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Design and evaluation of potential viral methyltransferase inhibitors
Kocek, Hugo ; Nencka, Radim (advisor) ; Grantz Šašková, Klára (referee)
A global pandemic of SARS-CoV-2 confirmed the pandemic potential of the Coronaviridae family and pointed out the need for novel antiviral drugs. The SARS-CoV-2 pandemic has been tamed thanks to mRNA vaccines; however, monoclonal antibodies and small molecules such as nirmatrelvir (protease inhibitor), remdesivir (polymerase inhibitor), or molnupiravir (mutagen) are currently also available. It is worth noting that remdesivir and molnupiravir were previously investigated as antivirals against different pathogens. SARS-CoV-2 encodes 16 non-structural proteins, and two of them - methyltransferases (MTases) nsp14 and nsp16 - participate in RNA capping as the virus must mimic the host's mRNA to evade the cellular antiviral sensors (e.g., IFIT1) and replicate. These MTases are structurally very similar to those of SARS-Co-V; therefore, we might expect that inhibitors of SARS-CoV-2 MTases could be used in the future against different coronaviruses. For the reasons mentioned above, this thesis focuses on developing novel MTase inhibitors targeting SARS-CoV-2 nsp14 and nsp16. The design was based on S-adenosyl-L-homocysteine (SAH; endogenous inhibitor of MTases) and an in silico compound library was constructed with various replacements for SAH's amino acid moiety. The potential inhibitory activity was...
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Immune stimulation by lipid nanoparticles
Aulichová, Veronika ; Grantz Šašková, Klára (advisor) ; Pačesová, Andrea (referee)
Lipid nanoparticles are the most advanced non-viral delivery system of nucleic acids. They enable safe and efficient transport of nucleic acids to the targeted cells and represent a key enabling technology of RNA therapeutics. RNA therapeutics represent a rapidly emerging field of genetic medicine; however, due to the nature of RNA molecules and their susceptibility to nuclease degradation, the delivery system is crucial for its translation to the clinic. This thesis explores the ability of lipid nanoparticles based on the XMAN6 ionizable lipidoid to elicit an immune response against mRNA-encoded antigen. XMAN6 is a lead lipidoid from a new class of adamantane-based ionizable lipidoids developed in our team. The mRNA-LNP formulations were first tested in vitro to evaluate their functionality and cytokine production and then in vivo using a mouse model. LNPs were applied via two different routes and in different experimental set ups, and their safety was evaluated by analyzing mouse activity. The data showed that XMAN6 is a promising lipidoid for in vivo delivery of mRNA. The mRNA-LNPs successfully induced the antibody response against the encoded antigen by intraperitoneal and intramuscular application. XMAN6 LNPs showed to be suitable for in vivo use, as they were well tolerated and caused only...
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Exploring novel strategies targeting HBV
Šmilauerová, Kristýna ; Grantz Šašková, Klára (advisor) ; Černý, Jan (referee)
An effective and safe vaccine against Hepatitis B virus already exists, yet morbidity and mortality of this illness are still high. The key to developing a reliable treatment is a deep knowledge of the virus' life cycle and functions of all its components. In the presented work we explored an interactome of the Core protein of the Hepatitis B virus. Using proximity-dependent biotin identification technique (BioID) coupled to mass spectrometry we have identified a list of potential candidates that are either significantly enriched (in total 105 proteins) or less abundant in the presence of the HBV Core protein in the cell (40 proteins). The list also includes known HBV Core interacting proteins SRPK1 and SRPK2, and p53 protein whose expression is known to be repressed due to the HBV Core interaction with the E2F1 transcription factor. Many of the newly identified possible HBV Core interacting proteins are involved in biological processes already known or are suspected to be influenced by the HBV such as translational and transporting processes or gene expression and macromolecule production. Overall, this work comprehensively characterizes the interaction landscape of the HBV Core protein in the live cells and might thus serve as a reliable start for in depth HBV-host interaction analysis. Key...
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Role of yeast WSS1 protease in DNA repair.
Adámek, Michael ; Grantz Šašková, Klára (advisor) ; Čáp, Michal (referee)
Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.
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Nanoparticles for gene editing
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Beranová, Jana (referee)
Early DNA-based therapies were tested for therapeutic applications, but they sooner or later revealed multiple hurdles and risks preventing their use in further clinical trials. Recently, they have been replaced by rapidly evolving gene editing using programmed nucleases capable of precise genome modifications by cleaving specific DNA sequences. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9 system are currently under investigation as potential therapeutics. However, their off-target effects must be controlled. Targeted delivery of nucleases in a form of mRNA seems as the most promising method. Various types of nanoparticles enable mRNA transfer and could be used to facilitate the nuclease application. Some of these nanoparticles together with characterization of the programmed nucleases are described in this thesis.
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Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
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Structure determination of helical domain of DNA damage-inducible protein 2
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human Ddi2 and his homologues are scarcely explored family of ubiquitin- like/ubiquitin-associated domain proteins (UBL/UBA proteins), containing domain which is structurally related to dimeric aspartyl proteases from retroviruses. The most studied of this family is Ddi1 protein from Saccharomyces cerevisiae, which functions within the ubiquitin- proteasome system. This key cellular system exploits tightly regulated enzymatic apparatus for highly selective posttranslational modifications of proteins with molecules of ubiquitin, which serves as intracellular signal determining the proteins fate, importantly its degradation in many cases. Ddi1 plays a role of proteasome adaptor within this context, helping the recognition of ubiquitylated proteins by the proteasome. Even though the function as a proteasome receptor is possible for human Ddi2 protein as well, its cellular role might have been altered during the evolution. One of the important steps in decoding its purpose in cell is exploration of function of its individual domains. This work focuses on structural study of neighbouring region of this protease domain, where the helix-rich region called HDD domain is located. This region of Ddi2 was cloned, expressed and subjected to the NMR measurements. Structural information based on the NMR data was...
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HIV - 1 Protease: Insights into Drug Resistance Development
Grantz Šašková, Klára ; Konvalinka, Jan (advisor) ; Forstová, Jitka (referee) ; Dohnálek, Jan (referee) ; Schiffer, Celia (referee)
Amino acid changes within HIV protease or its substrate that decrease the susceptibility to protease inhibitors represent a highly complex issue still not yet fully understood. Various mechanisms by which this often complicated pattern of mutations influence drug binding needs to be analyzed on a molecular level by a series of methods including experiments with recombinant viruses, biochemical enzyme analysis, structural and thermodynamical studies or molecular dynamics. Each result may help to complete the overall picture of protease inhibitor resistance evolution and therefore contribute to the design of more powerful 3rd generation HIV/AIDS drugs. This thesis presents several analyses of HIV resistance development on molecular level. We have focused on the nelfinavir resistance pathway, lopinavir mutation score, emergence of amino acid insertions in HIV protease gene and their contribution to protease inhibitor resistance and finally we analyzed a highly mutated protease species isolated from patients failing darunavir therapy. Since we are able to accomplish a wide combination of techniques, we could explain and put together some pieces of viral evolution considering the final steps of HIV life cycle and also provide knowledge necessary for novel inhibitor design. Aims of the Project There were...
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Novel substrates of cullin-RING ubiquitin ligases: identification and functional characterisation
Liďák, Tomáš ; Čermák, Lukáš (advisor) ; Grantz Šašková, Klára (referee) ; Mašek, Jan (referee)
Selective protein degradation by the ubiquitin-proteasome system is essential for cellular homeostasis and the regulation of diverse biological processes. The selectivity of this system is imparted by hundreds of ubiquitin ligases that specifically recognise substrates and catalyse their ubiquitination, thereby targeting them for degradation. Among ubiquitin ligases, multisubunit cullin-RING ubiquitin ligases constitute the largest group. However, despite significant advances in understanding their assembly, regulation, and molecular architecture, the substrates and functions of most of them remain unknown. This thesis focuses on two ubiquitin ligases from the cullin-RING ubiquitin ligase 4 (CRL4) subfamily: CRL4DCAF4 and CRL4DCAF12 . To identify their candidate substrates and to address their biological roles, several different approaches have been employed. First, proteomic screening revealed a wide range of candidate substrates. Next, detailed characterisation of the identified interactions and exploration of the condition under which candidate substrates undergo degradation was performed. Finally, knockout human cell lines and mice with a targeted disruption of genes encoding DCAF4 and DCAF12 were generated to explore the physiological roles of CRL4DCAF4 and CRL4DCAF12 . In summary, the herein...
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Cancerogenic and metastatic potential of cancer cells with non-functional CRL4 ubiquitination complex
Slámová, Monika ; Procházka, Jan (advisor) ; Grantz Šašková, Klára (referee)
Ubiquitination complex CRL4 (Cullin Ring Ligase) attracts a lot of attention due to its involvement in physiological and pathological processes, especially in the development of cancer. Cullin4 a/b proteins are reported to serve as oncoproteins in various malignancies. Due to their role in the regulation of cancer drugs targeting CRL4 have been identified, including thalidomide and its derivatives inhibiting one of the substrate receptors of the complex, the Cereblon protein. The adapter protein within the CRL4 complex - DDB1, which is involved i.a. in DNA repair, also has a role in cancer. However, the mechanism of this function has not yet been fully elucidated. The subject of this master thesis was to study the effects of elimination and suppression of CRL4 complex functions in the prostate cancer cell line LNCaP. Significantly variable changes in cell proliferation and migration have been observed if the complex functions were affected by thalidomide. The creation of the LNCaP cell line with conditionally suppressed DDB1 function was used to study tumor dynamics in a mouse model. Results show that suppression of DDB1 function has an inhibitory effect on tumor cell proliferation but increases their ability to invade adjacent tissues. Complete deletion of the DDB1 gene in the LNCaP cell line...
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