diplomsko delo
Nika Tomsič (Author), Miha Pavšič (Mentor)

Abstract

EpCAM je označevalec tumorskih in matičnih celic, ki sodeluje pri signaliziranju preko regulirane intramembranske proteolitične cepitve. V kristalni strukturi zunajceličnega dela EpCAM je bil opažen hidrofoben žep z vezanim kristalizacijskim aditivom N-decil-β-D-maltopiranozid. Struktura bi lahko predstavljala vezavno mesto za fiziološki hidrofoben ligand s podobnimi kemijskimi lastnostmi, to je daljšo hidrofobno verigo. Predhodne raziskave in silico so pokazale, da bi to lahko bila veriga palmitata. Namen našega dela je bil raziskati vezavo hidrofobnega liganda in vitro, pri tem pa smo kot testni ligand izbrali 11-(danzilamino)undekanojsko kislino (DAUDA). Po izražanju in čiščenju proteina v nemutirani in mutirani obliki (zaprt žep) z nikljevo kromatografijo in gelsko filtracijo smo izvedli teste vezave preko merjenja fluorescence DAUDA in merjenjem sprememb intrinzižne tirozinske fluorescence EpEX. Prvi sklop poskusov kaže, da se DAUDA šibko veže na protein ter da jo palmitat izpodrine. Rezultati tudi kažejo, da je vezava DAUDA neodvisna od oligomernega stanja EpEX. Vezave pri mutirani obliki EpCAM v nobenem primeru nismo zaznali oz. je bistveno šibkejša kot pri divjem tipu, kar kaže na to, da do vezave prihaja dejansko v hidrofobnem žepu ali njegovi neposredni okolici. Pri meritvah tirozinske fluorescence smo sicer ob vezavi palmitata opazili spremembo v absorpcijskem spektru, a je ne moremo nedvoumno pripisati vezavi tega liganda. Rezultati so dobra osnova za nadaljnje eksperimente, ki bi eventuelno lahko vodili do identifikacije fiziološko relevantnega liganda EpCAM.

Keywords

EpCAM;hidrofobni žep;DAUDA;palmitat;tirozinska fluorescenca;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [N. Tomsič]
UDC: 577.112(043.2)
COBISS: 127898115 Link will open in a new window
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Downloads: 2
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Other data

Secondary language: English
Secondary title: Characterization of EpCAM hydrophobic pocket potential for binding of lipid-like molecules
Secondary abstract: EpCAM is a tumor and stem cell marker involved in nuclear signaling via proteolytic cleavage. A hydrophobic pocket was observed in the crystal structure of EpCAM, into which the crystallization additive N-decyl-β-D-maltopyranoside was bound. The structure could represent a binding site for a physiological hydrophobic ligand with similar chemical properties, i.e. a longer hydrophobic chain. Preliminary in silico research suggested the palmitate chain as a possible ligand. The purpose of our work was to investigate hydrophobic ligand binding in vitro using 11-(danzylamino)undecanoic acid (DAUDA) as ligand. After the expression of the protein in the unmutated and mutated (closed pocket) form and purification by nickel chromatography and gel filtration, we performed binding tests by measuring DAUDA fluorescence and EpEX tyrosine fluorescence analysis, namely without and in the presence of palmitate as a competitor. The first set of experiments shows that DAUDA binds weakly to the protein and that palmitate displaces it. The results also show that the binding of DAUDA does not depend on the oligomeric state of EpEX. Binding on the mutated form of EpCAM was not detected in any case, which indicates that binding occurs in the hydrophobic pocket or in the adjacent area. A change in tyrosine fluorescence of EpEX was observed when palmitate was present, but it cannot be unequivocally attributed to the binding of this ligand. The results are a good basis for further experiments, which could eventually lead to the identification of a physiologically relevant EpCAM ligand.
Secondary keywords: EpCAM;hydrophobic pocket;DAUDA;palmitate;tyrosine fluorescence;Glikoproteini;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 31 str.
ID: 16391565