Biochemical and structural analysis of the E3 ligase TOPORS and the transcription factor ZBTB8A

master's thesis

• Title (croatian): Biokemijska i strukturna analiza E3 ligaze TOPORS-a i transkripcijskog faktora ZBTB8A
• Author: Lucija Mance
• Mentor: Vladimir Mrša (mentor)
• Committee member: Vladimir Mrša (predsjednik povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2022-07-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

SUMOylation is a posttranslational modification that regulates various aspects of protein functions, from stability and activity to localization in cellular compartments. Through its regulatory role, SUMOylation contributes to various eukaryotic cellular processes including DNA repair, transcription and nuclear transport. Followed by the cascade of enzymatic reactions, E3 ligases have a key role in the fast and efficient handover of SUMO to the molecules of a substrate. Although almost one-third of proteins are subject to SUMOylation, so far there are only two well-characterized human E3 ligases. The final effect of SUMOylation is also unclear for most substrates. To improve the understanding of SUMOylation, the focus of this project was on biochemical and structural characterization of one SUMO E3 ligase, TOPORS, and a set of SUMOylation substrates from the ZBTB family. An E3 ligase, TOPORS, unique because it displays both ubiquitin and SUMO ligase activities, was chosen because it has been poorly explored so far and its structure and mechanism remain unknown. During the project, we made preliminary bioinformatic and biochemical observations about its middle region which encompasses a putative DNA-binding PWI domain and a fragment that has the SUMO E3 ligase activity. As transcription factors are among the most frequently targeted proteins for SUMOylation, the second part of the project investigated ZBTB8A as a representative member of the large family of human transcription factors. While the effect of SUMOylation on this protein has not been determined during the project, we performed a structural analysis of a crystallized fragment of ZBTB8A, revealing that it organizes into filament structures via its BTB domain.

Abstract (croatian)

SUMOilacija je posttranslacijska modifikacija koja regulira različite aspekte funkcija proteina – od stabilnosti i aktivnosti, do lokalizacije u različite stanične odjeljke. Svojom regulatornom ulogom, SUMOilacija doprinosi različitim eukariotskim staničnim procesima, uključujući popravak DNA, transkripciju i transport u jezgru. Prethođene nizom enzimskih reakcija, E3 ligaze imaju ključnu ulogu u brzoj i učinkovitoj primopredaji SUMO proteina na molekule substrata. Iako je gotovo jedna trećina proteina podložna SUMOilaciji, do sada postoje samo dvije okarakterizirane humane E3 ligaze. Konačni učinak SUMOilacije također je nejasan za većinu supstrata. Kako bi se poboljšalo razumijevanje SUMOilacije, fokus ovog projekta bio je na biokemijskoj i strukturnoj karakterizaciji jedne SUMO E3 ligaze – TOPORS-a, i skupu supstrata za SUMOilaciju iz ZBTB obitelji. E3 ligaza TOPORS, jedinstvena jer pokazuje i ubikvitin i SUMO ligaznu aktivnost, odabrana je jer je do sada slabo istražena te joj struktura i mehanizam djelovanja nisu nepoznati. Tijekom projekta, napravili smo preliminarna bioinformatička i biokemijska istraživanja o srednjoj regiji TOPORS-a, koja obuhvaća navodnu DNA-veznu PWI domenu i fragment koji ima SUMO E3 ligaznu aktivnost. Budući da su transkripcijski faktori među najčešće ciljanim proteinima za SUMOilaciju, drugi dio projekta istraživao je ZBTB8A kao reprezentativnog člana velike obitelji humanih transkripcijskih faktora. Dok efekt SUMOilacije na ovaj protein nije utvrđen tijekom projekta, izvršili smo strukturnu analizu kristaliziranog fragmenta ZBTB8A, otkrivajući da tvori filamentozne strukture putem svoje BTB domene.

• Keywords: protein oligomerization; PWI; SUMOylation; TOPORS; ZBTB
• Keywords (croatian): oligomerizacija proteina; PWI; SUMOilacija; TOPORS; ZBTB
• Language: english
• URN:NBN: urn:nbn:hr:159:289306
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2024-04-29 09:26:37