PP15 - Interplay between lipid and protein carbonylation in a cardiomyocyte model of nitrosative stress

Abstract

Carbonylation of biomolecules, a stable oxidative modification yielding reactive carbonyl groups, is a well-accepted biomarker of oxidative damage, which has been linked to several oxidative stress (OS) related human diseases. Protein carbonylation can be induced via several independent mechanisms, including direct oxidation of amino acid residues or modification of nucleophilic residues with products of lipid and carbohydrate oxidation. Lipid-protein adducts are believed to be a major source of protein-bound carbonyls. Lipid peroxidation products (LPP) containing reactive carbonyls can modify proteins in two different ways via - (i) Schiff base formation with a loss of the carbonyl function or (ii) Michael addition resulting in protein carbonylation. Although oxidative modifications of lipids and proteins in biological systems are closely interconnected, they are rarely studied together. Here we combined a mass spectrometry (MS) based analysis of OS-derived LPP with proteomics targeting modified proteins for high-throughput identification of lipid-protein adducts in a dynamic cellular model of OS. The influence of nitrosative stress on carbonylation of proteins and lipids was investigated in primary cardiomyocytes treated with SIN-1 for different time intervals. Twenty-five carbonylated lipids were identified by LC-MS and considered as possibly modifying agents yielding the corresponding LPP-modified proteins. A combination of different proteomics techniques allowed identifying over 200 of these modified proteins. Systems biology analysis of modified proteins revealed alterations in Ca2+ -signalling pathways. Ca2+ mobilization was studied by activating voltage-dependent Ca2+ channels, ryanodine receptor 2 and inositol-trisphosphate receptor. Significant alterations in the activity of LPP-modified Ca2+ -channels were observed after 70 min of SIN-1 treatment. Thus, the combination of lipidomics, proteomics, and biochemical assays allowed connecting the molecular pattern of “carbonylation stress” to functional changes in the studied nitrosative stress cell model.