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

葡萄糖降解产物（GDPs）是在含糖产物热降解过程中形成的。人类接触的最重要来源是饮食和药物，它们含有作为渗透剂的高浓度葡萄糖溶液。gdp通常含有二羰基结构，因此与糖提取物相比具有更高的化学和生理反应性。例如，在腹膜透析患者中，使用富含gdp的透析液与局部纤维化、血管硬化和腹膜通透性丧失的发展有关，从长远来看，这导致停止治疗。进一步的研究表明，gdp的生理效应在很大程度上与3,4-二脱氧葡萄糖-3-烯（3,4- dge）的存在有关。虽然3,4- dge仅在低浓度下形成，但它显示出惊人的高细胞毒性和酶抑制活性。在这种情况下，酶失活是由gdp诱导的共价蛋白修饰引起的。因此，本项目的目的是阐明3,4- dge显著强酶抑制活性背后的分子机制。为此，我们计划在第一步研究3,4- dge的蛋白质修饰率比其他gdp高，在多大程度上引起酶抑制活性。在下一步，最重要的3,4- dge衍生蛋白修饰的结构将被阐明。先前的研究表明，与其他gdp或糖相比，3,4- dge的不饱和二羰基结构导致了半胱氨酸残基的主要修饰，从而形成了一类潜在的新型加合物结构。在该项目的第三部分，将调查3,4- dge特异性修饰是否以及为什么比先前确定的修饰更严重地损害蛋白质结构和蛋白质构象（从而也损害功能）。为此，模型酶RNAse A将与3,4- dge一起孵育，并通过非靶向和靶向质谱相结合的方法分析产生的蛋白质修饰的结构和结合位点。所鉴定的修饰对酶结构的影响将通过修饰后的RNase三级结构的三维可视化以及分子动力学计算来预测，这将是后续项目的主题。有了这些结果，将有可能更好地了解3,4- dge突出生理活性的分子机制。从长远来看，这一知识将促进针对3,4- dge造成的生理损害的缓解策略的发展。

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.