Medizinische Universität Graz - Research portal
Selected Publication:
Stryeck, S.
Deciphering the intricate interaction network in the ß-catenin destruction complex - the Axin-1 paradigm
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2019. pp. 147
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- Authors Med Uni Graz:
- Advisor:
- Madl Tobias
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- Abstract:
- During the last years, emerging evidence on the presence and importance of so-called membrane-less organelles in the cytoplasm but also in the nucleus of eukaryotes arose. Proteins capable of forming phase separated condensates (e.g. hnRNPA1, FUS, TDP-43) have already been linked to neurodegeneration due to formation of non-native protein aggregates in neuronal cells. The importance of liquid-liquid phase separation (LLPS) in cancer diseases still remains elusive. Therefore, I studied in my PhD project the protein Axin-1, which phase separates and which has already been linked to cancer diseases. It is a key element in the ß-catenin destruction complex, which regulates canonical Wnt signaling. In the literature, it has already been reported, that Axin-1 regulates its activity via self-binding. Such multivalent interactions are also a hallmark of LLPS. The central aim of this thesis is to contribute to the current understanding of Axin-1 auto-regulation, the formation of macromolecular assemblies within the cytoplasm (so-called puncta) and the effect of cancer mutations on those processes. Hereby I used a combination of structural biology and cell biology techniques in order to decipher intra-molecular interaction within the protein Axin-1. The key findings of the project were that three different regions Axin-1IDR interact with the Axin-1DIX. The interaction interface on the Axin-1DIX was the same for all three Axin-1IDR fragments, but it differs from the Axin-1DIX oligomerization interface. In contrast to current assumptions in literature, the Axin-1DIX interaction does not displace ß-catenin, since the binding sites on the Axin-1IDR differ. In addition, post-translation phosphorylation, which is an important regulatory modification of the protein, regulates its activity, but is not sufficient to dissolve puncta. Finally, a cancer variant of the protein with a mutation located in the Axin-1DIX, E816G, in the Axin-1DIX-Axin-1IDR interaction interface has been studied, and revealed an important role for the Axin-1IDR binding sites. During this thesis, intramolecular interactions within Axin-1 are studied, binding sites are mapped and effects of a cancer mutation in the binding interface are studied. Those data reveal important features for the phase separation of Axin-1 and are therefore essential in order to understand the importance of condensate formation in cancer. In the second chapter of my work I focus on a method development in NMR spectroscopy. Liver cirrhosis is one of the common end points of liver diseases. Besides other symptoms, increased gastric or intestinal permeability is one of the hallmarks of advanced liver damage. By oral administration of carbohydrate solutions and the detection thereof in urine, increased permeability can be diagnosed. However, techniques for sugar detection and quantification are limited, due to interfering substances or long measurement times. The main aim of this project was to develop a NMR-based method in order to enable a fast and robust quantification of sugars in urine. In our results we were able to show that JRES experiments are a useful tool for the reliable identification and quantification of the carbohydrates. This method builds a good alternative to commonly available techniques and might become an interesting approach for clinical applications.