Malfunctions and deregulation of signaling pathways within cells can lead to diseases and cancer. Among known signaling pathways affecting tumorigenesis is the Wnt signaling pathway, in which the key protein involved is β‑catenin. Through interaction with a transcriptional factor Lef-1, it promotes cell proliferation and cell cycle progression. Activation of transcription of similar genes can also be caused by the formation of a protein complex EpIC–FHL2–β‑catenin–Lef-1. After the regulated intramembrane proteolysis of the EpCAM protein, the EpIC part is released in the cytosol, where it interacts with β‑catenin and FHL2. In the cell nucleus, protein Lef-1 binds to the complex. Complex formation and interactions between proteins have already been confirmed, but current data regarding interactions is incomplete and requires further analysis.
In the scope of this master thesis, we aimed to characterize protein domains involved in interactions between mentioned proteins, using two different methods to identify interacting domains (HoldUp assay and analysis with OmniSEC system). First, we prepared vectors to express both fluorescently labelled and unlabelled proteins. During the expression in E. coli cells and purification of certain recombinant proteins, we encountered challenges. Additionally, the interaction between fluorescently labeled proteins resulted in a different stoichiometry of participating proteins compared to the interaction of unlabelled proteins. Hence, we did not perform the HoldUp assay. For OmniSEC analyses, we expressed and purified full-length and truncated FHL2 in β‑catenin. Some of the constructs exhibited high precipitation in used buffers, leading us to exclude them from the OmniSEC analysis due to the method’s sensitivity. Based on the obtained results, we can conclude that the third LIM domain of the FHL2 protein (LIM3) is important for its interaction with β‑catenin, but only the full-length FHL2 can form a stable interaction. The precipitation pattern of truncated forms of FHL2 suggests the possibility of an intramolecular interaction between the LIM0 and LIM2 domains, which prevents the intermolecular interaction of domains LIM1 and LIM2 of neighbouring molecules. In the absence of the LIM0 domain, dimerization and precipitation of the truncated forms of FHL2 can occur.
Results obtained in this master thesis provide insights into the interaction between FHL2 and β‑catenin, which are part of the EpIC–FHL2–β‑catenin–Lef-1 complex, however further research is necessary for a precise understanding of these interactions. This master thesis can serve as a basis for the expression and purification of and purifying used protein constructs, but the purification conditions still need to be optimized (using different buffers). The possibility of performing the HoldUp assay would provide essential data about interactions and enhance the design of targeted drug development.
|
