Eva Griesser

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

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.

Structural, biological and biophysical properties of glycated and glycoxidized phosphatidylethanolamines

Glycation and glycoxidation of proteins and peptides have been intensively studied and are considered as reliable diagnostic biomarkers of hyperglycemia and early stages of type II diabetes. However, glucose can also react with primary amino groups present in other cellular components, such as aminophospholipids (aminoPLs). Although it is proposed that glycated aminoPLs can induce many cellular responses and contribute to the development and progression of diabetes, the routes of their formation and their biological roles are only partially revealed. The same is true for the influence of glucose-derived modifications on the biophysical properties of PLs. Here we studied structural, signaling, and biophysical properties of glycated and glycoxidized phosphatidylethanolamines (PEs). By combining high resolution mass spectrometry and nuclear magnetic resonance spectroscopy it was possible to deduce the structures of several intermediates indicating an oxidative cleavage of the Amadori product yielding glycoxidized PEs including advanced glycation end products, such as carboxyethyl- and carboxymethyl-ethanolamines. The pro-oxidative role of glycated PEs was demonstrated and further associated with several cellular responses including activation of NFκB signaling pathways. Label free proteomics indicated significant alterations in proteins regulating cellular metabolisms. Finally, the biophysical properties of PL membranes changed significantly upon PE glycation, such as melting temperature (Tm), membrane surface charge, and ion transport across the phospholipid bilayer.

Protein Carbonylation and Glycation in Legume Nodules

In legume nodules, selective carbonylation and glycation of proteins occurs during nodule development and may have a role in the regulation of metabolism and senescence. Nitrogen fixation is an agronomically and environmentally important process catalyzed by bacterial nitrogenase within legume root nodules. These unique symbiotic organs have high metabolic rates and produce large amounts of reactive oxygen species that may modify proteins irreversibly. Here, we examined two types of oxidative posttranslational modifications of nodule proteins: carbonylation, which occurs by direct oxidation of certain amino acids or by interaction with reactive aldehydes arising from cell membrane lipid peroxides; and glycation, which results from the reaction of lysine and arginine residues with reducing sugars or their autooxidation products. We used a strategy based on the enrichment of carbonylated peptides by affinity chromatography followed by liquid chromatography-tandem mass spectrometry to identify 369 oxidized proteins in bean (Phaseolus vulgaris) nodules. Of these, 238 corresponded to plant proteins and 131 to bacterial proteins. Lipid peroxidation products induced most carbonylation sites. This study also revealed that carbonylation has major effects on two key nodule proteins. Metal-catalyzed oxidation caused the inactivation of malate dehydrogenase and the aggregation of leghemoglobin. In addition, numerous glycated proteins were identified in vivo, including three key nodule proteins: sucrose synthase, glutamine synthetase, and glutamate synthase. Label-free quantification identified 10 plant proteins and 18 bacterial proteins as age-specifically glycated. Overall, our results suggest that the selective carbonylation or glycation of crucial proteins involved in nitrogen metabolism, transcriptional regulation, and signaling may constitute a mechanism to control cell metabolism and nodule senescence.