Priprava in analiza rekombinantega signalnega kompleksa znotrajcelične domene EpCAM s proteini FHL2, β-kateninom in Lef-1

Prešern, Uroš (author), Gaber, Aljaž (mentor)

EpCAM je transmembranski glikoprotein, ki je pretirano izražen na površini številnih karcinomov. Eden od mehanizmov, preko katerih sodeluje pri razvoju in napredovanju raka, je regulirana intramembranska proteoliza, med katero se v citosol sprosti njegova znotrajcelična regija EpIC, ki tvori signalni kompleks s proteini FHL2, β-kateninom in Lef-1. Ta v jedru sproži transkripcijo onkogenov, ki spodbujajo proliferacijo celic. Da bi bolje razumeli delovanje omenjenega procesa, smo želeli pripraviti posamezne komponente signalnega kompleksa in analizirati interakcije med njimi. S tem namenom smo v E. coli izrazili rekombinanten β katenin, Lef-1, FHL2 in EpIC ter proteine očistili. Vzpostavili smo nov protokol izražanja in čiščenja Lef 1 in FHL2 ter optimizirali že obstoječ protokol izražanja in čiščenja β katenina ter različnih oblik EpIC v fuziji s sfGFP. Zaradi nagnjenosti FHL2 k tvorbi inkluzijskih teles, smo protein izrazili v fuzijski obliki z različnimi partnerji, ki izboljšujejo topnost proteina. Pri tem se je za najboljšo izbiro izkazal sfGFP, ker je omogočal visoke izplene izražanja in uspešno odstranitev fuzijskega partnerja med čiščenjem. Po pripravi posameznih proteinov smo izvedli analizo interakcij s SEC in prečnim povezovanjem. S SEC smo uspeli potrditi interakcijo med β-kateninom in FHL2, medtem ko interakcije EpIC z ostalimi komponentami nismo zaznali. S prečnim povezovanjem smo zaznali interakcijo med β kateninom in Lef-1 ter β-kateninom in FHL2. Za razliko od SEC smo zaznali tudi interakcijo EpIC s FHL2 in β-kateninom. Poleg opravljenih analiz vzpostavljen protokol priprave posameznih komponent omogoča izvedbo nadaljnjih raziskav o vlogah posameznih proteinov v kompleksu in interakcijah med njimi.

• Language: Slovenian
• Keywords: EpCAM, β-katenin, FHL2, Lef-1, proteinska interakcija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-136225
• COBISS.SI-ID: 105556483
• Publication date in RUL: 20.04.2022

Secondary language

• Language: English
• Title: Preparation and analysis of recombinant EpCAM intracellular domain signaling complex with FHL2, β-catenin and Lef-1

Abstract

EpCAM is a transmembrane glycoprotein that is overexpressed on the surface of various carcinoma cells. One of the mechanisms, of how EpCAM promotes cancer growth, is regulated intramembrane proteolysis, during which the intracellular domain of EpCAM (EpIC) is released in the cytosol. EpIC then forms a signaling complex with proteins FHL2, β-catenin, and Lef-1. In the nucleus, the signaling complex activates the transcription of oncogenes, which promote cell proliferation. To obtain a better understanding of this process, we wanted to prepare the components of the complex and analyze the interaction between them. Therefore, we expressed recombinant β-catenin, FHL2, Lef-1, and EpIC in E. coli and purify them. We established new protocols for the expression and purification of FHL2 and Lef-1. We also optimized already established protocols for the expression and purification of β-catenin and different variants of sfGFP fusions of EpIC. Because FHL2 is prone to the formation of inclusion bodies, we expressed the protein as a fusion with different solubility-enhancement tags. sfGFP proved to be the best choice since it provides high yields and enables the subsequent removal of the tag during purification. After we prepared purified proteins, we analyzed the interaction between them using SEC and cross-linking. The analysis with SEC showed the presence of the interaction between FHL2 and β-catenin, however, there was no interaction between EpIC and other proteins. After cross-linking we observed the interaction between FHL2 and β-catenin, and β-catenin and Lef-1. The results also showed the interaction of EpIC with β-catenin and FHL2. Besides the already performed analysis, the protocols for the preparation of individual components of the complex enables further research into their roles and interactions between them.

• Keywords: EpCAM, β-catenin, FHL2, Lef-1, protein interaction