Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

Protein carbonylation has been associated with various pathophysiological processes. A representative reactive carbonyl species (RCS), 4-hydroxy-2-nonenal (HNE), has been implicated specifically as a causative factor for the initiation and/or progression of various diseases. To date, however, little is known about the proteins and their modification sites susceptible to “carbonyl stress” by this RCS, especially in the liver. Using chemoprecipitation based on a solid-phase hydrazine chemistry coupled with LC–MS/MS bottom-up approach and database searching, we identified several protein-HNE adducts in isolated rat liver mitochondria upon HNE exposure. The identification of selected major protein targets, such as the ATP synthase β-subunit, was further confirmed by immunoblotting and a gel-based approach in combination with LC–MS/MS. A network was also created based on the identified protein targets, which showed that the main protein interactions were associated with cell death, tumor morphology and drug metabolism, implicating the toxic nature of HNE in the liver mitoproteome. The functional consequence of carbonylation was illustrated by its detrimental impact on the activity of ATP synthase, a representative major mitochondrial protein target for HNE modifications

Posttranslational carbonylation of mitochondrial proteins in the rat liver by 4-hydroxy-2-nonenal in vitro revealed protein targets associated with cell death, tumor morphology, drug metabolism and impact on oxidative phosphorylation.Figure optionsDownload high-quality image (167 K)Download as PowerPoint slideHighlights
► Proteomic identification of several protein adducts after exposing isolated rat liver mitochondria to 4-hydroxy-2-nonenal.
► Pathway analysis linked protein targets to cell death, tumor morphology and drug metabolism.
► Implication of the findings to the toxic impact of peroxidising lipid membranes on the liver mitoproteome.
► Demonstration of the functional consequence of reactive carbonyl stress on mitochondrial ATP synthase activity.