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Ligand-vezavne lastnosti hidrofobnega žepa EpCAM in njegov vpliv na regulirano intramembransko cepitev
ID Hočevar, Jošt (Author), ID Pavšič, Miha (Mentor) More about this mentor... This link opens in a new window

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Abstract
EpCAM je človeški transmembranski glikoprotein tipa I, ki se izraža na nizki ravni v epitelijskih celicah in na visoki ravni v številnih izvornih celicah. Poleg tega se izraža na visoki ravni v celicah različnih karcinomov. Zaradi slednje lastnosti je bil predlagan kot potencialna tarča pri zdravljenju nekaterih rakavih obolenj, razvita pa so bila številna terapevtska protitelesa. Obenem pa je naše razumevanje njegove vloge v celičnih procesih omejeno. Eksperimentalna določitev strukture zunajceličnega dela EpCAM je razkrila prisotnost hidrofobnega žepa na njegovi površini. Eden izmed mehanizmov, ki omogoča udeležbo proteina EpCAM v celičnih procesih, je regulirana intramembranska cepitev. Predpostavljamo, da hidrofoben žep igra vlogo pri regulaciji omenjenega mehanizma. V okviru diplomskega dela smo načrtali in pripravili mutantne oblike proteina EpCAM s fizično zaprtim hidrofobnim žepom in kvalitativno dokazali spremembe v regulirani intramembranski cepitvi. V okviru magistrskega dela smo te spremembe kvantificirali in analizirali spremembe v lokalizaciji mutantnih oblik. Slednje smo tudi izrazili v insektni celični liniji Sf9. Z očiščenimi proteini smo preučili ligand-vezavne lastnosti hidrofobnega žepa, tako in vitro kot tudi in silico. Opazili smo lokalizacijo mutantnih oblik na neznanih fibrilarnih strukturah in kvantificirali njihovo spremenjeno dovzetnost k regulirani intramembranski cepitvi. Dokazali smo tudi njihovo pravilno zvitje in vezanje z ligandi.

Language:Slovenian
Keywords:EpCAM, regulirana intramembranska cepitev, ligand
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2021
PID:20.500.12556/RUL-125113 This link opens in a new window
COBISS.SI-ID:54022403 This link opens in a new window
Publication date in RUL:04.03.2021
Views:984
Downloads:214
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Secondary language

Language:English
Title:Ligand-binding capabilities of EpCAM's hydrophobic pocket and its effect on regulated intramembrane proteolysis
Abstract:
EpCAM is a type I human transmembrane glycoprotein, expressed at low levels in epithelial cells and at high levels in a variety of stem cells. Additionally, high levels of expression have also been observed in various carcinomas. Due to the latter characteristic, EpCAM has been proposed as a potential target in treating certain types of cancer. Indeed, therapeutic antibodies have been developed. However, our understanding of EpCAM's involvement in cellular processes leaves much to be desired. After the structure of EpCAM's extracellular part had been experimentally determined, a hydrophobic pocket was observed on its surface. EpCAM's involvement in cellular processes is facilitated by regulated intramembrane proteolysis. We propose that the hydrophobic pocket could play a role in regulating the aforementioned process. Previously, we designed and prepared mutated forms of EpCAM with an obstructed pocket and qualitatively described changes in EpCAM's regulated intramembrane proteolysis. In this thesis, we quantified these changes and analyzed changes in the localization of mutant forms. We also expressed the former in an Sf9 insect cell line. We used the purified proteins to determine the ligand-binding capabilities of EpCAM's hydrophobic pocket, both in vitro and in silico. We observed the localization of mutant forms to unknown fibrous structures and quantified their changed susceptibility to regulated intramembrane proteolysis. Additionally, we provide proof of their correct folding and binding of ligands.

Keywords:EpCAM, regulated intramembrane proteolysis, ligand

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