Detekcija proteinskih interakcija domene MIU proteina SPRTN uslijed oštećenja DNA

master's thesis

• Title (english): Detection of protein interactions of the MIU domain of SPRTN due to DNA damage
• Author: Ivan Blažević
• Mentor: Ivana Marinović Terzić (mentor)
• Mentor: Nataša Bauer (komentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-02-23, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Nesmetano odvijanje osnovnih procesa poput replikacije, transkripcije te popravka DNA
esencijalni su za opstanak stanice. Prisustvo specifičnog tipa oštećenja pod nazivom
kovalentno vezanje proteina na DNA (engl. DNA-protein crosslink, DPC) onemogućava
odvijanje osnovnih staničnih procesa, čime se narušava stanična homeostaza. Zato što
akumulacija DPC u stanici doprinosi razvoju tumora te starenju, humana proteaza SPRTN,
koja je usko vezana uz proteolitički popravak DPC, predmet je brojnih istraživanja. U ovom
radu pozornost je stavljena na nekarakteriziranu domenu MIU proteina SPRTN. Testirali smo
interakcijske partnere domene MIU, te time pokušali predvidjeti njenu funkciju koju obavlja
za vrijeme proteolitičkog uklanjanja DPC. Tumorske stanične linije HeLa i HepG2 su
tretirane formaldehidom i UV-zračenjem u svrhu indukcije DPC. Korištenjem metode
pulldown ispitali smo interakciju domene MIU s proteinima XRCC1, CHK1, PMS2, GAPDH,
MLH1 te proteinima koji su obilježeni s ubikvitinom ili SUMO-2/3 posttranslacijskom
modifikacijom. Interakcija proteina je provjerena metodom Western blot. Domena MIU nije
vezala ni jedan testirani protein. Protein XRCC1 te PMS2 su definirani kao novi C-terminalni
interaktori proteina SPRTN dok je za CHK1 ustanovljeno da se veže za N-terminalni dio
proteina SPRTN.

Abstract (english)

The unobstructed performance of basic processes, such as DNA replication, transcription and
repaire, is essential for cell survival. The presence of a specific type of damage called DNAprotein crosslink (DPC) prevents the execution of basic cellular processes, thereby disrupting
cellular homeostasis. Due to the fact that an accumulation of DPC in cells contributes to
tumor development and aging, a human protease SPRTN, closely related to the proteolytic
repair of DPC, is the subject of numerous studies. This study focuses on the MIU domain, an
uncharacterized domain of SPRTN protein. We tested interaction partners of the MIU
domain, and thereby tried to predict its function during the proteolytic removal of DPC. For
the purpose of DPC induction, tumor cell lines HeLa and HepG2 were treated with
formaldehyde and UV radiation. Using the pulldown method, we examined interactions of the
MIU domain with XRCC1, CHK1, PMS2, GAPDH, MLH1 and proteins labeled with
ubiquitin or SUMO-2/3 post-translational modification. Protein interactions were verified by a
Western blot method. The MIU domain did not bind any tested protein. The XRCC1 and
PMS2 proteins were defined as new C-terminal interactors of SPRTN protein, while CHK1
was found to bind the N-terminal part of SPRTN protein.

• Keywords: kovalentno vezanje proteina; starenje; proteinske interakcije; obaranje proteina
• Keywords (english): DPC; aging; protein interaction; pulldown
• Language: croatian
• URN:NBN: urn:nbn:hr:217:557104
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2025-02-23
• Created on: 2023-03-30 11:36:14