20.500.12556/RUL-129644 Računalniška analiza oligomernega stanja in funkcijskih lastnosti človeškega ISOC2 Computational analysis of oligomeric state and functional properties of human ISOC2 ISOC2 spada v naddružino izokorizmatazi podobnih proteinov (Isochorismatase like proteins, IHL). Encimi iz naddružine IHL prepoznajo raznolike substrate, vendar večinoma katalizirajo cepitev C–N ali C–O vezi. So homooligomerni proteini z različnim številom podenot. Glede na število podenot so lahko monomeri, dimeri in tetrameri, v redkih primerih pa oktameri. Nekateri proteini znotraj naddružine IHL še niso bili biokemijsko karakterizirani, zato so njihove biološke funkcije neznane. ISOC1 in ISOC2 sta paraloga iz naddružine IHL, ki se pojavljata pri vretenčarjih, med drugim pri človeku. Oba proteina sta slabo karakterizirana in njuna funkcija še ni popolnoma pojasnjena. ISOC2 vsebuje 205 aminokislinskih ostankov. Znane so tri izooblike proteina, ki nastanejo z alternativnim izrezovanjem. Znano je, da protein interagira s tumorskim supresorjem p16. Namen dela je bil določitev oligomernega stanja proteina ISOC2 in njegova funkcijska karakterizacija. Ker kristalna struktura človeškega ISOC2 še ni znana, smo naredili modele njegove strukture, najprej tetramerne, kasneje še dimerne. Naredili smo analizo umestitve molekul iz knjižnice človeškega metaboloma, ki je razkrila dobro umeščanje nukleotidov v vezavni žep proteina. Eksperimentalno smo preverili v kakšnem oligomernem stanju se nahaja rekombinantni protein. Analiza s prečnim povezovanjem je razkrila, da je protein pretežno v dimernem stanju, nekaj pa se ga nahaja tudi v tetramernem stanju. Preverjali smo tudi, ali ima protein encimsko aktivnost. Izkazalo se je, da protein nima niti esterazne niti nukleazne aktivnosti. Z metodo pomika na gelu (EMSA) smo preverjali ali protein morda le interagira z nukleinskimi kislinami, vendar smo tudi to hipotezo zavrgli. ISOC2 belongs to the isochorismatase-like (IHL) protein superfamily. Enzymes from the IHL protein superfamily mostly catalyse the cleavage of C–N or C–O bonds but recognise different substrates. They are homo-oligomeric proteins with different numbers of subunits, depending on their identity. They may be monomers, dimers and tetramers or in rare cases octamers. Some proteins from the superfamily have not been biochemically characterised and their function remains unclear. Isochorismatase domain-containing protein 1 (ISOC1) and isochorismatase domain-containing protein 2 (ISOC2) are paralogous IHL superfamily proteins found in vertebrates including humans. The function of the two proteins has not yet been elucidated. ISOC2 contains 205 amino acids and occurs in three isoforms formed by alternative splicing. It is known to interact with the tumour suppressor p16. Our goal was to determine the oligomeric state of human ISOC2 and to uncover its putative enzymatic activity. Since the structure of ISOC2 is unknown, we first constructed homology models of tetrameric and dimeric ISOC2 structures. We then docked the human metabolome library to the model structures and discovered that nucleotides fit well into the presumed active site of the protein. We performed a cross-linking experiment with recombinant ISOC2 and showed that the protein is mainly a dimer, but a small amount of tetrameric ISOC2 was also observed. We also performed basic enzymatic characterization of the protein. Our experiments showed that the protein has neither esterase nor nuclease activity. In addition, an electrophoretic mobility shift assay showed that the protein does not interact with deoxyribonucleic acids. ISOC2 oligomerno stanje karakterizacija ISOC2 oligomeric state characterisation true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2021-09-06 12:40:10 2021-09-06 12:40:15 2022-09-08 04:09:48 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 14261 78765059 Fegus_Nastja_-_Racunalniska_analiza_oligomernega_stanja_in_funkcijskih_lastnosti_cloveskega_ISOC.pdf Fegus_Nastja_-_Racunalniska_analiza_oligomernega_stanja_in_funkcijskih_lastnosti_cloveskega_ISOC.pdf 1 ABDA8B230ED1E95204EB11825E29BADD c0d7f0382346954146a852ce6ecb3b0e43b39681f546ac1f4cd014400b874c69 942bd029-0efe-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=146660 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0