Identification of oxidized and S‐nitrosylated mitochondrial proteins in alcohol‐exposed rat liver

Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen and nitrogen species (ROS/RNS) and mitochondrial dysfunction. Despite many reports about the roles of ROS and RNS in alcohol‐induced cell damage, the proteins that are oxidized by ROS and S‐nitrosylated by RNS are poorly characterized. We hypothesized that certain Cys residues of target proteins are oxidized and S‐nitrosylated by alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. To test these hypotheses, we recently developed a sensitive method using biotin‐N‐maleimide (biotin‐NM) as a probe to positively identify oxidized and S‐nitrosylated mitochondrial proteins in ethanol‐sensitive hepatoma cells. In this study, the biotin‐NM‐labeled oxidized and S‐nitrosylated mitochondrial proteins from alcohol‐exposed rat livers were purified with streptavidin‐agarose and resolved on 2‐DE. Many protein spots that displayed differential intensities on 2‐D gels were identified by mass spectrometry. Our results revealed that many proteins such as aldehyde dehydrogenase 2 are both oxidized and S‐nitrosylated while some other proteins were only oxidized (eg 3‐ketoacyl CoA thiolase) or S‐nitrosylated (eg malate dehydrogenase). Activities of these oxidized and S‐nitrosylated proteins were significantly reduced in the alcohol‐exposed rat livers compared to the corresponding controls. Our results thus explain the underlying mechanisms for the mitochondrial dysfunction in alcohol‐mediated cell/organ damage.