Uloga dimerizacije mitohondrijskog proteina Nix u aktivaciji mitofagije

master's thesis

• Title (english): The role of mitochondrial protein Nix dimerization in mitophagy activation
• Author: Mija Marinković
• Mentor: Ivana Novak (mentor)
• Mentor: Inga Marijanović (mentor)
• Committee member: Inga Marijanović (predsjednik povjerenstva)
• Committee member: Ivana Novak (član povjerenstva)
• Committee member: Gordana Lacković-Venturin (član povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2013-02-08, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Nedavna istraživanja ukazuju da autofagija posreduje u selektivnom uklanjanju proteinskih agregata, organela poput mitohondrija i ribosoma ili patogena. Za selektivno uklanjanje mitohondija odgovoran je mitohondrijski protein Nix koji se ponaša kao receptor koji regulira mehanizam autofagije. Cilj ovoga istraživanja je ustanoviti ulogu dimerizacije proteina Nix u aktivaciji mitofagije. Ciljanom mutagenezom napravljena su dva tipa mutanti u proteinu Nix; koji tvore samo monomer i oni koji formiraju monomere i dimere. Za utvrđivanje sposobnosti vezanja autofagosomalnih proteina za divlji tip i mutante proteina Nix korištene su metode GST Pull-down i Western imunodetekcija. Imunofluorescencijska mikroskopija korištena je za određivanje stanične lokalizacije proteina Nix i za određivanje mitofagijske aktivnost u normalnim uvjetima kao i u prisutnosti mitohondrijskog otrova CCCP. Prema očekivanjima, rezultati pokazuju povećanu aktivnost mitofagije u stanicama koje prekomjerno eksprimiraju normalnu varijantu proteina Nix. Nasuprot tome, mutante koje gube sposobnost stvaranja dimera pokazuju smanjenu aktivnost mitofagije. Iz rezultata se zaključuje da je dimerizacija proteina Nix jedan od mogućih mehanizama kojim se povećava aktivnost mitofagije.

Abstract (english)

Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles such as mitochondria or ribosomes and microbes in cells. Recently, the role of mitochondrial protein Nix as a receptor for selective removal of mitochondria by autophagy was described. The aim of this research was to determine the role of Nix dimerisation in mitophagy activation. Using site directed mutagenesis, two types of mutants were made, with ability to form only monomers or to form both monomers and dimers. GST Pull-down and Western blot analysis were used to determine binding of different Nix forms to autophagosomal proteins. Immunofluorescence microscopy was applied to define Nix localization in cells and to quantify mitophagic activity in normal conditions and in the presence of mitochondrial poison CCCP. As expected, results showed increased mitophagy activity in the cells overexpressing wild type Nix that form dimers. Nix mutants without the ability to form dimers showed reduced mitophagic activity. In conclusion, Nix dimerisation is a possible additional mechanism how autophagic machinery is recruited to mitoichondria to enhance their removal.

• Keywords: autofagija; mitofagija; mitohondriji; dimerizacija; Nix
• Keywords (english): autophagy; mitophagy; mitochondria; dimerization; Nix
• Language: croatian
• URN:NBN: urn:nbn:hr:217:263545
• 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: Access restricted to students and staff of home institution
• Created on: 2017-04-27 10:16:51