Optimization of conditions for pull-downs of biotinylated proteins in cell line HEK293T

EpCAM je transmembranski glikoprotein, ki se v celici nahaja kot cis-dimer in igra pomembno vlogo pri celičnem signaliziranju preko regulirane intramembranske proteolize (RIP). EpCAM je podvržen dvema cepitvama – α- oz. β-cepitvi ter nato še γ-cepitvi. Dimerizacija EpCAM in njegova cepitev se med seboj izključujeta – proteazi, ki sta odgovorni za cepitev proteina, namreč ne moreta dostopati do cepitvenih mest, če se EpCAM nahaja v obliki dimera. Do sedaj še ni bilo ugotovljeno, kaj sproži disociacijo stabilnih dimerov EpCAM in posledično omogoči cepitev. Pri tem bi lahko sodelovali tudi drugi proteini oz. interakcije EpCAM z njimi – interaktom. V zadnjem desetletju se je razvila metoda bližnje ligacije (PLA), ki omogoča označevanje, izolacijo ter v kombinaciji z masno spektrometrijo tudi identifikacijo proteinov, ki interagirajo s proteinom, ki nas zanima (vaba). Z encimom biotin ligazo, ki ga spojimo z vabo, lahko biotiniliramo proteine, ki se nahajajo v njegovi neposredni bližini. Biotinilirane proteine nato z uporabo magnetnih kroglic s streptavidinom izoliramo z metodo »pull-down« ter identificiramo z masno spektrometrijo. V okviru diplomske naloge smo želeli optimizirati pogoje biotinilacije bližnjih proteinov EpCAM z biotin ligazo TurboID z uporabo heterodimerizacijskega sistema proteinov FRB in FKBP, ki dimerizirata v prisotnosti rapamicina. To nam za razliko od neposredne fuzije omogoča naknadno vezavo TurboID na EpCAM ter s tem večji vpliv na biotinilacijo s časovnega in prostorskega vidika. Najprej smo v bakterijskih celicah Rosetta-gami 2(DE3) izrazili protein FRB-TurboID in ga z nikljevo afinitetno kromatografijo uspešno izolirali ter ga uporabili za izvedbo PLA. Za PLA smo celice HEK293T transficirali s plazmidom, ki vsebuje zapis za FKBP-EpCAM-GFP. Celicam, ki po transfekciji izražajo FKBP-EpCAM-GFP, smo dodali FRB-TurboID, rapamicin ter biotin. Biotinilirane proteine smo nato izolirali z uporabo magnetnih streptavidinskih kroglic. Cilj diplomskega dela je bil določiti najmanjšo potrebno količino magnetnih kroglic z vezanim streptavidinom za metodo »pull-down« biotiniliranih proteinov v celični liniji HEK293T. Rezultati, ki smo jih dobili v okviru diplomskega dela, bodo uporabni za nadaljnje študije identifikacije interakcijskih partnerjev EpCAM, ki potencialno sodelujejo pri disociaciji cis-dimera proteina EpCAM in tako omogočajo njegovo cepitev.EpCAM is a transmembrane glycoprotein that occurs in cells as a cis-dimer and plays an important role in cell signaling pathways via regulated intramembrane proteolysis (RIP). EpCAM undergoes two proteolytic cleavages – α- or β-cleavage and then γ-cleavage. Dimerization of EpCAM and its cleavage are mutually exclusive – proteases that are responsible for the cleavages cannot access cleavage sites when EpCAM is in its dimeric form. It has not yet been discovered what triggers the dissociation of stable EpCAM dimers and enables its cleavage, but it is hypothesized that other proteins besides the proteases could also be involved in the process. Proximity ligation assay (PLA) is a method that has been developing in the last decade. It enables labelling, isolation and in combination with mass spectrometry identification of proteins, that interact with the protein of interest (bait). The assay is based on a bait fused to a promiscuous biotin ligase which biotinylated its adjacent proteins including its interaction partners. Biotinylated proteins can then be isolated using streptavidin beads and identified using mass spectrometry. In this work we wanted to optimize conditions of EpCAM proximity biotinylation with biotin ligase TurboID. We used FRB and FKBP heterodimerization system. These two proteins dimerize in the presence of rapamycin. This enables later binding of TurboID and EpCAM instead of direct fusion and with that greater impact on biotynilation from time and spatial aspect. We expressed protein His8-GST-HRV3C-FRB-TurboID in bacterial cells Rosetta-gami 2(DE3). Then we used nickel affinity chromatography to isolate FRB-TurboID. We transfected HEK293T cells with plasmid that codes for FKBP-EpCAM-GFP. We added FRB-TurboID and rapamycin to transfected cells. Later we isolated biotinylated proteins via streptavidin pull-down assay. The goal was to determine the lowest amount of streptavidin coated magnetic beads needed for the pull-down assay. The results will contribute to further studies on the identification of interaction partners of EpCAM that could take part in dissociation of cis-dimer and subsequently enable α- oz. β-cleavages