NINJ1 mediates plasma membrane rupture (PMR), the final step in immunogenic cell death (ICD), which results in the release of intracellular components. These components stimulate an immune response, thereby opening possibilities for the therapeutic use of regulated cell death (RCD), such as in the treatment of immunogenic tumours. The main aim of this master’s thesis was to develop variants of the NINJ1 protein that facilitate controlled cell death through plasma membrane disruption. In the experimental work, we focused on developing locked NINJ1 constructs by adding homo- or heterodimerising segments and domains to the N- and C-termini of NINJ1. These constructs were designed to enable inducible activation through different mechanisms. Their effectiveness was evaluated in comparison with the cytotoxicity of wild-type NINJ1. We transfected HEK293T cells and confirmed the expression of all designed constructs using western blotting and an enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was measured using a propidium iodide uptake assay. Our results confirmed that overexpression of NINJ1 induces PMR and consequently cell death. When testing the cytotoxicity of the locked NINJ1 constructs in comparison with the wild-type protein, no statistically significant differences were observed. Despite the negative results, recently published studies on NINJ1 structure enabled us to prepare guidelines for further construct optimisation, including tailoring activation to the tumour microenvironment.
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