Ninjurin-1 is a transmembrane protein that polymerizes and causes damage to the cell membrane after necrotic cell death, releasing intracellular components and thereby activating inflammation. Suppressing Ninjurin-1-mediated plasma membrane rupture could alleviate chronic inflammation. On the other hand, the regulated activation of immunogenic cell death through the action of Ninjurin-1 in the tumour microenvironment would provoke local inflammation, representing an innovative approach in antitumor therapy.
This work aimed to develop two different approaches for regulating Ninjurin-1-induced plasma membrane rupture via its C-terminus. In the first part, we attempted to isolate single-chain variable fragments that bind the C-terminus of Ninjurin-1 by phage display using Tomlinson I and J libraries. We performed three successive elution steps with increasingly stringent washing conditions, followed by an enzyme-linked immunosorbent assay to verify the success of the selection. The results showed that the phage display method failed to obtain a clone that would show strong affinity for Ninjurin-1. We then focused on investigating whether cell membrane damage can be regulated by attaching a globular protein domain to the C-terminus of Ninjurin-1 via a spacer. For this purpose, we prepared several Ninjurin-1-spacer-globular domain constructs using molecular cloning methods and, by introducing the constructs into a human cell line, monitored the influence of different components of the construct on the lytic activity of Ninjurin-1. Cell death was monitored by measuring propidium iodide uptake and the release of lactate dehydrogenase from the cells. We found that larger globular domains and shorter spacers inhibit Ninjurin-1-mediated plasma membrane rupture better. We then attempted to cleave the constructs at the cleavage site that we introduced into the spacer and thereby reactivate Ninjurin-1. We succeeded in this to a limited extent, so further optimization of the cleavage would be necessary in the future.
In this master's thesis, we attempted to develop two ways to regulate cell membrane damage mediated by Ninjurin-1. Although we were unable to develop antibody fragments with high affinity using phage display, in the second part, we investigated an innovative way to regulate the lytic activity of Ninjurin-1. We showed that by acting on the C-terminus of Ninjurin 1, we can effectively inhibit cell membrane damage, which will lead to the development of new approaches for the controlled induction of cell lysis, especially in cancer immunotherapy.
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