Comprehensive analysis of ROS-induced protein phosphorylation and oxidation in physiology and pathophysiology of pancreatic beta cells

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Dysfunction of pancreatic beta-cells is a hallmark of pathogenesis and pathophysiology of type 2 diabetes mellitus and characterised by an inappropriate insulin metabolism. Several reasons for beta-cell dysfunction have been identified; however, the increased formation of reactive oxygen species (ROS) due to chronic hyperglycemia has been identified as a major pathologic trigger. On the contrary, low amounts of ROS production in beta-cells are mandatory to maintain an appropriate insulin secretion. In either instance ROS-signalling seems to be mediated by the oxidation of cysteine residues resulting in changes of protein structure and function. Proteins that are frequently affected by ROS oxidation are protein kinases and phosphatases that are also essentially involved in the regulation of insulin synthesis, processing and secretion. The present project aims to analyse the effects of ROS on the physiology and pathophysiology of insulin secretion mediated by protein oxidation and phosphorylation and the interaction of both modification types in cell signalling. The effect of ROS on protein phosphorylation and oxidation will be analysed in vitro using primary rat beta-cells and immortalised INS-1E cells. Insulin secretion will be stimulated by glucose, glucose-like peptide 1 and its agonists as well as calcium with and without exposure to oxidants (i.e. hydrogen peroxide, menadion) and high-glucose stress. Cells will be harvested and lysed and proteins will be digested enzymatically. Subsequently, peptides will be labelled by isobaric tags and phosphorylated and cysteine containing peptides will be enriched by different chromatography techniques. Finally, peptide analysis will be accomplished by LC-MS/MS. Additionally, the posttranslational modification patterns of subcellular fractions (i.e. insulin vesicles and mitochondria) will be characterised after separation e.g. by ultracentrifugation. Finally, proteins that have been identified by this procedure as potent candidates will be confirmed and quantified by ELISA and immunoblotting. The systemic and comprehensive analysis of the association of protein phosphorylation and oxidation will considerably contribute to the understanding of physiological and pathophysiological processes in beta-cells associated with diabetes mellitus. Additionally, the results reveal great potential for the development of new therapeutic strategies and pharmacological intervention.