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Regulation of STING function during murine polyomavirus infection
Šnejdarová, Aneta ; Horníková, Lenka (advisor) ; Pimková Polidarová, Markéta (referee)
Stimulator of Interferon Genes (STING) is the adapter protein of an innate immunity signalling pathway, involved in detection of double-stranded DNA (dsDNA) in the cell cytoplasm, which leads to the expression of pro-inflammatory genes, including the production of type I interferon. Eventhough during the infection with a dsDNA virus, murine polyomavirus (MPyV), the STING protein is activated, the resulting interferon production is moderate. Therefore, it can be assumed that the function of the STING protein is regulated in MPyV-infected cells. The aim of this thesis was to investigate three mechanisms by which the regulation can occur, namely through protein interaction partners, post- translational modifications, or changes in the subcellular localization of the STING protein. A cell-line of mouse fibroblasts stably expressing the STING protein fused with the HA-tag was established to facilitate the research. Furthermore, two plasmids were prepared, that encode the STING protein fused with the green fluorescent protein, facilitating the monitoring of the localization of the protein in the cell, or with a composite tag containing an in vivo biotinylated BioEaseTM -tag enabling effective isolation of the STING protein. The results of colocalization observations and coimmunoprecipitation suggest that...
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The role of IFI16 protein in viral infection
Karchňák, Jan ; Šroller, Vojtěch (advisor) ; Poláková, Ingrid (referee)
6 Abstract Viruses are obligate intracellular parasites causing a large variety of diseases. Most of their hosts, including humans, have developed particular mechanisms, which are meant to tackle such diseases. First line of defense against viruses are pattern recognition receptors. These receptors are responsible for detection of pathogen associated molecular patterns (PAMPs) and of damage associated molecular patterns (DAMPs). Inteferon γ inducible protein 16 (IFI16) is one of these receptors and is responsible for detecting alien and damaged DNA both in the nucleus and cytoplasm. Its structure contains two sequence independent DNA binding HIN domains and one PYD domain, which mediates its protein-protein interactions. In the cell it functions as a regulator of cell cycle, differentiation and plays a role in cell aging. IFI16 also triggers activation of non-specific immune response and it directly acts as a restrictive factor for many viruses. During evolution these viruses have evolved mechanism which they us to evade its imunne activity or even use it to their advantage. Keywords: IFI16, STING, DNA sensing, interferons, restriction factor, pattern recogniton receptors
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Negative regulation of the IFI16 and cGAS DNA sensing pathways
Muhič, Samra ; Huerfano Meneses, Sandra (advisor) ; Kanwal, Madiha (referee)
DNA sensors are molecules with the ability to sense DNA constituting an important tool of innate immunity. They are initiators of various signalling pathways, one of them being the production of interferons, which induce not only an anti-viral cell state but also protect cells of treats not related to pathogens. At least, fourteen DNA sensors have been described so far, among them - IFI16 and cGAS. Both sensors signal via an adaptor protein STING resulting in the production of type I IFN. All three of these molecules (cGAS, IFI16, STING) are strictly regulated either by host-cells in order to prevent immune over-activation or by viruses for the immune evasion. This work focuses on the mechanisms of negative regulation of the three molecules: post-translational modifications such as phosphorylation, ubiquitination, SUMOylation, acetylation and methylation; protein-protein interactions; degradation by the proteasomal system or by autophagy. Not surprisingly, viruses encode proteins able to down-regulate IFN responses for example, some proteins of herpes viruses interact with cGAS, IFI16 or STING preventing their activation or leading to their degradation. Other proteins of herpes viruses cause the degradation of the mRNA of the sensors or the adaptor. Dengue protease factor NS2B degrades cGAS or the...
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Protein STING targeting for chronic hepatitis B treatment
Pimková Polidarová, Markéta ; Birkuš, Gabriel (advisor) ; Forstová, Jitka (referee) ; Rumlová, Michaela (referee)
Chronic hepatitis B (CHB) is an infectious disease caused by the hepatotropic hepatitis B virus (HBV). CHB affects millions of people worldwide, but the treatment options are still limited, and virus elimination is rarely achieved. The immunomodulatory approaches towards CHB treatment are recently being investigated. In this thesis, we focused on the anti-HBV potential of the cyclic- GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) DNA-sensing innate immunity pathway, which upon activation leads to the secretion of proinflammatory cytokines. We screened synthetic cyclic dinucleotide (CDN)-based STING agonists and their lipophilic prodrugs prepared in our laboratory in reporter cell lines and peripheral blood mononuclear cells (PBMC). We selected candidate compounds with superior cGAS-STING pathway activation properties compared to the natural eukaryotic STING agonist. We identified 3′,3′-c-di(2′-fluoro,2′- deoxyAMP) to have improved cGAS-STING pathway activation properties over the five most abundant STING isoforms found in the humans, including the variants that do not respond well to 3′,3′-CDNs. The symmetric bis-pivaloyloxymethyl prodrug of 3′,3′-c-di(2′-fluoro,2′-deoxyAMP) further increased the activity of parent CDNs by enhancing their intracellular entry. We showed that the...
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