diplomsko delo
Polona Skrt (Avtor), Uroš Petrovič (Mentor)

Povzetek

Virus SARS-CoV-2 je bil pri človeku prvič potrjen decembra 2019 in se za tem hitro razširil po svetu. Od preostalih koronavirusov se najbolj razlikuje v pomožnem proteinu ORF8, ki ima hkrati tudi enega od najbolj variabilnih zapisov v genomu virusa. S širjenjem virusa se pojavljajo vedno nove različice virusa in proteina ORF8, ki jih najdemo po svetu ter tudi na območju Slovenije. Te se pogosto kažejo v spremenjenem poteku okužbe ali razvoja bolezni COVID-19, kar nam daje iztočnico za raziskave molekularnega mehanizma delovanja virusnih proteinov. Kot interaktorji proteina ORF8 so bili napovedani številni proteini človeka, med drugimi tudi ADAM9, PLOD2, ITGB1, TGFB1 in IL17RA, ki vsi sodelujejo v fizioloških procesih, povezanih s simptomi okužbe z virusom SARS-CoV-2. V raziskavi smo se osredotočili na spremembe S24L, Y73C in L84S proteina ORF8, ki so bile najpogostejše v začetnih različicah virusa. Analiza interakcij je potekala najprej eksperimentalno, z metodo dvohibridnega sistema kvasovke, nato pa smo iste interakcije analizirali še z aktualnimi računskimi metodami molekulskega sidranja, kar je bil namen te diplomske naloge. Za uspešno bioinformatsko analizo smo pripravili modele struktur tarčnih proteinov, ki so bili napovedani z algoritmoma I-TASSER in AlphaFold ali eksperimentalno določeni, molekulsko sidranje ORF8 s proteini človeka pa je bilo izvedeno na spletnih strežnikih HADDOCK in ClusPro. V eksperimentalnem delu analize je uvedba mutacij v nekaterih primerih privedla do odstopanja rezultata od referenčnega, kar je služilo postavitvi hipoteze, da aminokisline, ki so spremenjene zaradi uvedenih mutacij in s tem vplivajo na eksperimentalen rezultat, sodelujejo v interakcijski površini proteinskega kompleksa. Hipotezo smo z uspešno pripravo modelov struktur protein-proteinskih interakcij in njihovo analizo potrdili ter tako pokazali komplementarnost eksperimentalnih in bioinformatskih metod v preučevanju molekularnih mehanizmov razvoja bolezni.

Ključne besede

SARS-CoV-2;proteini;ORF8;protein-proteinske interakcije;struktura proteinov;napoved proteinske strukture;molekulsko sidranje proteinov;računalnika orodja;diplomska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [P. Skrt]
UDK: 577.112(043.2)
COBISS: 79586563 Povezava se bo odprla v novem oknu
Št. ogledov: 256
Št. prenosov: 40
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Modelling of binary complexes of ORF8 SARS-CoV-2 protein with human targets
Sekundarni povzetek: Human infection with novel SARS-CoV-2 virus was first reported in December of 2019, with the subsequent rapid spread of the virus all over the world. SARS-CoV-2 differs greatly from the rest of coronaviruses in the accessory protein ORF8, which is encoded by one of the most variable parts of the viral genome. With the spread of infection, new variants of the virus and ORF8 emerged that could be found all over the world and also in Slovenia. New variants often result in an altered course of viral infection or COVID-19 disease development, which can be utilised in the research of molecular mechanisms of viral proteins. Many ORF8 interactors have been identified, among others also ADAM9, PLOD2, ITGB1, TGFB1 and IL17RA, which all take part in physiological processes that symptoms of infection with SARS-CoV-2 are attributed to. This research focused on ORF8 modifications S24L, Y73C and L84S, which appeared the most frequently in the earliest variants. Protein interaction analysis consisted of experimental yeast-two-hybrid screening, followed by current computational molecular docking methods, which were the focal point of this thesis. Algorithms I-TASSER and AlphaFold were used for protein structure prediction of unknown structures of target proteins together with experimentally solved structures of the rest of the target proteins, which were then used for protein docking on servers HADDOCK and ClusPro in order to successfully complete our bioinformatic analysis. In the experimental part of this research, certain mutations resulted in deviation from the referential result, which was used as a basis for the hypothesis that the modified amino acids which result in a deviated experimental result, are part of the interaction surface of the protein complex. Our hypothesis has been confirmed by successful modelling of mentioned protein-protein interactions and analysis of the produced models. We therefore showed complementarity of experimental and bioinformatic methods in research of molecular mechanisms of disease.
Sekundarne ključne besede: SARS-CoV-2;ORF8;protein-protein interactions;protein structure prediction;protein docking;
Vrsta dela (COBISS): Diplomsko delo/naloga
Študijski program: 1000371
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Strani: 47 str.
ID: 13331923