Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX

master's thesis

• Title (english): Identification of the kinase responsible for phosphorylation of NIX mitochondrial protein
• Author: Lara Šamadan
• Mentor: Ivana Novak Nakir (mentor)
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Sven Jelaska (član povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Committee member: Nataša Bauer (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-12-10, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Dosadašnja istraživanja ukazuju kako fosforilacija uvelike pospješuje vezanje mitohondrijskog proteina NIX za autofagosomalne receptore mitofagije LC3A i LC3B neophodnih prilikom inicijacije autofagije i regrutiranja autofagosoma. Cilj ovog istraživanja je identificirati kinazu odgovornu za fosforilaciju proteina NIX što bi moglo dodatno razjasniti mehanizam regulacije mitofagije. In silico analizom odabrane su kinaze IKKβ i GSK3β koje su testirane metodom Pull-down i imunoprecipitacijom na interakciju s fuzijskim proteinom EGFP-NIX i njegovim mutantama. Metodom Western blot provjerena je proteinska interakcija mitohondrijskog proteina NIX i dviju odabranih potencijalnih kinaza. Dobiveni rezultati pokazuju interakciju proteina NIX s kinazom GSK3β, dok interakcija s kinazom IKKβ izostaje te se iz rezultata zaključuje kako je mitohondrijski protein NIX u interakciji s kinazom GSK3β. S obzirom na ustanovljenu interakciju s kinazom GSK3β, ciljanom mutagenezom in vitro napravljene su dvije mutante, jedna s konstitutivno aktivnom kinazom GSK3β te druga čija je kinazna domena inaktivna. Buduća istraživanja s navedenim konstruktima trebala bi pokazati fiziološku važnost ove interakcije.

Abstract (english)

Previous research suggest that phosphorylation greatly enhances the binding of NIX mitochondrial protein to autophagosomal LC3A and LC3B mitophagy receptors necessary for autophagy initiation and cargo degradation. The aim of this study was to identify kinase responsible for phosphorylation of NIX protein, which could further clarify the mechanism of mitophagy regulation. Using In silico analysis, IKKβ and GSK3β kinases were selected and had been tested by Pull-down and immunoprecipitation for interaction with the fusion protein EGFP-NIX and its appropriate mutant variants. Western blot immunodetection was used to detect protein interaction of NIX and two selected potential kinases. The results show the interaction of NIX protein with kinase GSK3β, whereas interaction with kinase IKKβ was not confirmed. Taken together it can be concluded that of two tested kinases, NIX interacts in vitro with GSK3β. Given the established interaction with kinase GSK3β, two mutants, one with the constitutively active kinase domain and the other with inactive kinase domain, were generated by site-directed mutagenesis. Future studies using these mutants should provide the physiological importance of this interaction.

• Keywords: autofagija; mitofagija; NIX; fosforilacija; kinaza; GSK3β; IKKβ
• Keywords (english): autophagy; mitophagy; NIX; phosphorilation; kinase; GSK3β; IKKβ
• Language: croatian
• URN:NBN: urn:nbn:hr:217:644051
• 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: 2020-12-10
• Created on: 2019-10-10 08:17:51