magistrsko delo
Milena Stojkovska (Author), Marko Novinec (Mentor), Vera Župunski (Thesis defence commission member), Marina Klemenčič (Thesis defence commission member)

Abstract

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.

Keywords

proteini;proteinske interakcije;malomolekulski ligandi;afinitetna kromatografija;pirazolni derivati;ISOC2;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [M. Stojkovska]
UDC: 577.112:547.78(043.2)
COBISS: 65740035 Link will open in a new window
Views: 300
Downloads: 67
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: English
Secondary title: Identification of human cytoplasmic proteins that interact with two pyrazole derivatives
Secondary abstract: 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.
Secondary keywords: protein interactions;small molecule ligand;affinity chromatography;pyrazole derivatives;ISOC2;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 58 str.
ID: 12806423