The characterization of the protein sharpin

Medić, Nenad (2009) The characterization of the protein sharpin. Diploma thesis, Faculty of Science > Department of Biology.

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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.

Item Type: Thesis (Diploma thesis)
Keywords: sharpin, nuclear localization signal (NLS), nuclear export sequence (NES), leptinomycin B, ubiquitin
Supervisor: Đikić, Ivan
Co-supervisor: Matulić, Maja
Date: 2009
Number of Pages: 49
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 10 Sep 2014 12:43
Last Modified: 10 Sep 2014 12:43

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