Ninjurin-1 (NINJ1) is a transmembrane protein that mediates plasma membrane rupture (PMR) in some forms of lytic cell death, including pyroptosis. Ninjurin-2 (NINJ2), the second member of the ninjurin family in mammals, is not involved in cell membrane rupture. Within this MSc thesis, we wanted to investigate the mechanism by which NINJ1 mediates plasma membrane rupture. We designed and constructed point mutants and truncated versions of NINJ1. Based on the alignment of the amino acid sequences of NINJ1 and NINJ2, we also designed chimeras of NINJ1 and NINJ2, where we exchanged individual segments between them. We introduced the constructs into HEK 293T cells by lipofection, where we measured cell death via propidium iodide dye uptake and lactate dehydrogenase activity. Protein expression was evaluated by western blotting and expression on the cell surface by flow cytometry, for which we tagged the constructs with FLAG and HA tags. Using blue native gel electrophoresis, we showed that NINJ1 oligomerizes and observed higher-order oligomers, which were present to a lesser extent in NINJ2 samples. We have shown that the proteins that were able to mediate PMR are also capable of oligomerization. We have shown that the predicted region of the amphipathic helix and the cytosolic domain play an important role in mediating PMR, while transmembrane helices 1 and 2 and the extracellular C-terminus are interchangeable between NINJ1 and NINJ2. We were unable to determine the influence of the N-terminus (the adhesion motif of NINJ1) due to conflicting results.
Our results suggest an important role of the amphipathic helix and the cytosolic domain of NINJ1 in cell membrane rupture which, together with recent structural studies, may contribute to the development of innovative strategies to inhibit NINJ1-mediated PMR and inflammation that is perpetuated through this process.
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