The species-specific role of caspase-8 as a regulator of necroptosis

The molecular mechanisms of the necroptotic signaling pathway remain to a certain proportion unknown. Since necroptosis is associated to various diseases, a precise knowledge of the necroptotic signaling pathway is essential for the development of new therapeutic strategies. The aim of this dissertation was to therefore gain a deeper insight into molecular events of TNF-induced necroptosis. \nIn the first part of the thesis, murine L929Ts cells deficient for caspase-8 as well as human HT-29 cells deficient for caspase-8 or caspase-10 were generated by employing the CRISPR/Cas9 technology. It was shown that genetic ablation of caspase-8 in murine L929Ts cells enhances necroptosis, whereas genetic ablation of caspase-8 in human HT-29 cells protects the cells from TNF-induced necroptosis. It is noteworthy that humans express caspase-8 and caspase-10, while caspase-10 was lost in mice over the course of evolution. In the presented dissertation, it was demonstrated that genetic ablation of caspase-10 in human cells exacerbates necroptosis, supporting the hypothesis that caspase-10 in humans can substitute the function that is held by caspase-8 in mice. Furthermore, experiments aimed at the identification of differences in the composition of necrosomes in wild type vs. caspase-8- or caspase-10-deficient HT-29 cells found that caspase-10 associates with caspase-8 during TNF-induced necroptosis. Genetic ablation of caspase-8 disrupts the assembly of the necrosome while genetic ablation of caspase-10 promotes at least the recruitment of RIPK1 and RIPK3. Additionally, data obtained in this thesis revealed that the enzymatic activity of human and mouse caspase-8, as well as the enzymatic activity of caspase-10, are required for their function within the necroptotic signaling pathway. In summary, it can be stated that all data obtained in this thesis consistently point to a differential proteolytic regulation of necroptosis by caspase-8 in mice and in humans.