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Abstract

Wnt signaling pathways are a set of signal transduction cascades which are activated through the interaction of Wnt proteins with so-called Frizzled receptors [1-3]. These pathways are critically involved in many biological processes such as embryonic development, regeneration, organogenesis, cell division, cellular and tissue homeostasis, among many others [3, 4]. In addition, alterations of these signaling pathways have been linked to various types of diseases such as cancer [5-7], familial tooth agenesis [8], bipolar disease [9], Alzheimer's disease [10], and cardiac valve formation [11]. Wnt signaling components are accordingly promising drug targets to treat these diseases. Wnt pathways are probably among the best characterized receptor-ligand signaling pathways. Wnt proteins are therefore key players in biological signaling and promising drug targets to treat a plethora of diseases. Although several proteins involved in Wnt trafficking and secretion have been identified over the past years, little is known about the contribution of different lipid species into these processes. The trafficking and secretion of Wnts could be modulated by the type and number of acyl species covalently linked to Wnt proteins. Currently, the best described acyl modification is the palmiteoylation of a serine residue located around amino acids 205-215 mediated by the ER-resident O-acyltransferase Porcupine is responsible for this process. This lipid modification has been described for Wnt3a, Wnt5, xWnt8, and Wnt1, and it has been assumed to also take place in other members of the family of Wnt proteins. Despite the extensive data available, the debate around the lipid-modified amino acids in Wnt proteins has not yet reached a consensus. Recent results from O. Voloshanenko (M. Boutros group, DKFZ, Heidelberg, Germany) suggested that there may be other amino acid residues in Wnts that are lipidated, apart from this canonical serine residue. Furthermore, the specific saturation of the acylated chain that binds to Wnt remains inconclusive.

In this thesis, I aimed to define other putative acylation types and lipid-modified sites in Wnt proteins and to determine the role of these alternative lipidations in Wnt secretion and signaling. Furthermore, I evaluated the impact of Wnt signaling and Wnt secretion on the lipidome of HEK293T and HCT116 cells. To achieve this, I employed a combination of chemical biology tools, mutagenesis experiments, and mass spectrometric measurements. In particular, I focused on Wnt11 as a working model. I studied its acylation using clickable lipids such as palmitic acid alkyne (cC16:0) and palmitoleic acid alkyne (cC16:1n-7). One of the early observations was that palmitoylation and secretion of Wnt11 were not wholly abolished in Porcupine knockout cells or some mutant variants of Wnt11. However, these observations seem to depend on the type of clickable fatty acid used. Our results suggest a lipid modification of Wnt11 at serine 215 via the monounsaturated fatty acid cC16:1n-7, consistent with the previously predicted models. However, lipid modification with the saturated fatty acid cC16:0 showed variations in the experimental replicates, which did not fully resolve whether Wnt11 contains another modification site. Importantly, our experiments stress that unsaturation is a key feature for Wnt acylation. The relevance of covalent lipid binding for the secretion and signaling activity of Wnts has also been assessed. It was demonstrated that lipidation is essential for the signaling activity of Wnt11 but is not strictly necessary for its secretion. In addition, an impact of Wnt protein expression on the overall cellular lipidome of HEK293T and HCT116 cells has been tested, yielding preliminary observations on the crosstalk between the Wnt signaling and the overall cellular lipid homeostasis. This study is expected to contribute to our understanding of how post-translational lipid modifications influence Wnt cellular secretion, signaling and, conversely, how proteins of the Wnt signaling pathway affect the lipid composition of cells.