Priprava rekombinantnih ogrodnih proteinov β-katenin uničevalnega kompleksa in rekonstrukcija njihovih biomolekularnih kondenzatov

Vegelj, Nika (author), Gaber, Aljaž (mentor)

Kolorektalni rak je ena izmed najpogostejših in najsmrtonosnejših bolezni na svetu, pri čemer je v večini primerov za pojav bolezni odgovorna povečana aktivacija signalne poti Wnt. Signalna pot se aktivira z vezavo liganda Wnt, ki povzroči razpad uničevalnega kompleksa β-katenina, ki je odgovoren za uravnavanje ravni le-tega v celici. Če uničevalni kompleks razpade, se β-katenin kopiči v citoplazmi in preide v jedro, tam pa se veže na faktorje iz družine TCF/Lef, kar vodi v aktivacijo transkripcije genov navzdol od elementa WRE. Ogrodje uničevalnega kompleksa sestavljajo APC, aksin in dvl. Po vezavi β-katenina v uničevalni kompleks, glikogen sintaza kinaza 3β (GSK-3β) in kazein kinaza 1α (CK1α) fosforilirata serinske in treoninske ostanke v N-končni regiji β-katenina, kar vodi do njegove ubikvitinacije in nato proteasomske razgradnje. Da bi bolje razumeli delovanje uničevalnega kompleksa β-katenina, smo želeli pripraviti proteine aksin, dvl in APC ter in vitro analizirati biomolekularno kondenzacijo omenjenih proteinov. Zaradi težav pri pripravi ekspresijskih vektorjev z zapisom za protein APC, nam protein APC ni uspelo izraziti v insektnih celicah Sf9. V bakterijskih celicah E. coli BL21 [DE3] smo z dodatkom plazmida pRARE uspeli izraziti rekombinantna proteina aksin in dvl. Med pripravo lizatov je prišlo do dodatnih cepitev s celičnimi proteazami, kar smo za protein aksin uspešno rešili z uporabo ustreznega lizirnega pufra, ki je vseboval dodatek inhibitorjev proteaz in NaCl, medtem ko pri proteinu dvl tega problema nismo uspeli odpraviti. Zaradi pretežno neurejene strukture obeh proteinov, smo oba izrazili v fuziji z različno kombinacijo dveh fuzijskih partnerjev, ki izboljšujeta topnost, in sicer sfGFP in MBP. Za primernejšo se je izkazala oznaka MBP, ki je omogočila uspešno izražanje obeh proteinov, vendar nam je z optimizacijo izražanja uspelo izraziti le protein aksin v topni frakciji in hkrati preprečiti dodatne cepitve. Aksin smo uspešno izolirali in le delno očistili z metodo nikelj afinitetne kromatografije. Pridobljeni rezultati služijo kot osnova za nadaljnje raziskave pri optimizaciji izražanja proteinov aksin, dvl in APC, ki bodo uporabljeni za nadaljne raziskave biomolekularne kondenzacije uničevalnega kompleksa β-katenina.

• Language: Slovenian
• Keywords: aksin, dvl, APC, signalna pot Wnt, uničevalni kompleks β-katenina
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-152929
• COBISS.SI-ID: 178455811
• Publication date in RUL: 12.12.2023

Secondary language

• Language: English
• Title: Preparation of recombinant β-catenin destruction complex framework proteins and reconstruction of their biomolecular condensates

Abstract

Colorectal cancer is one of the most common and deadliest diseases worldwide, with the majority of cases attributed to increased activation of the Wnt signaling pathway. The signaling pathway is activated by binding the Wnt ligand, leading to the disintegration of the β-catenin destruction complex responsible for regulating its cellular levels. Upon complex disintegration, β-catenin accumulates in the cytoplasm and translocates to the nucleus, where it binds to TCF/Lef family factors, initiating the transcription activation of genes downstream of the WRE element. The destruction complex framework consists of APC, axin, and dvl. Following β-catenin binding to the destruction complex, glycogen synthase kinase 3β (GSK-3β) and casein kinase 1α (CK1α) phosphorylate serine and threonine residues in the N-terminal region of β-catenin, leading to its ubiquitination and subsequent proteasomal degradation. To better understand the functioning of the β-catenin destruction complex, we aimed to prepare axin, dvl, and APC proteins and analyze the biomolecular condensation of these proteins in vitro. Due to difficulties in preparing expression vectors encoding the APC protein, we were unable to express APC in Sf9 insect cells. However, we successfully expressed recombinant axin and dvl proteins in E. coli BL21 [DE3] bacterial cells by adding the pRARE plasmid. During lysate preparation, additional cleavages occurred with cell proteases, which we resolved for axin by using an appropriate lysis buffer containing protease inhibitors and NaCl, while we were unable to eliminate this issue for the dvl protein. Due to the mostly unstructured nature of both proteins, we expressed them in fusion with different combinations of two fusion partners, sfGFP and MBP, to improve solubility. The MBP tag proved more suitable, allowing the successful expression of both proteins. However, optimization efforts only resulted in axin expression in the soluble fraction, preventing additional cleavages. Axin was successfully isolated and partially purified using nickel affinity chromatography. The obtained results serve as a foundation for further research in optimizing the expression of axin, dvl, and APC proteins for subsequent studies on the biomolecular condensation of the β-catenin destruction complex.

• Keywords: axin, dvl, APC, Wnt signalling pathway, β-catenin destruction complex