Background and previous work
Wnt/β-catenin signalling plays multiple roles in embryonic development, tumourigenesis and stem cell biology and is linked to multiple processes in ageing, including DNA damage and ROS response, metabolic regulation and cellular senescence. A high incidence of hyperglycaemia is observed in old individuals resulting in enhanced oxidative phosphorylation and elevated ROS levels. Interestingly, increased glucose levels modulate β-catenin transcriptional activity. High glucose levels also induce the hexosamine pathway (O-GlcNAcylation) and AGE production. β-Catenin has been identified as an O-GlcNAcylated protein. Moreover, ROS and metabolic stress induce FOXO transcription factors and appear to redirect β-catenin from T-cell factor (TCF) transcription factors to FOXO, thereby enhancing FOXO target gene transcription. Currently, it is unclear how elevated glucose levels/glycation and Wnt/β-catenin signalling are interconnected. In this context, we observed that induction of AGE formation results in a dose-dependent reduction of β-catenin transcriptional activity. In a second approach we observed a role of the hexosamine pathway in regulating Wnt signalling. From these observations we conclude that Wnt signalling is a target of AGE modification(s) and of hexosamine pathway-dependent PTMs.
In this respect, we want to (1) identify components of the Wnt signalling pathway that are targeted by the corresponding PTMs and determine the specific sites of modification, (2) analyse the effect of these PTMs on modulators of β-catenin signalling and its crosstalk with FOXO, and (3) investigate the role of these modifications in stress response and senescence/ageing.
AGE-modified and O-GlcNAcylated Wnt-pathway proteins in human fibroblasts or cells
expressing mutated Wnt-pathway components will be identified by co-immunoprecipitation and Western blotting (SP2, SP8). After enrichment of modified cellular proteins they will be subjected to mass spectrometric analysis (SP1, SP5). Alternatively, if only insufficient amount of proteins can be isolated from cells, AGE-modified recombinant proteins will be analysed. Effects of Wnt-pathway component PTMs will be studied with respect to altered binding to known interaction partners, stability and solubility and modulations in known cross-talks with other signalling pathways with a focus on FOXO signalling (SP4, SP9,SP10, SP13). To study the role of the identified PTMs, proteins with mutated modification sites will be expressed in cells subjected to different stress conditions especially increased
ROS and effects on Wnt/β-catenin signalling will be analysed. If possible, we will generate specific antibodies against peptides including the identified posttranslationally modified.