Optimizacija priprave zunajceličnega dela disintegrinske metaloproteaze TACE in analiza cepitve proteina EpCAM in vitro

Van Midden, Katarina Petra (avtor), Lenarčič, Brigita (mentor)

Disintegrinska metaloproteaza TACE (znana tudi pod imenom ADAM17) je pomemben regulator različnih signalnih poti, povezanih z razvojem, imunostjo, vnetjem in nastankom tumorjev. Cepi več kot 90 substratov, ki obsegajo citokine, rastne faktorje in adhezijske molekule. Kljub mnogim raziskavam molekularni mehanizmi cepitve nekaterih substratov še vedno niso pojasnjeni. Eden izmed takih substratov je tudi dimerni transmembranski glikoprotein EpCAM, ki se pogosto povišano izraža pri karcinomih in je zato pomemben diagnostični marker in tarča za protirakave terapije. Cepitev s TACE je prvi korak v signalni poti, ki se prične z regulirano intramembransko proteolizo in v končni fazi sproži prepisovanje onkogenov. S celičnimi študijami in analizo z masno spektrometrijo je bilo pokazano, da se cepitveni mesti nahajata v žlebu med obema podenotama dimera in sta zato proteazi TACE težko dostopni. Mehanizem cepitve proteina EpCAM s TACE tako še ni pojasnjen. Da bi odkrili, ali dimerizacija EpCAM ovira cepitev s TACE in ali so za prepoznavo substrata in cepitev potrebni še kakšni drugi faktorji, smo pripravili različne konstrukte celotnega proteina in zunajcelične domene TACE ter jih izrazili v insektni celični liniji Sf9. Optimizirali smo izražanje in čiščenje topne ektodomene TACE (TACE-EX), ter pripravljen konstrukt uporabili za prvo analizo cepitve različnih oblik ektodomen proteina EpCAM in vitro. S cepitvami smo uspeli pokazati, da lahko TACE prepozna in cepi EpCAM na predvidenih mestih, ter da dimerizacija proteina EpCAM ovira cepitev s TACE. To je tudi prva eksperimentalna potrditev hipoteze, da se mora za cepitev EpCAM nahajati v monomerni obliki.

• Jezik: Slovenski jezik
• Ključne besede: TACE, EpCAM, regulirana intramembranska proteoliza, izražanje in čiščenje proteinov
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-118175
• COBISS.SI-ID: 26524931
• Datum objave v RUL: 25.08.2020

VAN MIDDEN, Katarina Petra, 2020, Optimizacija priprave zunajceličnega dela disintegrinske metaloproteaze TACE in analiza cepitve proteina EpCAM in vitro [na spletu]. Magistrsko delo. [Dostopano 5 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=118175

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Optimization of preparation of the extracellular region of disintegrin metalloprotease TACE and cleavage analysis of protein EpCAM in vitro

Izvleček

The disintegrin metalloprotease TACE, also known as ADAM17, is an essential regulator of diverse pathways that include development, immunity, inflammation and cancer progression. It cleaves an array of more than 90 different substrates such as cytokines, growth factors and adhesion molecules. Even though TACE has been extensively studied, molecular mechanisms that govern cleavage of some substrates have still not been elucidated. One of such substrates is a transmembrane glycoprotein EpCAM, which was identified as a tumour-associated antigen due to its high expression level in rapidly growing epithelial tumours. Cleavage with TACE is the first step in a signalling pathway that leads to oncogene expression. In vivo studies in combination with mass spectrometry have shown that TACE cleaves EpCAM at two sites, which are located at the interface of the two subunits in a dimer - the native oligomeric state of EpCAM. The cleavage sites are thereby inaccessible to TACE. To better understand mechanisms by which TACE is able to cleave EpCAM we designed various full length and extracellular constructs of TACE and expressed them in a Sf9 insect cell line. We optimized the expression and purification methods for the construct containing the whole wild-type ectodomain (TACE-EX) and performed the first in vitro cleavage of different EpCAM ectodomain constructs. We were able to show that TACE is able to recognize and cleave EpCAM in vitro at proposed cleavage sites and that dimerization of EpCAM obstructs cleavage because of steric hindrance. This is also the first experimental proof of the hypothesis that EpCAM has to be in a monomeric state to be cleaved.

• Ključne besede: TACE, EpCAM, regulated intramembrane proteolysis, protein expression and purification