Ninjurin 1 (NINJ1) is a transmembrane protein that mediates plasma membrane rupture in certain forms of necrotic cell death and promotes inflammation, most notably during secondary necrosis after apoptosis, pyroptosis, and ferroptosis. It is involved in a wide range of diseases and pathological conditions, making it a promising therapeutic target. However, few inhibitors of NINJ1 have been identified to date. In this master's thesis, we aimed to develop de novo designed small minibinder proteins against NINJ1 to modify NINJ1-mediated plasma membrane rupture. These were engineered to selectively bind to the amphipathic region of NINJ1, thereby inhibiting its oligomerization and subsequent membrane rupture. Using in silico design tools, we designed minibinders and prepared expression vectors for each protein. Additionally, we prepared constructs encoding the target protein, mouse NINJ1, as well as two similar proteins: mouse NINJ2 and human NINJ1. All proteins, targets and minibinders, were produced in a bacterial expression system, purified, and analyzed using a binding assay to identify the most promising candidates for further investigation. For six selected minibinders, we determined their secondary structure and assessed their effects on plasma membrane rupture during different forms of cell death by measuring the release of lactate dehydrogenase and propidium iodide uptake. Our results indicate that these minibinders enhance plasma membrane rupture during pyroptosis but suppress it during ferroptosis. Given the growing body of evidence implicating ferroptosis in various diseases, particularly neurodegenerative disorders, these findings highlight the therapeutic potential of novel ferroptosis and inflammation inhibitors, such as designed minibinder proteins.
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