20.500.12556/RUL-126447 Identifikacija človeških citoplazemskih proteinov, ki interagirajo z dvema derivatoma pirazola Identification of human cytoplasmic proteins that interact with two pyrazole derivatives Identifikacija malih molekul, ki interagirajo z biološkimi makromolekulami, je eden temeljnih pristopov pri iskanju novih zdravilnih učinkovin. Pri tem ponavadi izhajamo iz znane makromolekule in odkrivamo male molekule, ki z njo interagirajo. Alternativno pa lahko iščemo tudi makromolekule, ki interagirajo z neko izbrano malo molekulo, katere biološka (fenotipska) aktivnost je lahko znana ali pa ne. Najbolj razširjena tehnika, ki jo lahko uporabimo v ta namen, je afinitetna kromatografija. Cilj magistrske naloge je bil identificirati proteine, ki specifično interagirajo z dvema podobnima malima molekulama, 4-(2-aminoetil)-1-cikloheksil pirazol-5-olom (ligand 1) in 4-(2-aminoetil)-1-fenil pirazol-5-olom (ligand 2), ki sta bili sintetizirani na Katedri za organsko kemijo FKKT UL. Mali molekuli smo imobilizirali na afinitetni nosilec NHS-activated Sepharose™ 4 Fast flow in pripravljeni koloni uporabili za izolacijo tarčnih proteinov. Preiskovani vzorec je bil lizat celic človeških monocitov U937, gojenih v suspenzijski kulturi. Analize NaDS-PAGE so pokazale, da se liganda med seboj razlikujeta v naboru interagirajočih proteinov. Eluate s kolone z ligandom 1 smo analizirali z masno spektrometrijo in na ta način identificirali enega od interagirajočih proteinov kot ISOC2 – slabo poznan človeški protein, ki vsebuje izohorizmatazno domeno in interagira z zaviralcem tumorjev p16INK4a. Če je ISOC2 regulator p16INK4a, je potencialna nova tarča za razvoj nove strategije za zdravljenje raka in staranja, ligand 1 pa prva znana mala molekula, ki se nanj specifično veže. Za namen podrobnejše karakterizacije interakcije in vitro smo v okviru tega dela pripravili tudi sistem za izražanje človeškega ISOC2 v bakteriji E. coli. Analiza rekombinantnega proteina s kromatografijo z ločevanjem po velikosti je pokazala, da je rekombinanten protein najverjetneje homodimer, po čemer se razlikuje od svojih homologov, ki so večinoma homotetrameri. Understanding the interactions between small molecules and macromolecules from different perspectives is important for the advancement of basic science and drug development. Towards this goal, we usually begin with a known macromolecule and discover small molecules that interact with it. Alternatively, looking for macromolecules that interact with a selected small molecule, whose biological (phenotypic) activity may or may not be known. Affinity chromatography is the most widely used technique for isolating specific target proteins from a complex proteome. The aim of the master's thesis was to identify proteins that specifically interact with two similar small molecules, 4- (2-aminoethyl) -1-cyclohexyl pyrazol-5-ol (ligand 1) and 4- (2-aminoethyl)-1-phenyl pyrazole-5-ol (ligand 2), which were synthesized at the Department of Organic Chemistry UL FCCT. Both molecules were individually immobilized on NHS-activated Sepharose™ 4 Fast flow agarose and the prepared columns were used to isolate target proteins. The test sample was a lysate of U937 human monocyte cells cultured in suspension. SDS-PAGE analyzes showed that the ligands differed from each other in the set of interacting proteins. Eluates from the ligand 1 column were sent for mass spectrometry analysis, thus identifying one of the interacting proteins as ISOC2, a little-known human protein that has an isochorimatase domain and interacts with the tumor inhibitor p16INK4a. If ISOC2 were a regulator of p16INK4a, it might be a new target for development of a novel strategy for the treatment of cancer and aging. For the purpose of more detailed characterization of the in vitro interaction, we developed a system for expressing the human ISOC2 form in E. coli. Analysis of the recombinant protein by size separation chromatography showed that it was most likely a homodimer, distinguishing it from its homologues, which are mostly homotetramers. proteinske interakcije malomolekulski ligand afinitetna kromatografija pirazolni derivati ISOC2 protein interactions small molecule ligand affinity chromatography pyrazole derivatives ISOC2 true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-04-22 14:34:59 2021-04-22 14:35:02 2022-09-05 03:55:19 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 6713 65740035 Stojkovska_Milena_-_Identifikacija_cloveskih_citoplazemskih_proteinov,_ki_interagirajo_z_dvema_d.pdf Stojkovska_Milena_-_Identifikacija_cloveskih_citoplazemskih_proteinov,_ki_interagirajo_z_dvema_d.pdf 1 6C82CC21DC3E814F7D4BB74D262C3AAA deefe73ef731418883ac25ce8ee6694843e128dd2ab4fe8259be444577cbbdd1 2887b3af-a367-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=142419 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0