µL||-||2.40 µL|
|3X Gel Buffer||1.34 µL||1.34 µL||1.16 µL||1.50 µL|
|Glycerol||-||0.47 µL||0.40 µL||-|
|10% AMPS||26.0 µL||26.0 µL||23.0 µL||70.0 µL|
|TEMED||4.0 µL||4.0 µL||3.50 µL||9.0 µL|
|2-D BN-PAGE Gel Buffer||2.98 mL|
|10% AMPS||60.0 µL|
The authors would like to thank Dr. Paul S. Brookes, University of Rochester, for advice and assistance in establishing the BN-PAGE technique in our laboratories. This work was supported by AA15172 (SMB) and AA13395 (VDU). The MALDI-TOF mass spectrometer in the UAB mass spectrometry shared facility was purchased with funds provided by NCRR Grant S10 RR-11329. Operation of the mass spectrometry shared facility came, in part, from the UAB Comprehensive Cancer Center Core Support Grant P30 CA-13148.
1Adjust the pH of these solutions at 4°C because gels are run in the cold room (4°C).
2The 3X gel buffer can be stored at room temperature. However, the pH must be adjusted at 4°C because gels are run in the cold room (4°C).
3These gel volumes can be proportionally increased to prepare a resolving gel that has an extended length. This is typically done if the 1-D BN-PAGE gels are to be run overnight to improve the resolution (i.e. separation) of the complexes. Similarly, when gels are run overnight the Cathode buffer can be switched to a “blue-free” buffer, i.e. 50 mM Tricine, 15 mM BisTris, pH 7.0, with no added Coomassie Blue G-250. This helps to minimize the high blue background when 1-D BN-PAGE gels are destained for imaging and densitometry purposes.
4For the separation of large membrane protein complexes it is recommended that the concentration of the Coomassie Blue G-250 in the sample be ¼ of the lauryl maltoside concentration for electrophoresis (17,18). Therefore, based on the given stock solutions and protocol for the extraction of inner membrane proteins this works out to be approximately 6.3 µL of a 5% (w/v) Coomassie Blue G-250 solution. Addition of a molar excess of dye to protein also helps to remedy one problem of native electrophoresis, i.e. the tendency of membrane proteins to form aggregates in the presence of detergents. However, the problem of aggregation is minimized by the inclusion of the Coomassie Blue G-250. This dye binds to the hydrophobic regions on the proteins surface, which induces a negative surface charge thereby reducing protein aggregation. In addition, the anionic nature of Coomassie Blue G-250 induces the needed “charge shift” on proteins such that they will migrate to the anode at pH 7.5 (17,18).