Background and previous work
AGEs are associated to changes during ageing and related pathologies. Recently, we established a new class of AGEs with amide structure in vivo, including acetyl-, glycerinyl-, lactoyl-, formyl- and glycolic acid lysine amides. The formation can be explained by isomerisation or -dicarbonyl cleavage. Hence, Maillard glycation processes also lead to accelerated acylation of proteins and of amino acids. N-acetyl lysine can also be found in histone core proteins and has been established as an important tool of enzymatic gene expression regulation. This means that fundamentally important enzymatic processes are paralleled by non-enzymatic pathways. The physiological importance of such nonenzymatic acylation pathways is not understood.
To answer our working hypothesis that the profile of amide-AGEs changes with ageing and correlated pathologies, we will address the following topics: (1) What are the relevant amide-AGE structures in vivo? (2) What is their mechanistic background of formation? (3) How do they correlate to other PTMs researched within the RTG?
The range of novel AGEs with amide structure has to be extended by chemical synthesis. Targets will be selected based on fragmentation of physiological relevant α-dicarbonyl compounds. A coupled HPLC-MS/MS method has to be developed for the detection in biological samples to identify both novel but also major known AGEs and low molecular weight biomarkers thus allowing comprehensive argumentations in collaborative studies. As amide-AGEs are acid-sensitive, both acid and enzymatic hydrolyses are needed to address the notion that major AGEs belong to the class of amides. This work-up has to be fine-tuned in close consultation with project partners to include the specific biological matrices used within the RTG. The mechanistic background of AGE formation will be elucidated by isotopic labelling experiments but also targeted analyses to identify the specific sites in modified model proteins (specific collaboration with SP3 on modulation of protein hydrolysis by proteasomes, with SP2 on the modification of tight junction proteins in respect to the competition of glycation to glycosylation to influence the blood brain barrier, with SP8 on protein crosslinking, with SP9 on mechanisms of glycation within the Klotho/FOXO signalling cascade). The detection and correlation of non-enzymatic protein modification in the various aging models will be done in collaboration with most RTG groups but specifically with SP7 on protein glycation induced by metabolic stress in animals and SP4 on correlation to stem cell self-renewal and differentiation.