Određivanje prisutnosti disulfidnih veza u NGR preusmjeravajućem slijedu ugrađenom u vlakno adenovirusnog vektora

master's thesis

• Title (english): Assessing existence of disulfide bonds in ngr retargeting motif inserted in fiber of adenovirus vector
• Author: Davor Nestić
• Mentor: Andreja Ambriović Ristov (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2015-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Adenovirusi se u genskoj terapiji tumora koriste kao vektori za prijenos genskog materijala do tumora ili stanica koje podržavaju rast tumora kao što su endotelne stanice u angiogenezi. Ugradnjom preusmjeravajućeg slijeda u kapsidni protein replikacijski defektnog adenovirusa može se povećati specifičnost vezanja adenovirusnog vektora na ciljnu stanicu. Preusmjeravajući slijed NGR se veže na aminopeptidazu N koja je jako eksprimirana na endotelnim stanicama u angiogenezi, čime predstavlja moguću metu za usmjeravanje adenovirusnog vektora s ugrađenim NGR peptidnim slijedom, a sve s ciljem inhibicije angiogeneze. Pokazano je da aminokiselinski okoliš koji omeđuje NGR slijed ima ulogu u specifičnosti preusmjeravanja adenovirusnih vektora, a posebnu ulogu ima ciklizacija posredovana disulfidnim vezama. Cilj ovog rada bio je utvrditi postojanje disulfidnih veza u preusmjeravajućem slijedu C539NGRC543VSGC547AGRC551 ugrađenom u HI petlju vlakna AdFNGR te ako postoje, identificirati spektrometrijom masa koji su cisteini uključeni u stvaranje disulfidnih veza. Nadalje, cilj je bio utvrditi važnost stvaranja disulfidnih veza na preusmjeravanje i djelotvornost transdukcije stanica rabdomiosarkoma čovjeka (RD) i stanica tumora dojke čovjeka (MDA-MB-435S) obradom AdFNGR ditiotreitolom. Spektrometrijom masa je pokazano da cistein na poziciji 539 ne stvara disulfidne veze s preostala tri cisteina u preusmjeravajućem slijedu C539NGRC543VSGC547AGRC551. Ovim eksperimentalnim pristupom nije se dokazalo postoji li disulfidna veza između cisteina C543, C547 i C551. Obrada AdFNGR ditiotreitolom dokida povećanu djelotvornost transdukcije RD i MDA-MB-435S stanica u odnosu na Adwt, što upućuje na moguće postojanje disulfidnih veza u preusmjeravajućem slijedu koje omogućuju povoljniju konformaciju HI petlje u AdFNGR i posljedično tome bolje vezanje i internalizaciju adenovirusa u stanicu.

Abstract (english)

Adenoviruses are used for cancer gene therapy as vectors for introducing the foreign genetic material to the tumors or cells that support tumor growth such as endothelial cells undergoing angiogenesis. Incorporation of targeting motif into the capsid protein of replication deficient adenovirus can increase the specificity of binding of the adenoviral vector on the target cell. Targeting motif NGR binds to aminopeptidase N which is highly expressed in endothelial cells undergoing angiogenesis, thereby represents a possible target for retargeting the adenoviral vector containing NGR peptide motif with the aim of inhibiting angiogenesis. It was shown that the amino acid environment has an influence in the specificity of retargeting adenoviral vectors, and that cyclization mediated by disulfide bonds has an important role. The aim of this work was to investigate the existence of disulfide bonds in retargeting motif C539NGRC543VSGC547AGRC551 inserted in HI loop of the fiber AdFNGR and in case of their existence, identify by mass spectrometry which cysteines are involved in disulfide bond formation. Further, the aim was to investigate the significance of disulfide bond formation on retargeting and transduction efficiency of rhabdomiosarcoma (RD) and breast cancer (MDA-MB-435S) cell lines with AdFNGR pretreated with dithiothreitol. Mass spectrometry has shown that cystein at position 539 does not form disulfide bond with any of the three cysteines in retargeting motif C539NGRC543VSGC547AGRC551, but this experimental approach did not prove whether there is a disulfide bond between cysteines C543, C547 and C551. Treatment of AdFNGR by dithiothreitol abolish increased transduction efficiency of RD and MDA-MB-435S cell lines as compared to Adwt, suggesting a possible existence of disulfide bonds in retargeting motif which allows a more favourable conformation of HI loop in AdFNGR and consequently better binding and internalization of adenovirus in the cell.

• Keywords: adenovirus tipa 5; NGR; disulfidni most; preusmjeravajući slijed; angiogeneza
• Keywords (english): adenovirus type 5; NGR; disulfide bond; retargeting motif; angiogenesis
• Language: croatian
• URN:NBN: urn:nbn:hr:159:778042
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-04-28 08:26:10