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The impact of nucleophosmin gene mutations on its interaction potential
Šašinková, Markéta ; Brodská, Barbora (advisor) ; Čáp, Michal (referee) ; Starková, Júlia (referee)
Nucleophosmin 1 (NPM1) is predominantly localized in the nucleolus and occurs mainly in oligomers formed through its N-terminal domain (NTD). As a transport facilitator and chaperone, NPM1 has a wide range of interacting partners including tumor suppressors p53 and p14Arf. Characteristic C-terminal mutations in NPM1 are reported in approximately 30 % of acute myeloid leukemia (AML) cases and cause aberrant cytoplasmic localization of mutated (mut) NPM1. As a result, many NPM1-interacting proteins, including wild type (wt) NPM1, are relocalized to the cytoplasm. In order to analyze interactions and the oligomeric state of NPM1, we have introduced and optimized several in vitro techniques - native and semi-native polyacrylamide gel electrophoresis and immunoprecipitation - as well as in vivo confocal microscopy and time-resolved fluorescence approaches. Using these methods, we revealed that mutations at the C-terminal domain of NPM1 prevent it from binding nucleolar protein nucleolin (NCL), which has previously been shown to interact with the central part of NPM1, and that drug-induced relocation of mutNPM1 to close proximity of NCL does not induce mutNPM1-NCL complex formation. We proved a lowered stability of mutNPM1-formed oligomers as compared to the wtNPM1 ones, which could be useful for NPM1...
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