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Abstract

S-acylation is a reversible post-translational modification involving the covalent attachment of a fatty acid to a cysteine residue of a protein. This modification can influence protein conformation, localization, and protein-protein interactions. Due to its reversibility, S-acylation functions as a regulatory switch in various cellular processes, including T cell activation. Morrison et al. explored the S-acylome of resting and activated T cells using mass spectrometry and identified 88 proteins exhibiting changes in their state of S-acylation upon T cell stimulation. This thesis further investigates three of these proteins and their possible role in the regulation of T cell signaling by reversible S-acylation: 1. Cytoskeleton associated protein 4 (CKAP4) is dynamically deacylated at cysteine100 upon Dickkopf1 (DKK1) binding in pancreatic cancer cells and is associated with key processes of T cell signaling such as calcium mobilization and cell migration. However, its role in T cell activation has not been explored before. In this study, the deacylation switch of CKAP4 could be confirmed and fundamental groundwork was laid to study the localization of CKAP4 in T cells by demonstrating that CKAP4 is only present in small amounts at the T cell plasma membrane. Moreover, CKAP4-3xALFA-Halo cells were generated enabling live-tracking of the protein following T cell activation. 2. Family with sequence similarity 49 member B (FAM49B) plays a role in cytoskeletal remodeling and Erk phosphorylation taking place during T cell signaling. Although S-acylation of FAM49B has been observed before, its exact site and functional relevance remain unclear. Here, S-acylation of FAM49B could be corroborated and preliminary data suggested that cysteine-10 is a potential S-acylation site. 3. Retinitis pigmentosa 2 activator of ARL3 GTPase (RP2) is implicated to play a role in the transport of lymphocyte-specific protein tyrosine kinase (Lck), a key enzyme in T cell signaling. While S-acylation of RP2 at cysteine-3 is documented, its functional role is not fully understood and there is no established link between S-acylation of RP2 and T cell activation. For RP2 it was found, that overexpression in T cells induced increased levels of Erk phosphorylation in a S-acylation-dependent manner, pointing towards its involvement in T cell activation. Moreover, CKAP4, FAM49B and RP2 knock-outs in T cells were generated and tools to study their role in T cell activation were established. In summary, this thesis provides new insights into the regulatory role of S-acylation in T cell activation, sets the stage for further studies of S-acylation-switches of proteins by establishing important experimental tools, and highlights avenues for future research of this reversible modification in immune signaling.