20.500.12556/RUL-145087 Določanje proteinov v okolici STAU1 in STAU2 v celicah HEK293 Identification of proteins in the proximity of STAU1 and STAU2 in HEK293 cells Protein staufen je RNA-vezavni protein, ki skupaj z molekulo RNA tvori ribonukleoproteinske komplekse kot so na primer stresne granule, ki omogočajo preživetje stresnih pogojev. Spremembe RNA-vezavnih proteinov lahko povzročijo številne nevrodegenerativne bolezni, za katere je značilno propadanje nevronov. V naši raziskavi smo z metodo označevanja okolice z biotinom označili proteine, ki interagirajo ali so v bližini proteina staufen. Uporabili smo dve biotin-ligazi: BirA* in TurboID. Ko smo dokazali, da se je fuzijski protein sintetiziral v celicah HEK293 in je bila biotinilacija proteinov uspešna smo izolirane proteine poslali na analizo z masno spektrometrijo. Rezultati so pokazali, da večina proteinov, ki smo jih določili, sodeluje pri vezavi RNA ali pri strukturi ribosoma, kar nekaj proteinov pa se veže z molekulo DNA. Večje razlike med proteini v okolici proteinov staufen 1 in staufen 2 nismo zaznali. Uporaba različnih biotin-ligaz pa nam je dala vpogled v časovno dinamiko interakcij. Pokazali smo, da se protein staufen nahaja v okolici ribosoma in molekule RNA. Staufen protein is an RNA-binding protein that forms ribonucleoprotein complexes with the RNA, e.g., stress granules which help survive stress conditions. Changes of RNA-binding proteins can cause a number of neurodegenerative diseases characterised by neuronal degeneration. In our study, we used a proximity labelling method to label proteins that interact or are in the vicinity of the Staufen protein. Two biotin ligases were used: BirA* and TurboID. After confirming that the fusion protein was correctly expressed and that the biotinylation of the proteins was successful, we sent the isolated proteins for analysis by mass spectrometry. We concluded that most of the proteins we identified are involved in RNA binding or ribosome structure, and few proteins bind to DNA. We did not detect any major difference between the proteins surrounding Staufen 1 and Staufen 2 proteins. Using different biotin-ligases gave us insight into the temporal dynamics of the interactions. We showed that the Staufen protein is located near the the ribosome and the RNA molecule. nevrodegenerativne bolezni protein staufen interaktor biotin-ligaza neurodegenerative diseases staufen protein interactor biotin ligase true false false [K. Lenarčič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-04-05 07:15:02 2023-04-05 07:15:12 2023-04-19 09:06:34 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577.2 217622 149656067 MAG_Lenarcic_Katarina_2023.pdf MAG_Lenarcic_Katarina_2023.pdf 1 751DC04B19BE46EF8603891FA2E29624 77aa4391f262e5dc08d7cf3e6be7259219034b7d4a38eac3e864f011301bb722 b84de3e8-d370-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=167727 Biotehniška fakulteta 0 0 0