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Structural studies of selected protein complexes involved in signal transduction
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Bouřa, Evžen (referee) ; Pavlíček, Jiří (referee)
Protein-protein interactions are critical for most physiological and pathophysiological processes. Detailed characterization of these interactions is therefore essential not only to understand the nature of these events, but also to design strategies to target these interactions. This work focuses on the study of the structure and interactions of several proteins and their complexes. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the p38/JNK protein kinase pathways, thereby directing cells toward an inflammatory response or apoptosis. ASK1 interacts with thioredoxin (TRX), a small dithiol oxidoreductase, which inhibits ASK1, but the mechanism of this inhibition has not been clarified. CaMKK1 and CaMKK2 are Ca2+ /calmodulin (CaM)-dependent protein kinases that regulate cellular energy balance, memory, and inflammation, among others. Both are inhibited by 14-3-3 proteins, but despite their domain and sequence similarities, the extent of 14-3-3 protein- mediated inhibition is different. Estrogen receptor alpha (ERα) is a nuclear receptor involved in breast cancer. Tamoxifen, an ERα antagonist, is used to treat this disease, but resistance often develops. 14-3-3 proteins interact with ERα and inhibit its transcriptional activity,...
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Structural studies of an abasic site DNA damage repair and DNA interstrand cross-link formation
Landová, Barbora ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee) ; Schneider, Bohdan (referee)
DNA damage refers to any alteration or modification in the DNA structure that deviates from its natural state. Abasic site (Ap site) is one of the most common DNA lesions resulting from spontaneous depurination/depyrimidination or enzymatic base excision. When left unrepaired it can lead to a cascade of genetic mutations, potentially causing diseases like cancer. Understanding DNA repair mechanisms is vital for medical research and applications. Bacterial MutM is a DNA repair glycosylase, removing DNA damage generated by oxidative stress and preventing mutations and genomic instability. MutM belongs to the Fpg/Nei family of procaryotic enzymes, sharing structural and functional similarities with their eukaryotic counterparts, such as NEIL1-NEIL3. Here, I present two crystal structures of MutM from pathogenic Neisseria meningitidis: MutM holoenzyme and MutM bound to DNA. The free enzyme exists in an open conformation, while upon binding to DNA, both the enzyme and DNA undergo substantial structural changes and domain rearrangement. One of the DNA lesion repaired by MutM is the Ap site, which, if not repaired, may spontaneously lead to the formation of an abasic site interstrand crosslink (Ap-ICL) with an adjacent adenine in the opposite strand. NEIL3 glycosylase is known to remove Ap-ICL. With a...
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Biophysical Characterization of Coronaviral nsp14 Inhibition
Trembulaková, Pavla ; Bouřa, Evžen (advisor) ; Dejmek, Milan (referee)
RNA virus SARS-CoV-2 caused worldwide pandemic of severe disease COVID-19 which lasted more than a year. Repair mechanisms of this virus during replication process significantly reduce efficiency of nucleotide analog drugs, eg. remdesivir. Nonstructural protein (nsp) 14 and nsp10 form a complex which acts as an exonuclease enzyme and will be furthrer referred to as an ExoN complex. This complex can probably cause lower efficiency of incorporation of nucleotide analogs compared to viruses without exonuclease enzymes. The two-protein complex with active site on nsp14 containing two magensium ions seems like a good target for testing potent inhobitors. Among possible inhibitors of SARS-CoV-2 exonuclease complex are isobavachalcone and sofalcone. According to published studies, those small organic molecules chelate magensium ions in active site of exonulease part in nsp14. This results in inactivation of ExoN complex active site in nsp14 structure and disables the catalytic function which acts as repairing element in RNA synthesis process. This tesis focuses on characterization of inhibition of protein complex nsp14 and nsp10 in presence of small selected molecules, isobavachalcone and sofalcone. Exonuclease activity assays in presence of various RNA substrates were performed. Furthermore, there have been...
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Methyltransferases of human +ssRNA viruses
Skořepa, Jan ; Bouřa, Evžen (advisor) ; Otava, Tomáš (referee)
The aim of this bachelor's thesis is a structural and functional description of viral methyltransferases of important human +ssRNA viruses from the families Coronaviridae and Flaviviridae. Over 400 million people are infected with diseases such as Dengue, Yellow fever, or Japanese encephalitis (Flaviviridae) every year. During the current pandemic of the SARS-CoV-2 virus (Coronaviridae), more than 750 million people have already been infected worldwide. Methyltransferases are involced in the synthesiz of the cap structure at the 5' end of the viral RNA, which increases its stability and facilitates translation. A detailed structural understanding of proteins NS5 (Flaviviridae methyltransferase), nsp14 and nsp16 (Coronaviridae methyltransferases) is necessary for the subsequent development of their inhibitors. As antivirals, these could help with the treatment of viral diseases caused by coronaviruses and flaviviruses.
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Characterization of a recombinant exonuclease nsp14 of the SARS-CoV-2
Vaško, Michal ; Bouřa, Evžen (advisor) ; Kohoutová, Klára (referee)
Coronaviruses are enveloped viruses with a genome composed of a single-stranded positive-sense RNA molecule. With genome sizes, in some species, exceeding 30 kb, they represent the largest known RNA viruses. They infect birds and mammals, including humans, causing a wide spectrum of diseases. Most human coronaviruses predominantly cause mild respiratory infections. However, in the last two decades, there have been two extensive epidemics and a global pandemic of severe respiratory illnesses, often ending in fatalities, caused by coronaviruses. The most recent and extensive of these was caused by the coronavirus SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus). Within the coronavirus genome, there is a gene encoding a non-structural protein 14 (nsp14), which possesses 3'-5' exonuclease and methyltransferase enzymatic activities. The exonuclease nsp14 participates in the repair of misincorporated nucleotides during viral genome replication, and its presence is exceptionally rare among RNA viruses. The exonuclease activity of nsp14 is significantly enhanced by the binding of another viral protein, nsp10, which lacks enzymatic activity but acts as a critical cofactor for several enzymatically active coronavirus nsps. Both nsp14 and nsp10 are highly conserved and sequence-similar among...
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Viral proteins that facilitate RNA replication
Černý, Prokop ; Bouřa, Evžen (advisor) ; Faltová, Lenka (referee)
In recent years, the pathogenic human coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a global pandemic of highly infectious disease COVID--19 (coronavirus disease 2019). According to the World Health Organization, as of August 20, 2023, there have been nearly 770 million confirmed cases and nearly 7 million deaths associated with the disease. However, only a small number of effective antiviral drugs are currently known to help treat the disease in already infected patients. Therefore, in this work, the interaction of host 14-3-3 proteins with the nucleocapsid protein of SARS-CoV-2 was studied as a potential biological target for the rational design of new antiviral drugs. The importance of this interaction in the virus life cycle has not been fully elucidated, but it probably has an important regulatory function affecting multiple processes in the infected cell. The SARS-CoV-2 N protein contains several binding motifs for 14-3-3 proteins in its sequence, two of which (at positions S197 and T205), most likely responsible for the protein- protein interaction, were studied in this work. Microscale thermophoresis was performed to determine the affinity of each of these phosphorylation sites on the nucleoprotein for the 14-3-3ζ protein. The key phosphorylated residue...
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Structural and functional characterization of inhibition of a coronaviral methyltransferase.
Ivanovská, Dana ; Bouřa, Evžen (advisor) ; Faltová, Lenka (referee)
Coronaviral methyltransferases participate in the modification of the 5'-end of viral RNA. Their enzymatic activity not only ensures efficient mRNA translation, but also allows the virus to escape the recognition of the innate immune system. This work is focused on the SARS-CoV- 2 methyltransferases (the methyltransferase domain of the nonstructural protein 14, MT14, the nonstructural protein 16 and its cofactor - the nonstructural protein 10, nsp16/10), which represent attractive molecular targets for therapeutic intervention. The aim of this work was to structurally characterize the coronaviral methyltransferases in complex with various small molecules. The recombinantly prepared proteins were purified and subsequently subjected to crystallization trials. The obtained crystals of the nsp16/10 heterodimer in complex with sinefungin were soaked in a solution containing a S-adenosyl-L-homocysteine analogue. Crystals suitable for X-ray crystallography of MT14 in complex with two different inhibitors were obtained by optimizing the identified primary crystallization conditions. The aquired structural data of the MT14 inhibitory complexes will serve as a basis for the design of new small molecule inhibitors targeting the S-adenosyl-L-methionine binding site. Keywords: methyltransferase, nsp14, nsp16,...
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Structural and functional study of viral RNA polymerases
Dubánková, Anna ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee) ; Plevka, Pavel (referee)
Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...
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The crystal structure of PI4 kinase
Bäumlová, Adriana ; Bouřa, Evžen (advisor) ; Obšil, Tomáš (referee) ; Bařinka, Cyril (referee)
Phosphatidylinositol 4-kinases (PI4K/PI4-kinases) catalyse the production of phosphatidylinositol 4-phosphate (PtdIns4P), the first step in the generation of higher phosphoinositides. PtdIns4P is an essential precursor in the production of second messengers, Ins(1,4,5)P3 and diacylglycerol, in a receptor activated phospholipase C signalling pathway. Moreover, PtdIns4P itself regulates conserved compartment-specific biological processes, mainly via recruiting a broad spectra of effector proteins. Because PI4-kinases have a central position in PtdIns4P synthesis on a surface of intracellular membranes, they are implicated in a wide range of PtdIns4P-induced processes such as lipid transport and metabolism, intracellular trafficking processes and cargo sorting, membrane and cytoskeleton remodelling events, signal transduction and many others. In mammals, two types of PI4-kinases were identified: type II and type III. Both types do not bear high sequence similarity to each other and, therefore, they possess diverse biochemical properties. In order to elucidate their structural relationship to other lipid kinases, structural analysis is highly demanded. The structural characterisation of individual PI4-kinases could also clarify the catalytic mechanism of PtdIns4P synthesis. Furthermore, information...
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