Karakterizacija interakcije proteina BLNK i receptorne tirozin-kinaze MET uporabom ligacije proteina posredovane pocijepanim inteinom

master's thesis

• Title (english): Characterization of BLNK protein interaction with receptor tyrosine-kinase MET using split intein-mediated protein ligation
• Author: Robert Valla
• Mentor: Igor Štagljar (mentor)
• Mentor: Nataša Bauer (sumentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Committee member: Marin Ježić (član povjerenstva)
• Committee member: Petra Korać (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-07-19, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Receptorna tirozin-kinaza MET je protoonkogen uključen u razne fiziološke procese, sudjeluje u tkivnoj homeostazi i regeneraciji te u brojnim patofiziološkim stanjima, primjerice različitim tipovima raka. Protein BLNK je središnji adaptorni protein nizvodno od signalne kaskade B-staničnog receptora. Protein BLNK omogućuje stvaranje multimolekularnih signalnih kompleksa, što prethodi sazrijevanju limfocita B. Protein BLNK je novo opisani interackijski partner receptora MET te bi interakcija receptora MET i proteina BLNK mogla imati jedinstvenu biološku značajnost. U ovom istraživanju je u svrhu karakterizacije interakcije receptora MET i proteina BLNK korištena nova metoda istraživanja proteinskih interakcija zvana ligacija proteina posredovana pocijepanim inteinom (engl. SIMPL). Interakcija receptora MET i proteina BLNK je potvrđena te je istražena ovisnost interakcije o fosforilaciji. Uspostavljena je i okarakterizirana stanična linija sa stabilnom ekpresijom receptora MET i taj se stabilni sustav MET-SIMPL može upotrijebiti kao platforma za probir lijekova. Identificirane su aminokiseline odgovorne za interakciju receptora MET i proteina BLNK. Metoda SIMPL ELISA također je upotrebljena za određivanje veznih mjesta proteina GRB2 na protein BLNK te za istraživanje uloge proteina GRB2 u interakciji receptora MET i proteina BLNK. Dobiveni rezultati pružaju bolji uvid u funkcionalne i mehanističke značajke interakcije receptora MET i proteina BLNK i proširuju razumijevanje biologije receptorne tirozin-kinaze MET.

Abstract (english)

MET receptor tyrosine-kinase is a proto-oncogene involved in various physiological processes, such as tissue homeostasis and regeneration, as well as numerous pathophysiological conditions, such as different types of cancers. BLNK is a central adaptor protein downstream of the B-cell receptor signaling cascade, which enables the formation of multimolecular signaling complexes preceding the maturation of B lymphocytes. BLNK protein is a newly discovered interaction partner of MET, and MET-BLNK interaction may have a unique biological significance. A novel method to study protein-protein interactions called split intein-mediated protein ligation (SIMPL) was used in this research to characterize MET-BLNK interaction. MET-BLNK interaction was orthogonally confirmed and its phosphorylation-dependent nature was investigated using the SIMPL assay. The stable MET-SIMPL system was generated, characterized, and utilized as a drug screening platform. The amino acid residues on MET and BLNK responsible for MET-BLNK interaction were identified using SIMPL assay. SIMPL ELISA was used to determine the binding sites for GRB2 protein on BLNK and assess the role of GRB2 in MET-BLNK interaction. These results provided insight into functional and mechanistic features of MET-BLNK interaction and have deepened the understanding of MET receptor tyrosine-kinase biology.

• Keywords: MET; BLNK; proteinske interakcije; prekrajanje proteina; SIMPL
• Keywords (english): MET; BLNK; PPI; protein splicing; SIMPL
• Language: croatian
• URN:NBN: urn:nbn:hr:217:560473
• 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: 2023-07-19
• Created on: 2021-07-22 12:48:06