ABSTRACT
UNLABELLED: Hepatitis C virus (HCV) infection produces chronic liver injury that is significantly exacerbated by alcohol consumption. While multiple mechanisms contribute to this synergy, a viral-induced loss of antioxidant responses has been shown to play an important role. This study examined the effects of HCV infection and alcohol on the regulation of the transcription factor FOXO3, an important regulator of Mn-superoxide dismutase (SOD2) expression, a tumor suppressor, and a component of the hepatic antioxidant response system. FOXO3 was activated by either HCV or alcohol alone but suppressed by the combination. To understand this paradoxical result, we applied a capillary isoelectric focusing (IEF) method to determine the pattern of FOXO3 posttranslational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used antiphosphoserine, acetyl-lysine, methylarginine, and ubiquitin antibodies to identify the PTM patterns present in each species. HCV caused multiple changes including phosphorylation of FOXO3 at S-574, a novel c-Jun N-terminal kinase (JNK) site, which promoted nuclear translocation and transcription. Ethanol suppressed arginine-methylation of FOXO3 promoting nuclear export and degradation of the JNK phosphorylated form. Human liver biopsy samples showed the presence of the HCV-specific form of FOXO3 in HCV-infected livers but not in normal liver or nonalcoholic steatohepatitis. CONCLUSION: The development of this novel IEF method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 PTMs that contribute to pathogenesis. This approach will allow further dissection of the role of protein PTMs in viral liver disease.
Subject(s)
Alcohol Drinking/metabolism , Forkhead Transcription Factors/metabolism , Hepatitis C/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Protein Processing, Post-Translational , Amino Acid Substitution , Cell Line, Tumor , Ethanol/pharmacology , Forkhead Box Protein O3 , Forkhead Transcription Factors/drug effects , Humans , Isoelectric Focusing , Methylation/drug effects , Phosphorylation/drug effects , Solvents/pharmacologyABSTRACT
Viral infections frequently alter mitochondrial function with suppression or induction of apoptosis and enhanced generation of reactive oxygen species. The mechanisms of these effects are varied, and mitochondria are affected by both direct interactions with viral proteins and by secondary effects of viral-activated signaling cascades. This chapter describes methods used in our laboratory to assess the effects of the hepatitis C virus core protein on mitochondrial ROS production, electron transport, and Ca(2+) uptake. These include measurements of the effects of in vitro incubation of liver mitochondria with purified core protein and assessment of the function of mitochondria in cells and tissues expressing core and other viral proteins. These methods are generally applicable to the study of viral-mitochondrial interactions.
