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Proteolitično procesiranje receptorja za urokinazni aktivator plazminogena s cisteinskimi katepsini
Kolarič, Matej (Author), Turk, Boris (Mentor) More about this mentor... This link opens in a new window

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Abstract
Za boljše prognoze rakavih obolenj je ključno razumevanje molekularnobioloških bolezenskih procesov. V magistrski nalogi smo proučevali proteolitično interakcijo proteina uPAR in zunajceličnih katepsinov, kar bi lahko imelo velik vpliv na napredovanje in metastaziranje raka. Da bi identificirali cepitveno mesto katepsina na proteinu uPAR, smo zanj pripravili cDNA konstrukt, kateremu smo dodali zapis za reporterski peptid. Fuzijski protein smo izrazili v človeških celičnih linijah MDA-MB-231, HEK-293 in HeLa. Transficirane celice HeLa, ki so od vseh celic izražale največjo količino fuzijskega proteina, smo obdelali s cisteinskimi katepsini K, L in S. Ugotovili smo, da vsi odcepijo ektodomeno uPAR. Od testiranih katepsinov je uPAR najbolj učinkovito cepil katepsin S. S tandemsko masno spektrometrično analizo smo določili cepitveni mesti D299 in Q301, ki sta posledica katepsina S. Cepitveni mesti se ujemata z velikostjo odcepljene ektodomene uPAR, ki smo jo zaznali z imunološko detekcijo. Po primarni strukturi ustrezata znani proteolitični specifičnosti katepsina S, njun položaj na terciarni struktur proteina uPAR pa pokaže, da se nahajata na sterično dostopni regiji.

Language:Slovenian
Keywords:uPAR, rak, proteoliza, cisteinski katepsini, tandemska masna spektrometrija
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2020
COBISS.SI-ID:31281155 This link opens in a new window
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Downloads:94
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Secondary language

Language:English
Title:Proteolytic processing of urokinase plasminogen activator receptor with cystein cathepsins
Abstract:
The key for achieving a better outcome in cancer patients is a better understanding of cancer's molecular biology. In this master's thesis we investigated the proteolytic interaction of the uPAR protein and extracellular cathepsins, which could play a role in cancer progression and metastasis. To determine the cathepsin cleavage site on the uPAR protein, the cDNA fragment for uPAR was cloned and the reporter peptide was added to it. The fusion protein was expressed in MDA-MB-231, HEK-293 and HeLa human cell lines. The transfected HeLa cells, which were expressing the most uPAR of all tested cell lines, were treated with cysteine cathepsins K, L and S. We determined that all the cathepsins cleaved uPAR's ectodomain, with the most active cathepsin being cathepsin S. Via a tandem mass spectrometric analysis, we determined cathepsin S cleavage sites D299 and Q301 on the uPAR. The cleavage sites are in accordance with ectodomains masses obtained by immunological detection. According to uPAR's primary structure, the cleavage sites are in accordance with cathepsin S's proteolytic specificity and according to uPAR's tertiary structure, the cleavage sites are sterically accessible.

Keywords:uPAR, cancer, proteolysis, cysteine cathepsins, tandem mass spectrometry

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