Proteomic analysis of 4-hydroxynonenal (4-HNE) modified proteins in liver mitochondria from chronic ethanol-fed rats

Chronic ethanol-mediated oxidative stress and lipid peroxidation increases the levels of various reactive lipid species including 4-hydroxynonenal (4-HNE), which can subsequently modify proteins in the liver. It has been proposed that 4-HNE modification adversely affects the structure and/or function of mitochondrial proteins, thereby impairing mitochondrial metabolism. To determine whether chronic ethanol consumption increases levels of 4-HNE modified proteins in mitochondria, male rats were fed control and ethanol-containing diets for 5 weeks and mitochonclrial samples were analyzed using complementary proteomic methods. Five protein bands (approx. 35, 45, 50, 70, and 90 kDa) showed strong immunoreactivity for 4-HNE modified proteins in liver mitochondria from control and ethanol-fed rats when proteins were separated by standard 1D SDS-PAGE. Using high-resolution proteomic methods (2D IEF/SDS-PAGE and BN-PAGE) we identified several mitochondrial proteins immunoreactive for 4-HNE, which included mitofilin, dimethylglycine dehydrogenase, choline dehydrogenase, electron transfer flavoprotein alpha, cytochrome c(1), enoyl CoA hydratase, and cytochrome c. The electron transfer flavoprotein a consistently showed increased 4-HNE immunoreactivity in mitochondria from ethanol-fed rats as compared to mitochondria from the control group. Increased 4-HNE reactivity was also detected for dimethylglycine dehydrogenase, enoyl CoA hydratase, and cytochrome c in ethanol samples when mitochondria were analyzed by BN-PAGE. In summary, this work identifies new targets of 4-HNE modification in mitochondria and provides useful information needed to better understand the molecular mechanisms underpinning chronic ethanol induced mitochondrial dysfunction and liver injury. (C) 2014 The Authors. Published by Elsevier B.V.