diplomsko delo
Aljaž Simonič (Author), Miha Pavšič (Mentor)

Abstract

EpCAM in Trop2 sta paralogna homodimerna transmembranska proteina, ki sta bila odkrita kot antigen na površini celic karcinoma debelega črevesja in kot antigen na površini celic trofoblasta. EpCAM se pojavlja pri vseh vretenčarjih, Trop2 pa je nastal z retrotranspozicijo EPCAM in je prisoten pri plazilcih, ptičih in sesalcih. Izražena sta v epitelijskih celicah in sta pomembna za razvoj epitelijskih tkiv, regulirata celično adhezijo in sodelujeta pri proliferativni signalizaciji. Pogosto sta močno izražena v karcinomih. S programom Alphafold Multimer smo predvideli strukture EpCAM iz hišne miši, domače kokoši, navadne krempljarke in navadne cebrice ter simulirali dinamiko njihovih zunajceličnih delov, skupaj s simulacijo dinamike kristalne strukture zunajceličnega dela človeškega EpCAM. Prav tako smo s programom Alphafold Multimer predvideli strukture človeškega homodimera EpCAM, homodimera Trop2 in heterodimera EpCAM:Trop2, simulirali njihove zunajcelične dele skupaj s kristalnima strukturama zunajceličnega dela homodimerov EpCAM in Trop2 ter na podlagi teh simulacij z metodo MM/GBSA izračunali spremembo proste energije pri tvorbi dimera. Ugotovili smo, da je N-končna domena žabjega EpCAM fleksibilnejša od ostalih. Interakcijska površina med podenotama je bila v primerjavi z ostalimi manjša v EpCAM iz navadne krempljarke, medtem ko ima tiroglobulinska zanka v EpCAM iz navadne zebrice manjšo interakcijsko površino z nasprotno podenoto. Izračuni proste energije so napovedali najmočnejšo interakcijo med podenotama v zunajceličnem delu EpCAM iz domače kokoši, najšibkejšo pa v EpCAM iz navadne cebrice. Hidrofobni vezavni žep v EpCAM iz navadne cebrice je manjši od ostalih, v EpCAM iz navadne krempljarke pa je rigidnejši – oboje bi lahko vplivalo na vezavne lastnosti žepa. Ugotovili smo tudi, da sta podenoti v heterodimeru povezani močneje kot v homodimeru EpCAM in podobno močno kot v homodimeru Trop2 in da bi posledično tvorba heterodimera lahko bila termodinamsko ugodna. V laboratoriju z nativno elektroforezo in s prečnim povezovanjem heterodimera nismo opazili, vendar zaradi omejitev uporabljenega pristopa na podlagi laboratorijskih rezultatov ne moremo zagotovo izključiti obstoja heterodimera.

Keywords

EpCAM;Trop2;proteinske interakcije;prosta energija;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [A. Simonič]
UDC: 577.112(043.2)
COBISS: 160078339 Link will open in a new window
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Downloads: 12
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Other data

Secondary language: English
Secondary title: Molecular dynamics of EpCAM from different species and its interaction with the paralogous protein Trop2
Secondary abstract: EpCAM and Trop2 are paralogous homodimeric transmembrane proteins that were first discovered as an antigen on the surface of colorectal cancer cells and an antigen on the surface of trophoblast cells, respectively. EpCAM is present in all vertebrates, while Trop2 arose by retrotransposition of EPCAM and is present in reptiles, birds and mammals. They are expressed on epithelial cells and participate in epithelial development, regulate cell adhesion and participate in proliferative signalling. Using Alphafold Multimer, we predicted the structures of EpCAM dimers from house mouse, chicken, African clawed frog and zebrafish and performed long molecular dynamics simulations of their extracellular parts, together with the simulation of the crystal structure of the human extracellular part of EpCAM. Using Alphafold Multimer we also predicted the structures of human EpCAM homodimer, Trop2 homodimer and EpCAM:Trop2 heterodimer. We performed short molecular dynamics simulations of extracellular parts of these structures, alongside simulations of crystal structures of extracellular parts of human EpCAM and Trop2 homodimers, and calculated the free energy difference upon dimer formation for these structures using MM/GBSA. We determined that the N-terminal domain of African clawed frog EpCAM exhibits greater flexibility compared to EpCAMs from other species. Dimer interface was smaller in African clawed frog EpCAM compared to others, while thyroglobulin loop in zebrafish EpCAM formed smallest interface with the opposite subunit. Interaction between subunits is the strongest in chicken and the weakest in zebrafish. Hydrophobic binding pocket in zebrafish EpCAM is smaller than others, while in African clawed frog it is more rigid – both differences could result in different binding properties. We also discovered that the subunits in the putative heterodimer are more strongly bound than in EpCAM homodimer and approximately as strong as in Trop2 homodimer. Consequently, heterodimer formation could be thermodynamically favourable. In the laboratory, we didn’t observe the heterodimer using native electrophoresis and crosslinking, but we couldn’t conclusively exclude the possibility of heterodimer formation due to the limitations of our approach.
Secondary keywords: EpCAM;Trop2;molecular dynamics;protein-protein interactions;free energy;Beljakovine;Molekularna dinamika;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: 45 str.
ID: 19478712