20.500.12556/RUL-171449 Priprava in ovrednotenje delovanja majhnih vezavnih proteinskih posrednikov Ninjurina 1 Preparation and functional evaluation of minibinder proteins targeting Ninjurin 1 Ninjurin 1 (NINJ1) je transmembranski protein, ki posreduje poškodbo celične membrane pri nekaterih oblikah nekrotične celične smrti in spodbuja vnetje, med drugim pri sekundarni nekrozi po apoptozi, piroptozi in feroptozi. Povezan je s številnimi boleznimi in bolezenskimi stanji in je potencialno dobra terapevtska tarča, ampak trenutno poznamo zelo malo načinov, kako zavreti njegovo delovanje. V sklopu magistrske naloge smo želeli spremeniti potek z ninjurinom 1 posredovane poškodbe membrane z uporabo de novo načrtovanih majhnih vezavnih proteinskih posrednikov, ki naj bi se tarčno vezali na amfipatični del proteina in s tem preprečevali njegovo fibrilacijo ter posledično poškodbo plazmaleme. Z uporabo računalniških orodij smo minivezavne proteinske posrednike načrtovali in nato pripravili konstrukte za posamičen vezavni protein. Pripravili smo tudi konstrukte, ki kodirajo tarčo, mišji ninjurin 1 in dva sorodna proteina, mišji ninjurin 2 ter humani ninjurin 1. Vse proteine in tarče smo pripravili v bakterijskem ekspresijskem sistemu, jih izolirali in vzpostavili test vezave, na podlagi katerega smo naredili ožji izbor minivezavnih proteinov za nadaljnje delo. Šestim izbranim minivezavnim proteinom smo določili sekundarno strukturo in testirali njihov vpliv na poškodbo membrane med indukcijo različnih tipov celične smrti, ki smo jo spremljali preko meritev sproščanja laktat dehidrogenaze iz celic in vstopa propidijevega jodida v celice. Naši rezultati nakazujejo, da minivezavni proteini stimulirajo poškodbo membrane med piroptozo, nasprotno pa jo med feroptozo zavirajo. Vse več študij nakazuje na vpletenost feroptoze v različnih boleznih, predvsem nevrodegenerativnih, kar poudarja pomen zaviralcev feroptoze in vnetja, kot so tudi načrtovani minivezavni proteini. 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. Ninjurin 1 NINJ1 poškodba celične membrane minivezavni proteini celična smrt Ninjurin 1 NINJ1 plasma membrane rupture minibinders cell death true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-08-27 07:15:06 2025-08-27 07:15:19 2025-09-02 03:42:32 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 261527 246750979 50624.pdf 50624.pdf 1 03FEFEAE4CB01C61D0A8F75187DAD9B0 29cb2705df5701990301d0a41c31c249d24a5d8f799cac3e60aed45efdb1e573 b3d4e31b-8304-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=214775 Biotehniška fakulteta 0 0 0