diplomsko delo
Anamarija Agnič (Author), Miha Pavšič (Mentor)

Abstract

Epitelijska celična adhezijska molekula (EpCAM) je homodimerni transmembranski glikoprotein, vključen v procese celičnega signaliziranja. Fiziološka prisotnost povišane količine EpCAM na površini zarodnih celic epitelijskih tkiv in manjšega števila na diferenciranih celicah priča o njegovem pomenu pri razvoju in regeneraciji tkiv. Če v celicah normalnega diferenciranega tkiva pride do porušenja ravnotežja obsega cepitve EpCAM, je to znak za tumorsko spremenjenost celic. EpCAM je povečano izražen na površini rakavo spremenjenih celic številnih adenokarcinomov in metastazirajočih celic in je kot tak pomemben tumorski označevalec v prognostiki nekaterih rakavih obolenj. Zaradi razlike v obsegu izražanja na površini normalnih in rakavo spremenjenih celic je EpCAM potencialno primerna tarča imunoterapevtikov. Zunajcelični del EpCAM (EpEX) je podvržen proteolitičnim cepitvam šedaznih encimov, pri katerih se topna oblika EpEX sprosti v zunajcelični prostor. Zaporedje nadaljnjih cepitev privede še do sprostitve znotrajceličnega dela (EpIC), ki se nadalje vključi v β-kateninsko signalno pot. Povečan obseg cepitve se v končni fazi odrazi v prepisovanju genov, ki spodbujajo migracijo in proliferacijo celic. Predpostavlja se, da so v cepitev vključeni zaenkrat še neidentificirani interakcijski partnerji, ki opravljajo vlogo regulatorjev cepitve. Eden izmed pristopov za študij vpliva regulatorjev na cepitev je identifikacija regij na površini EpCAM, ki so vključene v omenjene interakcije in posledično v cepitev. Študije cepitve in vivo predhodno načrtovanih mutantnih proteinov s seti mutacij na različnih delih površine so za nekatere izmed njih pokazale signifikantno odstopanje obsega cepitve glede na divji tip proteina. Z izražanjem zunajceličnega dela izbranih mutantov in s CD-spektrometrijo bi lahko preverili pravilnost njihovega zvitja in na podlagi tega sklepali, ali je spremenjen obseg cepitve res posledica prekinitve interakcije z vezavnimi partnerji in ne nepravilnega zvitja ali drugačnega znotrajceličnega razmeščanja. Diplomsko delo je sestavljeno iz dveh delov: predstavitve pristopov za identifikacijo ključnih cepitev transmembranskih proteinov in priprave DNA-konstruktov z zapisi za mutantne oblike EpCAM, ki bi jih uporabili za analizo pravilnosti zvitja. Mutacije smo v zapis vnašali z metodo mestnospecifične mutageneze Quik-Change, ki temelji na načrtovanju mutantnih začetnih oligonukleotidov in PCR. Njeno uspešnost smo povečali z ločitvijo začetnih oligonukleotidov v posamezni vzporedni reakcijski mešanici. Pripravljeni DNA-konstrukti bodo uporabni za nadaljnje delo v smislu podrobnih analiz cepitev EpCAM in vitro.

Keywords

proteini;epitelijska celična adhezijska molekula;EpCAM;celična adhezija;celično signaliziranje;regulirana intramembranska proteoliza;proteaze;mestnospecifična mutageneza;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [A. Agnič]
UDC: 577.112(043.2)
COBISS: 29473027 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Preparation of EpCAM mutants for cleavage studies with TACE protease
Secondary abstract: The epithelial cell adhesion molecule (EpCAM) is a homodimeric transmembrane glycoprotein, involved in cell signalling pathways. Physiologically present higher expression levels of EpCAM on the surface of stem cells and lower on the surface of differentiated cells indicate its role in the tissue development and regeneration of the organism. The collapse in the balance of the cleavage abundance is a sign for the tumorigenic transformation of cells. Higher levels of EpCAM expression are typical for many types of adenocarcinoma and metastatic cells and therefore relevant as a tumor marker in the prognostics for some types of cancers. The difference in EpCAM expression levels in normal and cancer cells implies its potential role as an immunotherapeutic target. The extracellular domain of EpCAM (EpEX) is subjected to proteolytic cleavages mediated by proteolytical enzymes, called sheddases. Such cleavages result in a release of a soluble EpEX into the matrix. The sequence of following cleavages leads to the release of an intracellular domain (EpIC), which is further integrated in the signalling processes of β-catenin pathway. The increased extent of cleavages ultimately leads to more abundant transcription of genes, correlated to cell migration and proliferation. The cleavages by sheddases are presumably regulated by yet unidentified regulators/interaction partners. One of the approaches for studying the effect of such regulators is by the selective mutagenesis of EpEX surface amino acids and the analysis of the effect those mutantions have on the extend of the cleavages through diminished or abolished interaction with the particular regulator. The results of one of such analysis imply that the cleavage rate of some mutants by proteases significantly differs from that of the wild type. Secondary structure analysis using CD spectrometry would assure that increased rate of the cleavage is not a result of a misfolded protein due to the introduced mutations but a biologically relevant consequence of the absence of an important cleavage/RIP regulator. This work is combined of a short review on current approaches addressing the identification of key cleavages during the shedding process and of the cloning of DNA constructs of EpCAM mutants for further analysis of proper folding. Mutations were introduced into the DNA sequence using the method of the site-directed mutagenesis Quik-Change which is based on the mutant primers design and the polymerase chain reaction. We increased its efficiency by separating primers into two separated reaction mixtures. The prepared DNA constructs will be further used for the detailed in vitro analysis of the EpCAM cleavage.
Secondary keywords: regulated intramembrane proteolysis;site-directed mutagenesis;
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: 41 str.
ID: 12031208