Molecular Mechanisms of the Physiological Activtiy of 3,4-Dideoxyglucosone-3-ene (3,4-DGE)

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Glucose degradation products (GDPs) are formed to a significant extent during the thermal degradation of sugar-containing products. The most important sources of human exposure are the diet as well as drugs, which contain highly concentrated glucose solutions as osmotic agent. GDPs often contain dicarbonyl structures, thus leading to a higher chemical and physiological reactivity compared to the sugar educt. In peritoneal dialysis patients, for example, the application of dialysis fluids rich in GDPs was associated with the development of local fibrosis, vascular sclerosis and the loss of peritoneal permeability, which, in the long term, results in the discontinuation of therapy. Further studies revealed that the physiological effects of GDPs are largely related to the presence of 3,4-dideoxyglucosone-3-ene (3,4-DGE). Although 3,4-DGE is formed only in low concentrations, it shows a strikingly high cytotoxicity and enzyme inhibitory activity. Enzyme inactivation is caused in this case by GDP-induced covalent protein modifications.The aim of the present project is, therefore, to elucidate the molecular mechanisms behind the prominently strong enzyme inhibitory activity of 3,4-DGE. For this purpose, we plan to investigate in the first step to which extent the enzyme inhibitory activity is caused by a higher protein modification rate of 3,4-DGE compared to other GDPs. In the next step, the structure of the most important 3,4-DGE-derived protein modifications will be elucidated. Previous studies indicate that the unsaturated dicarbonyl structure of 3,4-DGE leads, in contrast to other GDPs or sugars, to a predominant modification of cysteine residues and, thus, to the formation of a potentially novel class of adduct structures. In the third part of the project, it will be investigated, if and why 3,4-DGE-specific modifications impair protein structure and protein conformation (and consequently also function) more severely than the previously identified modifications. For this purpose, the model enzyme RNAse A will be incubated with 3,4-DGE and the structures and binding sites of arising protein modifications will be analyzed by a combination of untargeted and targeted mass spectrometric methods. Effects of the identified modifications on the enzyme structure will be predicted by three-dimensional visualization of the tertiary structure of modified RNase as well as by molecular dynamic calculations, which will be the subject of a follow-up project. With these results in hand, it will be possible to better understand the molecular mechanisms of the prominent physiological activity of 3,4-DGE. In the long term, this knowledge will promote the development of mitigation strategies against physiological damages caused by 3,4-DGE.