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master's thesis
Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX
Zagreb: University of Zagreb, Faculty of Science, 2018. urn:nbn:hr:217:644051
Šamadan, Lara
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

Cite this document

Šamadan, L. (2018). Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:644051

Šamadan, Lara. "Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:644051

Šamadan, Lara. "Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:644051

Šamadan, L. (2018). 'Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX', Master's thesis, University of Zagreb, Faculty of Science, accessed 13 May 2024, https://urn.nsk.hr/urn:nbn:hr:217:644051

Šamadan L. Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2018 [cited 2024 May 13] Available at: https://urn.nsk.hr/urn:nbn:hr:217:644051

L. Šamadan, "Identifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2018. Available at: https://urn.nsk.hr/urn:nbn:hr:217:644051

Metadata
TitleIdentifikacija kinaze odgovorne za fosforilaciju mitohondrijskog proteina NIX
Title (english)Identification of the kinase responsible for phosphorylation of NIX mitochondrial protein
AuthorLara Šamadan
MentorIvana Novak Nakir (mentor)
MentorIvana Ivančić Baće (mentor)
Committee memberIvana Ivančić Baće (predsjednik povjerenstva)
Committee memberSven Jelaska (član povjerenstva)
Committee memberNada Oršolić (član povjerenstva)
Committee memberNataša Bauer (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2018-12-10, 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
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:644051
Study programmeTitle: Molecular Biology
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Embargo expiration date: 2020-12-10
Terms of useLicence In copyright icon
Created on2019-10-10 08:17:51