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Studies of precursor forms of SARS-CoV-2 3-CL protease
Nováková, Veronika ; Majerová, Taťána (advisor) ; Novotný, Marian (referee)
The SARS-CoV-2 virus (Severe acute respiratory syndrome coronavirus 2), the causative agent of the disease COVID-19 (coronavirus disease 2019), is an enveloped virus with a positive RNA genome. After the binding of the virus to the cell receptor and the release of the genome into the cytoplasm, the viral genome is immediately translated. It is translated into a polyprotein consisting of non-structural proteins necessary for viral replication. A part of this polyprotein is also protease 3-CL, which autocatalytically cleaves itself out from this polyprotein and further releases (and thus activates) individual proteins. Due to this key function in virus replication, 3-CL protease has become an important target for the design of specific antiviral drugs. As part of this thesis, we focused on the study of the precursor form of this protease, i.e. the protease that is still embedded in the polyprotein. The precursor form of the 3-CL protease was mimicked by attaching a 100 amino acid extramembrane C-terminal fragment of the nsp4 protein to the N-terminus of the mature form of the protease. The aim was to find out, how the sequence added to the N-terminus of the matured protease affects its enzymatic characteristics. As a part of this work, mutations of the N-terminal autoprocessing site were identified,...
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Ubiquitin-proteasome system in studies of its inhibition and its utilization in the cell-based assay measuring viral protease activity
Fürst, Eliška ; Majerová, Taťána (advisor) ; Čermák, Lukáš (referee)
and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...
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Ubiquitin-proteasome system in studies of its inhibition and its utilization in the cell-based assay measuring viral protease activity
Fürst, Eliška ; Majerová, Taťána (advisor) ; Čermák, Lukáš (referee)
and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...
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