Karakterizacija interakcije humanih proteina SPRTN I p97 metodom supročišćavanja afinitetnom kromatografijom

master's thesis

• Title (english): Characterization of SPRTN protein interaction with p97 using pull-down assay
• Author: Vedrana Vukasović-Lončar
• Mentor: Igor Stuparević (mentor)
• Committee member: Višnja Gaurina-Srček (predsjednik povjerenstva)
• Committee member: Marta Popović (član povjerenstva)
• Committee member: Igor Slivac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2022-09-22, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Križne veze DNA i proteina (engl. DNA-protein crosslinks, DPCs) česta su vrsta oštećenja DNA koja nastaju kovalentnim i ireverzibilnim vezanjem proteina na DNA. Riječ je o velikim lezijama koje sterički ometaju procese koji se odvijaju na DNA poput replikacije, transkripcije, rekombinacije i popravka. Nepopravljeni DPC-ovi narušavaju stabilnost genetičkog materijala i funkciju stanica. U nedavno otkrivenom proteolitičkom putu popravka križnih veza DNA i proteina sudjeluju proteaza Wss1 u kvascu i SPRTN u metazoama. Gen koji kodira za SPRTN esencijalan je u sisavaca te mutacijama uzrokuje SPARTAN ili Ruijs-Aalfs sindrom karakterizaran preuranjenim starenjem, kromosomskom nestabilnošću i karcinomom jetre u mladoj dobi. SPRTN stupa u interakciju s p97/VCP AAA ATPazom koja potencijalno sudjeluje u proteolitičkom putu popravka. U ovom radu pripravljeni su konstrukti različitih duljina humanih proteina SPRTN i p97 koji su prekomjerno eksprimirani u bakteriji Escherichia coli, pročišćeni afinitetnom kromatografijom i prema potrebi gel-filtracijom. Potom je određeno njihovo mjesto interakcije metodom supročišćavanja afinitetnom kromatografijom. Rezultati ukazuju na to da su SHP motiv SPRTN-a i N-D1 domene p97 ključne za interakciju između SPRTN i p97 proteina. Dobiveni rezultati biti će iskorišteni za daljnju strukturnu analizu SPRTN:p97 kompleksa metodom rendgenske kristalografije i krio-elektronske mikroskopije.

Abstract (english)

DNA-protein crosslinks (DPCs) are a frequent type of DNA lesion caused by covalent and irreversible binding of proteins to DNA. These are large lesions that sterically interfere with processes that occur on DNA such as replication, transcription, recombination and repair. Unrepaired DPCs affect the stability of genetic material and cell function. The proteases Wss1 in yeast and SPRTN in metazoans are involved in the recently discovered repair of DNA-protein crosslinks by proteolysis. The gene encoding SPRTN is essential in mammals and its mutations cause SPARTAN or Ruijs-Aalfs syndrome, which is characterized by premature ageing, chromosomal instability and early liver cancer. SPRTN interacts with p97/VCP AAA ATPase which may be involved in the proteolytic repair pathway. In this work, constructs of different lengths of the human SPRTN and p97 proteins were overexpressed in Escherichia coli, purified by affinity chromatography and, if necessary, by gel-filtration. Pull-down assays were performed to identify their interaction sites, indicating that the SPRTN SHP motif and the p97 N-D1 domains are crucial for this interaction. These results thus provide valuable information for further structural analyses of the SPRTN:p97 complex by X-ray crystallography and cryo-electron microscopy.

• Keywords: križne veze DNA i proteina; SPRTN proteaza; p97 ATPaza; popravak DNA; supročišćavanje afinitetnom kromatografijom
• Keywords (english): DNA-protein crosslinks; SPRTN protease; p97 ATPase; DNA repair; pull-down assay
• Language: croatian
• URN:NBN: urn:nbn:hr:159:046928
• 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: 2023-02-13 13:29:17