Interakcije EpCAM kot determinante signaliziranja preko regulirane proteolize

doktorska disertacija

Tomaž Žagar (Avtor), Miha Pavšič (Mentor), Brigita Lenarčič (Član komisije za zagovor), Gregor Anderluh (Član komisije za zagovor), Kristina Sepčić (Član komisije za zagovor)

Povzetek

EpCAM je dimerni transmembranski protein tipa I, ki je pomemben za normalen potek embrionalnega razvoja in vzdrževanje integritete črevesnega epitelija v razvijajočih in odraslih organizmih. Njegovo izražanje je pogosto povečano pri številnih karcinomih, zato služi kot pomemben tumorski označevalec in terapevtska tarča. Začetni eksperimenti so nakazovali, da EpCAM deluje kot celična adhezijska molekula, vendar so kasnejše študije to spoznanje ovrgle, v ospredje raziskovanja pa je prišla njegova signalna vloga. EpCAM je udeležen v več signalnih poteh, med katerimi je najpomembnejša z RIP aktivirana Wnt/β-kateninska signalna pot, ki je odgovorna za večino onkogenih učinkov EpCAM, kot so povečana proliferacija, migracija in invazija. RIP se začne s cepitvijo ektodomene EpCAM (EpEX) s proteazama TACE ali BACE1, čemur sledi znotrajmembransko proteolitično procesiranje z γ-sekretazo in sprostitev znotrajceličnega dela EpCAM (EpICD), ki sodeluje v jedrnem signaliziranju in induciranju izražanja proteinov, ki so povezani s signalno potjo.Cepitev EpEX je ključni korak za začetek RIP, vendar dejavniki, ki vplivajo na ta proces, niso dobro poznani. Namen doktorskega dela je bil raziskati dva izmed teh potencialnih dejavnikov: oligomerno stanje EpCAM in njegove potencialne interakcijske partnerje. EpCAM obstaja v dimerni obliki, pri čemer poglavitno dimerizacijsko površino tvorijo podenote EpEX, ki sicer dimerizirajo tudi same po sebi brez preostalega dela EpCAM. Cepitvena mesta proteaz TACE in BACE1, ki sta odgovorni za cepitev EpEX, se nahajajo znotraj njegove dimerizacijske površine, zaradi česar so v dimernem stanju EpCAM proteazam nedostopna. Za uspešno cepitev mora dimer EpCAM disociirati na monomerne podenote, kar smo dokazali s poskusom cepitve izoliranega monomernega in kovalentno povezanega dimernega mutanta EpEX z ektodomeno TACE. Izkazalo se je, da oligomerno stanje EpEX odločilno vpliva na njegovo cepitev, saj je za cepitev z ektodomeno TACEdovzetna izključno monomerna oblika EpEX. Poleg vpliva oligomernega stanja EpCAM na cepitev EpEX smo preučili še morebiten vpliv njegovih potencialnih interakcijskih partnerjev, pri čemer smo se osredotočili na interakcijske partnerje samega EpEX, saj ta predstavlja poglavitni del površine EpCAM. Eksperiment smo razdelili na dva dela. V prvem delu smo identificirali potencialne interakcijske površine na EpEX, v drugem delu pa njegove potencialne interakcijske partnerje. Potencialne interakcijske površine smo poskusili identificirali s presejalno mutagenezo aminokislinskih ostankov na površini EpEX, s čimer smo želeli selektivno ošibiti interakcije s potencialnimi interakcijskimi partnerji, učinek zmanjšane interakcije na obsežnost cepitve pa smo preučili s kvantitativno analizo obsežnosti cepitve EpCAM s TACE ali BACE1 v celični liniji kolorektalnega karcinoma HCT8. Pri šestih od osemindvajsetih mutantov se je obsežnost cepitve EpEX statistično signifikantno razlikovala od divjega tipa, in sicer je bila pri vseh mutantih povečana. Mutirani aminokislinski ostanki, vključeni v teh šest setov mutacij, tako predstavljajo potencialne stične površine, preko katerih se na EpCAM vežejo proteini, ki zaviralno vplivajo na cepitev EpEX. Pri tem je potrebno izpostaviti, da se kar štirje od šestih signifikantnih setov mutacij nahajajo na N-končni domeni EpEX, kar nakazuje na to, da N-končna domena morda predstavlja pomembno interakcijsko površino. Analiza cepitve mutantov EpCAM je dodatno razkrila, da sta cepitvi s TACE ali BACE1 ter matriptazo – proteazo, ki cepi EpEX znotraj tiroglobulinske zanke – medsebojno izključujoči. Ta ugotovitev je na videz protislovna, saj je z matriptazo cepljeni EpEX nezmožen dimerizirati, kar povzroči destabilizacijo dimera EpCAM in izpostavitev cepitvenih mest za proteazi TACE in BACE1, zaradi česar bi pričakovali sinergističen učinek na cepitev EpEX. Na tej točki tako ni jasno ali so opaženi pojavi posledica spremenjene interakcije EpCAM neposredno s proteazami ali so v ozadju kakšni drugi dejavniki, kot je spremenjeno razmeščanje proteinov ali lokalizacija v druge membranske domene. Drugi del eksperimenta je vključeval identifikacijo potencialnih interakcijskih partnerjev EpCAM s testom bližnje ligacije z biotinilacijo z uporabo nespecifične biotin ligaze TurboID in masno spektrometrijo v celični liniji kolorektalnega karcinoma HCT8. V ta namen smo pripravili sistem za specifično označevanje proteinov na celični membrani, ki temelji na posredni vezavi TurboID na EpCAM po tem, ko je ta v celici že sintetiziran in premeščen na celično membrano. Takšen pristop omogoča časovno in prostorsko nadzorovano označevanje EpCAM bližnjih proteinov na celični membrani. Z eksperimentom smo pridobili nabor kandidatov interakcijskih partnerjev EpCAM, med katerimi so tudi nekateri membranski proteini, ki se vključujejo v obstoječe vedenje o vlogi EpCAM, nekateri pa odpirajo tudi nove možnosti za funkcijske raziskave, zato so potrebne nadaljnje validacijske študije. To delo predstavlja dobro izhodišče za nadaljnje raziskave dejavnikov, ki vplivajo na cepitev EpEX in potencialno regulacijo signaliziranje EpCAM preko RIP, ter odpira vrata do temeljitejšega razumevanja vloge EpCAM v celici, predvsem z vidika njegovega pomena pri nastanku in razvoju raka.

Ključne besede

EpCAM;epitelijska celična adhezijska molekula;signaliziranje;RIP;regulirana intramembranska proteoliza;mutageneza;interakcijske površine;interakcijski partnerji;bližnje označevanje na osnovi biotinilacije;TurboID;masna spektrometrija;doktorske disertacije;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2024
Tipologija:	2.08 - Doktorska disertacija
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[T. Žagar]
UDK:	577.112:577.2(043.3)
COBISS:	191520003
Št. ogledov:	42
Št. prenosov:	18
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	EpCAM interactions as determinants of signaling via regulated proteolysis
Sekundarni povzetek:	EpCAM is a dimeric type I transmembrane protein that plays a crucial role in normal embryonic development and the maintenance of intestinal epithelial integrity in developing and adult organisms. Its expression is frequently upregulated in various carcinomas, making it an important tumor marker and therapeutic target. Initial experiments suggested that EpCAM functions as a cell adhesion molecule; however, subsequent studies have refuted this notion, shifting the focus of research to its signaling role. EpCAM is involved in several signaling pathways, the most important of them being the RIP-activated Wnt/β-catenin signaling pathway, responsible xiiiort hi majority of EpCAM oncogenic effects, such as increased proliferation, migration, and invasion. RIP is initiated by the EpCAM ectodomain (EpEX) shedding by proteases TACE or BACE1, followed by intramembrane proteolytic processing by γ-secretase and release of the EpCAM intracellular domain (EpICD), which is involved in nuclear signaling and induction of pathway-related protein expression. EpEX shedding is a critical step in initiating RIP, yet the factors influencing this process are not well understood. The aim of this doctoral work was to investigate two factors, possibly involved in EpEX shedding: the oligomeric state of EpCAM and its potential interaction partners. EpCAM exists in a dimeric form, with the main dimerization surface formed between EpEX subunits, which can also dimerize independently without the rest od EpCAM. The cleavage sites of TACE and BACE1, responsible for EpEX cleavage, are located within its dimerization interface, rendering them inaccessible to proteases in the dimeric state of EpCAM. For successful cleavage, the EpCAM dimer must dissociate into monomeric subunits which was demonstrated by the cleavage experiment using isolated monomeric and covalently linked dimeric EpEX mutants with the TACE ectodomain. The cleavage assay revealed that EpEX oligomeric state is crucial for its cleavage, as only the monomeric form of EpEX is susceptible to cleavage by the TACE ectodomain. In addition to the impact of the oligomeric state of EpCAM on EpEX shedding, we also investigated the possible effect of its potential interaction partners, focusing on the interaction partners of the EpEX itself, which represents the major interaction surface of EpCAM. The experiment was divided into two parts. In the first part, we identified potential interaction surfaces on the EpEX, and in the second part, we identified its potential interaction partners. Potential interaction surfaces were identified through site-directed screening mutagenesis of amino acid residues on the surface of EpEX, aiming to selectively weaken interactions with potential interaction partners. The effect of reduced interaction on the extent of EpEX shedding was assessed quantitatively by analyzing the shedding extent of EpCAM mutants in the colorectal carcinoma cell line HCT8. Among the twenty-eight mutants tested, the shedding extent significantly differed from the wild type in six mutants, with an increased shedding extent observed in all of them. The mutated amino acid residues within these six mutation sets represent potential contact surfaces through which proteins diminishing EpEX shedding bind to EpCAM. Notably, four of the six significant mutation sets lie in the N-terminal domain of EpEX, suggesting that the N-terminal domain may represent an important interaction surface. Cleavage analysis of EpCAM mutants further revealed that cleavages by TACE or BACE1 and matriptase, a protease that cleaves EpEX within the thyroglobulin loop, seem to be mutually exclusive. This finding appears contradictory since matriptase-cleaved EpEX is unable to dimerize, which leads to the destabilization of the EpCAM dimer and exposure of TACE and BACE1 cleavage sites and should have a synergistic effect on shedding. It is currently unclear whether the observed effect is a result of altered EpCAM interaction with proteases directly xvort it involves other factors such as altered protein trafficking or localization within other membrane domains. The second part of the experiment involved the identification of potential interaction partners of EpCAM using the biotinylation-based proximity labeling technique utilizing the promiscuous biotin ligase TurboID and mass spectrometry performed in the colorectal carcinoma cell line HCT8. xvort his purpose, we developed a system for specific labeling of proteins on the cell membrane, based on the indirect binding of TurboID to EpCAM after its synthesis and translocation to the cell membrane. This approach allows for temporally and spatially controlled labeling of EpCAM-proximal proteins on the cell surface. The experiment yielded a set of candidate interaction partners of EpCAM, including membrane proteins that are involved in known EpCAM functions, as well as proteins that open new possibilities for functional research, necessitating further validation studies. This work provides a solid foundation for further investigations into the factors influencing EpEX shedding and the potential regulation of EpCAM signaling through RIP. It paves the way for a more comprehensive understanding of the role of EpCAM in the cell, particularly regarding its significance in cancer development and progression.
Sekundarne ključne besede:	EpCAM;signaling;RIP;mutagenesis;interaction surfaces;interaction partners;biotinylation-based proximity labeling;TurboID;mass spectrometry;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS):	Doktorsko delo/naloga
Študijski program:	1000381
Konec prepovedi (OpenAIRE):	1970-01-01
Komentar na gradivo:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Strani:	90 f.
ID:	23267955