Disulfidne veze u preusmjeravajućim motivima RGD ili NGR ugrađenim u adenovirusu tipa 5

master's thesis

• Title (english): Disulfide bondsin retargeting motifs RGD or NGR inserted into the adenovirus type 5
• Author: Kristijan Pažur
• Mentor: Andreja Ambriović Ristov (mentor)
• Mentor: Inga Marijanović (mentor)
• Committee member: Inga Marijanović (predsjednik povjerenstva)
• Committee member: Andreja Ambriović Ristov (član povjerenstva)
• Committee member: Ana Galov (član povjerenstva)
• Committee member: Domagoj Đikić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2013-09-30, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Replikacijski defektni adenovirusi tipa 5 (Ad5) koriste se kao vektori za gensku terapiju tumora. Preusmjeravanje Ad5 postiže se ugradnjom preusmjeravajućih motiva u proteine kapside, najčešće u HI-petlju vlakna ili u hipervarijabilnu regiju heksona 5 (HVR5). Integrini αv često su povećano eksprimirani na stanicama tumora u odnosu na normalno tkivo, a preusmjeravajući motiv kojim se mogu preusmjeravati Ad5 na αv integrine na površini stanice je RGD. U genskoj terapiji tumora mogu se ciljati i stanice koje podržavaju rast tumora kao što su endotelne stanice u angiogenezi koje stvaraju nove krvne žile u tumoru. Endotelne stanice u angiogenezi eksprimiraju aminopeptidazu N, a preusmjeravajući motiv koji se veže na APN je NGR. Poznato je da različiti aminokiselinski okoliš, posebice postojanje cisteina oko RGD i NGR motiva ugrađenih u HI-petlju vlakna Ad5 utječe na afinitet vezanja za odgovarajući receptor. Cilj ovog rada bio je istražiti postoje li disulfidne veze u dvama replikacijski defektnim Ad5 vektorima: (i) Ad5HCRGDC s preusmjeravajućim motivom CRGDC ugrađenim u HVR5 i (ii) Ad5ARAP s motivom CNGRCVSGCAGRC ugrađenim u HI-petlji vlakna Ad5. Pokazano je da obrada Ad5HCRGDC ditiotreitolom ne mijenja djelotvornost transdukcije stanica rabdomiosarkoma (RD), što znači da ne utječe na vezanje na αv integrine. Potvrda da cisteini unutar CRGDC motiva ne stvaraju disulfidne veze dobivena je spektrometrijom masa. Obrada Ad5ARAP ditiotreitolom dokida sposobnost preusmjeravanja ovog virusa na APN eksprimiran na stanicama RD, što znači da cisteini smješteni oko NGR preusmjeravajućeg motiva u CNGRCVSGCAGRC stvaraju disulfidnu/e vezu/e, istovremeno važnu/e za vezanje na APN. Analiza peptidnog okoliša preusmjeravajućih motiva omogućuje bolje razumijevanje odnosa strukture i funkcije preusmjeravajućih motiva što pomaže u dizajnu preusmjerenih adenovirusa s ciljem genske terapije tumora.

Abstract (english)

Replication deficient adenoviruses type 5 (Ad5) are widely used vectors for cancer gene therapy. The retargeting of Ad5 is achieved by incorporation of a targeting motifs into the adenovirus capsid proteins, mostly in the HI-loop of fiber or hexon hypervariable region 5 (HVR5). Increased expression of αv integrins has been frequently shown in tumor cells as compared to normal cells. The targeting motif within Ad5 that retargets its transduction to cells expressing αv integrins is RGD. One of the strategies of tumor gene therapy is inhibition of angiogenesis. Endothelial cells in angiogenesis express aminopeptidase N (APN) which binds targeting motifs containing NGR. It is known that different amino acid environment and/or cysteine residues flanking targeting peptides RGD and NGR influence the affinity and specificy of binding to corresponding receptors. The aim of this work was to investigate the existance of disulfide bond/s in two replication deficient Ad5 vectors: (i) Ad5HCRGDC containing CRGDC retargeting motif inserted in HVR5 and (ii) Ad5ARAP containing CNGRCVSGCAGRC retargeting motif inserted in HI loop of the fiber protein. It was shown that treatment with dithiothreitol had no effect on Ad5HCRGDC transduction efficacy of human embryonal rhabdomyosarcoma (RD) cell line i.e. it did not influence binding to αv integrins. The proof that cysteines in the CRGDC motif do not form disulfide bond was given by mass spectrometry. Treatment of Ad5ARAP by ditiothreitol abolished retargeting to APN that is expressed in RD cell line. Therefore, cysteines surrounding NGR retargeting motif within CNGRCVSGCAGRC form disulfide bond/s crucial for APN binding. The analysis of amino acid residues surrounding retargeting motifs enables better understanding of the relation between their structure and function which helps in design of improved retargeting adenoviruses for tumor gene therapy.

• Keywords: adenovirus tip 5; disulfidne veze; RGD i NGR preusmjeravajući slijed
• Keywords (english): adenovirus type 5; disulfide bond; RGD and NGR retargeting motif
• Language: croatian
• URN:NBN: urn:nbn:hr:217:256226
• 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: Access restricted to students and staff of home institution
• Created on: 2017-04-27 12:09:40