Interakcije onkoproteina E6 HPV-a s vezujućim partnerima: utjecaj na stabilnost i stanične funkcije E6

Abstract

Human papillomaviruses (HPVs) are a group of small DNA viruses that cause various human malignancies, with cervical cancer being the most significant disease associated with a persistent HPV infection. Only a small number of HPV types has been shown to be responsible for these malignancies. These HPVs are referred as high-risk (HR) types, with HPV-16 and -18 being the most prominent ones. Two major viral oncoproteins, E6 and E7, directly contribute to the development of cancers by interfering with various cellular signaling pathways. A number of HPV-16 variants has been identified in different geographical locations, with some variants exhibiting higher oncogenic potential than others. The first part of thesis focuses on the analysis of the HPV-16 E6 D25N L83V variant, which was shown to be strongly associated with the development of cervical cancer. It was shown that this variant exhibits an increased capacity for interacting with E6AP ubiquitin ligase and consequently degraded it more efficiently, in comparison to the other analyzed mutants HPV-16 E6 D25N and E6 L83V. The HPV-16 E6 mutants' abilities to degrade key cellular target proteins, including the p53 tumor suppressor and PDZ-domain containing substrates, were investigated through in vitro and overexpression degradation assays. The analyses revealed no significant differences in the degradatory activities among the evaluated E6 mutant oncoproteins. Furthermore, the second part of this thesis demonstrates that multiple α-E6 oncoproteins can bind to MAML1 via LXXLL motif, resulting in an increased α-E6 protein stability. β-E6 oncoprotein stability was also shown to be dependent on the interaction with MAML1, whilst the absence of MAML1 led to both HPV-8 E6 and HPV-18 E6 oncoprotein rapid turnover at the proteasome. The study proposed a model by which most of β-E6s interact exclusively with MAML1, whereas it appears that two cellular pools of HR α-E6 are present, one forms a complex with MAML1, while the other one interacts with E6AP. Although HR α-E6/MAML1 complex does not affect the targeting of cellular substrates such as p53 and DLG1, co-expression of MAML1 and E6AP with HR α-E6 modulates MAML1's normal cellular activities leading to a significant increase in cellular proliferation. Silencing MAML1 decreases wound closure in HeLa cells, suggesting its role in the regulation of cellular migration in HPV-positive cells and maintenance of the transformed phenotype. Overall, this doctoral thesis provides novel insights into the functions of both α- and β-E6 oncoproteins and their roles in HPV-induced pathogenesis.Humani papilomavirusi (HPVs) pripadaju skupini malih DNA virusa koji uzrokuju različita maligna oboljenja, pri čemu je rak vrata maternice najznačajnija bolest povezana sa dugotrajnom HPV infekcijom. Samo mali broj HPV tipova uzrokuje zloćudne bolesti. Spomenuti tipovi nazivaju se visokorizičnim (HR), među kojima su HPV-16 i HPV-18 najistaknutiji. Dva glavna virusna onkoproteina E6 i E7 svojim djelovanjem izravno pridonose razvoju raka djelujući na različite stanične signalne puteve. Postoje mnoge varijante HPV-16 zastupljene na različitim geografskim lokacijama te se pokazalo da neke od varijanti imaju veći onkogeni potencijal. Prvi dio doktorske disertacije usmjeren je na analizu HPV-16 E6 D25N L83V varijante koja je usko povezana s nastankom raka vrata maternice. Utvrđeno je da varijanta D25N L83V ostvaruje povećanu interakciju sa ubikvitinskom ligazom E6AP te je posljedično najučinkovitija u poticanju njezine razgradnje u usporedbi sa ostalim ispitanim mutantima HPV-16 E6 D25N i E6 L83V. Svojstva mutanata da izazovu razgradnju ključnih staničnih ciljnih proteina, uključujući tumor-supresor p53 i stanične proteine koji sadrže PDZ-domene ispitana su in vitro esejima razgradnje i esejima razgradnje provedenim korištenjem kulture stanica. Međutim, nisu otkrivene značajne razlike u aktivnostima razgradnje među ispitivanim HPV-16 E6 mutantima. Drugi dio doktorske disertacije otkriva da se različiti α-E6 onkoproteini vežu za MAML1 putem LXXLL strukturalnog motiva, što rezultira povećanom stabilnošću onkoproteina E6. Nadalje, stabilnost onkoproteina β-E6 također ovisi o interakciji s MAML1, dok utišavanjem MAML1 dolazi do brže proteasomske razgradnje onkoproteina HPV-8 E6 i HPV-18 E6 u HPV-pozitivnim stanicama. Studija predlaže molekularni model prema kojemu većina onkoproteina β-E6 stupa u interakciju isključivo s MAML1, dok su prisutna dva stanična skupa HR α-E6, od kojih jedan tvori kompleks s MAML1, a drugi stupa u interakciji s E6AP. Iako kompleks HR α-E6/MAML1 nema ulogu u razgradnji važnih staničnih ciljnih proteina kao što su p53 i DLG1, zajednička ekspresija MAML1 i E6AP sa HR α-E6 mijenja uobičajene stanične aktivnosti MAML1 dovodeći do značajnog povećanja stanične proliferacije. Dodatno, utišavanje MAML1 smanjuje „proces cijeljenja rane“ u HeLa stanicama ukazujući na njegovu ulogu u regulaciji stanične migracije HPV-pozitivnih stanica i održavanju transformiranog fenotipa. Sveobuhvatno, doktorska disertacija donosi nove uvide o funkcijama onkoproteina α- i β-E6 i njihovoj ulozi u patogenezi izazvanoj HPV infekcijom