Investigation of the structural constraints on Wnt ligands evolution through mutagenesis

Abstract

The Wnt/β-catenin pathway is a signaling pathway that controls many cellular decisions and behaviors during embryonic and adult development. Dysfunction of this signaling pathway can lead to diseases. The challenge in treating these diseases is to find specific treatments that act in a targeted way, without disrupting the Wnt/β-catenin pathway in the rest of the body. Previous studies have focused specifically on blood-brain barrier (BBB) dysfunction and therapeutic strategies to treat it. The Wnt7a/b protein is an important signal for the formation and maintenance of the BBB and could be an interesting therapeutic asset, assuming that it is specific to the BBB. Martin, M. et al (2022) identified variants in Wnt7a that exhibit specific agonism for Gpr124/Reck, which is a complex involved in Wnt7a/b signaling at the BBB. These agonists only activate the Wnt/β-catenin pathway in the presence of the Gpr124/Reck complex, making them specific to Gpr124/Reck contexts, such as the BBB. Why these agonists lost the ability to activate the Wnt/β-catenin pathway in the absence of the Gpr124/Reck complex remains to be determined, and is the aim of this master thesis. The first hypothesis formulated in this study is based on the relative instability of Gpr124/Reck agonists. The mutation of a single surface residue in the different agonists could lead to a decrease in the stability of the proteins, making them too weak to activate signaling in the absence of the stabilizing Reck. Since the Wnt7a protein is naturally unstable, working on the stability of its agonists is laborious. The corresponding mutations were therefore introduced in the more stable Wnt3 protein in order to compare the stability of the variants with the wild type (WT) protein. We show that a prototypical Wnt3 variant is less stable than the Wnt3WT protein, reinforcing the hypothesis stated by Martin, M. et al (2022). Furthermore, we also document that the Wnt3 variant exerts a dominant negative effect on the WT protein. This dominant-negative effect, the mechanism of which remains to be explained, was also observed between the Wnt7aWT protein and one of its agonists. Finally, a divergence between the different Wnt ligands is observed with respect to the functional importance of the surface residues. Indeed, the point mutation of an equivalent surface residue will not have the same impact on the different Wnt ligands. The stability between the Wnt ligands also seems to vary, with Wnt7a proteins being much less stable than other Wnt ligands such as Wnt1 and Wnt3.