20.500.12556/RUL-170203 Analiza interakcij med lipidnimi membranami in kondenzati proteina G3BP2 in vitro oziroma stresnimi granulami kot celičnimi analogi kondenzatov na primeru proteina G3BP1 Investigating the interactions between lipid membranes and G3BP2 protein condensates in vitro, and stress granules as cellular analogues of condensates, using G3BP1 V celicah se s procesom ločitve tekočih faz (LLPS) tvorijo številni nemembranski organeli. Mednje uvrščamo stresne granule, ki se v citosolu tvorijo, ko je celica izpostavljena različnim oblikam stresa. Kronična izpostavljenost celic stresu ter posledična deregulacija procesa LLPS vodita v odmiranje živčnih celic in razvoj nevrodegenerativnih bolezni. Ena izmed ključnih komponent stresnih granul je vezavni protein aktivatorja Ras GTPaze 2 (G3BP2), ki ima napram svojemu bolj znanemu paralogu G3BP1 višjo raven izražanja v nevronih. V okviru tega magistrskega dela smo preučevali tvorbo kondenzatov proteina G3BP2 v in vitro sistemu. Osredotočili smo se na to, kako sestava kondenzatov vpliva na njihove fizikalno-kemijske lastnosti ter interakcije z lipidnimi membranami. Kondenzate smo tvorili v prisotnosti molekul RNA ali polietilenglikola. Ugotovili smo, da so kondenzati, ki se tvorijo v prisotnosti polietilenglikola, manjših dimenzij ter imajo večji delež nemobilne frakcije. Lipidne membrane omočijo, vendar stopnja omočenja ni odvisna od naboja membrane. Po drugi strani RNA-kondenzati lipidnih membran ne omočijo. Ker so RNA-kondenzati po sestavi in mehanizmu tvorbe bolj podobni stresnim granulam, smo v celičnih sistemih preverili, kaj omogoča njihovo interakcijo z lipidnimi membranami. Zato smo ugotavljali, ali je znan adapterski protein aneksin A11 (ANXA11) prisoten v stresnih granulah, ki interagirajo s poškodovanimi endolizosomskimi ter lizosomskimi membranami. V ta namen smo uporabili celično linijo FlpIn SH-SY5Y mScarlet I-G3BP1-myc. Potrdili smo, da se ob poškodbi endolizosomskih in lizosomskih membran poveča interakcija med poškodovanimi organeli in stresnimi granulami ter da skoraj 90 % stresnih granul, ki interagirajo z poškodovanimi membranami, vsebuje ANXA11. In cells, numerous membraneless organelles are formed through the process of liquid liquid phase separation (LLPS). Among these are stress granules, which assemble in the cytosol when the cell is exposed to various forms of stress. Chronic exposure to stress and the resulting dysregulation of LLPS lead to the death of neurons and the development of neurodegenerative diseases. One of the key components of stress granules is the Ras GTPase-activating protein-binding protein 2 (G3BP2), which, compared to its better known paralog G3BP1, exhibits higher expression levels in neurons. In this master’s thesis, we studied the formation of G3BP2 condensates in an in vitro system. We focused on how the composition of the condensates influences their physicochemical properties and their interactions with lipid membranes. The condensates were formed in the presence of either RNA molecules or polyethylene glycol (PEG). We found that condensates formed in the presence of PEG were smaller in size and had a larger immobile fraction. They were capable of wetting lipid membranes, but the extent of wetting was not dependent on membrane charge. In contrast, RNA-condensates did not wet lipid membranes. Since RNA-condensates are more similar in composition and formation mechanism to stress granules, we further examined what enables their interaction with lipid membranes in cellular systems. Specifically, we investigated whether the known adaptor protein annexin A11 (ANXA11) is present in stress granules that interact with damaged endolysosomal and lysosomal membranes. To this end, we used a FlpIn SH SY5Y cell line expressing mScarlet I-G3BP1-myc. We confirmed that upon endolysosomal and lysosomal membrane damage, there is an increased interaction between damaged organelles and stress granules, and that nearly 90 % of stress granules interacting with damaged membranes contain ANXA11. ločitev tekočih faz stresne granule G3BP1/2 lipidne membrane liquid-liquid phase separation stress granules G3BP1/2 lipid membranes true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-07-02 15:15:16 2025-07-02 15:15:24 2025-07-11 13:27:32 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-06-20 25370 242306563 Kodra_Ana_marija_-_Analiza_interakcij_med_lipidnimi_membranami_in_kondenzati_proteina_G3BP2_in_v.pdf Kodra_Ana_marija_-_Analiza_interakcij_med_lipidnimi_membranami_in_kondenzati_proteina_G3BP2_in_v.pdf 1 3911EFF2FCD5377C344E02321895E33F 19ffee4e0c5af9ac2077c450f57b3c91068ec70e943d942ed6078696c3c5ac0e b9b6ee2e-5744-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=212156 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0