Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress

Abstract

Reactive oxygen and nitrogen species (ROS/RNS) play an important role in the regulation of cardiac function. Increase in ROS/RNS concentration results in lipid and protein oxidation and is often associated with onset and/or progression of many cardiovascular disorders. However, interplay between lipid and protein modifications has not been simultaneously studied in detail so far. Biomolecule carbonylation is one of the most common biomarkers of oxidative stress. Using a dynamic model of nitroxidative stress we demonstrated rapid changes in biomolecule carbonylation in rat cardiomyocytes. Levels of carbonylated species increased as early as 15 min upon treatment with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1), and decreased to values close to control after 16 h. Total (lipids+proteins) vs. protein-specific carbonylation showed different dynamics, with a significant increase in protein-bound carbonyls at later time points. Treatment with SIN-1 in combination with inhibitors of proteasomal and autophagy/lysosomal degradation pathways allowed confirmation of a significant role of the proteasome in the degradation of carbonylated proteins, whereas lipid carbonylation increased in the presence of autophagy/lysosomal inhibitors. Electrophilic aldehydes and ketones formed by lipid peroxidation were identified and relatively quantified using LC-MS/MS. Molecular identity of reactive species was used for data-driven analysis of their protein targets. Combination of different enrichment strategies with LC-MS/MS analysis allowed identification of more than 167 unique proteins with 332 sites modified by electrophilic lipid peroxidation products. Gene ontology analysis of modified proteins demonstrated enrichment of several functional categories including proteins involved in cytoskeleton, extracellular matrix, ion channels and their regulation. Using calcium mobilization assays, the effect of nitroxidative stress on the activity of several ion channels was further confirmed.

Mitochondrial protection by simvastatin against angiotensin II‐mediated heart failure

“…Recent studies have demonstrated that the lysosome pathway plays a main role in lipid degradation to FAs (Griesser et al, 2017;Settembre & Ballabio, 2014). Moreover, Dupont et al (2014) demonstrated that LD accumulation might promote autophagy, because LDs supply lipid precursors for incipient autophagosome membrane formation.…”

Protein Carbonylation and Glycation in Legume Nodules

“…Due to the low abundance of modified peptides, the derivatization step was necessary because it facilitated the enrichment of carbonylated peptides by biotin-avidin affinity chromatography and provided specific mass increments in the corresponding spectra, thus significantly improving the identification rates by liquid chromatography-tandem mass spectrometry (LC-MS/MS;Bollineni et al, 2013). This methodology, combined with search engine-assisted protein identification using the mass increments of the different posttranslational modifications (PTMs) as variables (Bollineni et al, 2014a;Griesser et al, 2017), allowed the identification of 238 carbonylated plant proteins. We only considered rank 1 peptides identified with high/medium confidence in at least five biological replicates.…”

Irreversible oxidative post-translational modifications in heart disease

“…Oxidized lipid products are potent electrophiles which subsequently further react with various amino acids, predominantly nucleophiles such as lysine, arginine and histidine, causing carbonylation of proteins and triggering their proteolysis. While oxidation of lipids occurs minutes after induction of oxidative stress, carbonylation of proteins takes place in the timescale of approximately 1 h and is stable for several hours before the carbonylated protein is degraded [47,54]. An overview of effects of reversible and irreversible oxPTMs on protein function and stability is provided in Figure 3.…”

“…The two most abundant LPP causing protein carbonylation are malondialdehyde and 4-hydroxynonenal derived from oxidative degradation of lipid substrates. These LPP can react either directly with amino acid side chains to form a Schiff base or with nucleophiles in a Michaeladdition mechanism ( Figure 5) [13,54]. Irrespective of the mechanism, subsequent reaction of the product can lead to intra-and intermolecular crosslinks with other proteins (see next section).…”

“…Therefore, only a thorough mass spectrometric analysis that takes the large variety of different possible products into account can potentially depict the entirety of protein carbonylation. Very recently, to our knowledge the so far most complete assessment of protein carbonylation by LPP was applied to a rat cardiomyocyte model by Griesser et al [54], identifying constituents of the cytoskeleton, proteins of the extracellular matrix and proteins involved in cell adhesion as major targets.…”