Karakterizacija proteina sharpina

master's thesis

• Title (english): The characterization of the protein sharpin
• Author: Nenad Medić
• Mentor: Ivan Đikić (mentor)
• Mentor: Maja Matulić (mentor)
• Committee member: Mirjana Kalafatić (predsjednik povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2009, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Sharpin, protein prvotno otkriven u postsinaptičkoj gustoći štakora, posjeduje homologe kod miša i čovjeka te je eksprimiran u varirajućim količinama i u drugim organima osim mozga. Konkretna uloga i funkcija sharpina još nije razjašnjena, ali dosadašnja istraživanja upućuju na ulogu u sinaptičkoj funkciji te u signalnom putu NFκB. Recentna istraživanja također upućuju na sharpinovu povezanost s ubikvitinsko-proteosomskim sustavom. U neuronima hipokampusa, te u HeLa stanicama, u kojima je endogeno eksprimiran, sharpin je prisutan i u citoplazmi i u jezgri. Bioinformatički alati koji na temelju dosad poznatih konsenzusnih jezgrinih lokalizacijskih signala (NLS), odnosno jezgrinih izlaznih sekvenci (NES) predviđaju postojanje istih dali su pretpostavku o prisutnosti hidrofobnog, leucinom bogatog NES-a unutar sharpinove primarne sekvence. Mutacijom triju leucina unutar konsenzusne izlazne sekvence zamijećeno je nakupljanje sharpina u jezgri. Isti učinak postignut je tretiranjem stanica leptinomicinom B, specifičnim inhibitorom jezgrinog eksporta proteina koji posjeduju leucinom bogatu izlaznu sekvencu. Ovi rezultati govore o prisutnosti leucinom bogate jezgrene izlazne sekvence kod sharpina te o njezinoj ulozi u regulaciji sharpinove jezgrene lokalizacije.

Abstract (english)

Sharpin, originally discovered as a novel protein of the rat postsynaptic density, has both human and mouse homologues and is expressed in varying amounts in other organs, besides the brain. Its function has not yet been clearly resolved, however, research performed so far suggest sharpin's role in the synaptic function and the NFκB signalling pathway. Recent research also implies a possible connection to the ubiquitin-proteasome system. In hippocampal neurons, as well in HeLa cells, which express it endogenously, sharpin is present in both the cytoplasm and the nucleus. Bioinformatical tools, based on consensus sequences from all known nuclear localization signals (NLSs) and nuclear export sequences (NESs) are able to predict such sequences in protein primary structures. This kind of a bioinformatical analysis found no known NLS in sharpin but predicted the existence of a hydrophobic, leucine rich NES. Using site directed mutagenesis, we mutated three leucines in the predicted consensus sequence into alanines. As a result, we noticed an increase in the nuclear localization of sharpin, as compared to the control. We obtained similar results after incubating HeLa cells with leptinomycin B, a known inhibitor of leucine-rich sequence dependant nuclear export. These results suggest the presence of leucine-rich NES in sharpin, and its role in regulating sharpin's nuclear localization.

• Keywords: sharpin; jezgrin lokalizacijski signal (NLS); jezgrina izlazna sekvenca (NES); leptinomicin B; ubikvitin
• Keywords (english): sharpin; nuclear localization signal (NLS); nuclear export sequence (NES); leptinomycin B; ubiquitin
• Language: croatian
• URN:NBN: urn:nbn:hr:217:764078
• 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
• Created on: 2018-03-13 13:36:29