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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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Creating an in-vitro model system for studying phase separation in virus assembly
WIENER, Katharina
This thesis focuses on the study of the ?NS protein of the avian reovirus and as this virus is causing considerable annual losses in the poultry industry, understanding its replicative cycle on a molecular basis is highly anticipated. One area of interest is hereby the formation of so-called viroplasms via liquid-liquid phase separation (LLPS) of the ?NS protein. The exact processes involved in LLPS, however, remain mostly unknown. By establishing an in-vitro model system we tried to elucidate the environmental conditions needed for phase separation, in order to gain a better understanding of the viral replicative cycle, as well as LLPS in general.
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Production of cysteine cathepsins and structural characterization of their interaction with peptidomimetic inhibitors
Wichterle, Filip ; Mareš, Michael (advisor) ; Knejzlík, Zdeněk (referee)
Cysteine cathepsins participate in many pathological processes such as cancer and neurodegen- erative, cardiovascular, or autoimmune diseases. This work is focused on cathepsins B, L, and V (CatB, CatL, CatV), which represent attractive targets for the development of inhibitors as potential chemotherapeutics and diagnostic tools. The aim of this study was to prepare these cathepsins and structurally characterize their complexes with selected synthetic peptidomimetic inhibitors. Recombinant CatB and CatL were prepared in the yeast Pichia pastoris, and the expression conditions were optimized for the production of cathepsin zymogens. A chromato- graphic purification protocol was designed for CatB and CatL, while CatV was purified using a previously developed procedure. The obtained enzymes were used to prepare complexes with six peptidomimetic inhibitors equipped with a carbamate, vinyl sulfone, or azanitrile warhead, which selectively target CatB, CatL, and CatV, respectively. Their inhibition parameters were determined in a kinetic assay and initial crystallization conditions were identified. After opti- mizing the crystallization conditions for CatB with three carbamate inhibitors, crystals suitable for X-ray crystallography were obtained. Based on the crystal structures of these complexes, the...
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Nové inhibitory HIV proteasy: návrh, synthesa a testování aktivity
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
More than 20 years after its discovery HIV protease still remains one of the primary targets in HIV treatment. Currently there are 9 approved protease inhibitors on the market. However, due to immense replication rate and the high error prone nature of reverse transcriptase, resistance to each of them has already been described. Therefore, the search for new protease inhibitors with different binding mode is still active. A novel type of protease inhibitors (1, 4-benzodiazepine analogs) was recently discovered in our laboratory. Even though this new class of inhibitors is highly potent (Ki' in range of 10-9 ), it also has several undesirable qualities, such as low solubility and a high number of stereogenic centers. Primary objective of this study was to try to prepare more soluble compounds with lower number of possible stereoisomers, enzymologically characterize its binding to the wild-type and mutated HIV protease and to determine its structure in the complex with the enzyme. A small library of 1, 4-benzodiazepine inhibitors of HIV protease was synthesized and fully characterized using NMR spectroscopy and mass spectroscopy. The number of stereogenic centers was successfully reduced from 4 to 2 without loosing activity of the inhibitor. The improvement in solubility was always associated with a...
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Study of receptor-ligand pair NKR-P1F and Clrg
Kotýnková, Kristýna ; Man, Petr (advisor) ; Schneider, Bohdan (referee)
Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...
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Preparation and biochemical characterization of protease inhibitor equistatin
Polatová, Daniela ; Mareš, Michael (advisor) ; Bořek Dohalská, Lucie (referee)
Equistatin from the sea anemone Actinia equina contains a protein domain Eqd2 which inhibits aspartic peptidases and has not been characterized in detail. Recombinant Eqd2 was produced in the yeast expression system, and a protocol for its chromatographic purification was designed. The inhibitory specificity of Eqd2 was determined using a fluorescence inhibition assay, showing that Eqd2 is a highly selective inhibitor of cathepsin D-like and pepsin-like aspartic peptidases of family A1. Furthermore, size exclusion chromatography was used to analyze the Eqd2-peptidase complex and Eqd2 oligomerization in solution. Initial screening of crystallization conditions for Eqd2 was performed towards its structural analysis. This work provides important new information about Eqd2 as a unique type of natural inhibitors of aspartic peptidases. Its interaction mechanism can be exploited in the development of synthetic mimetics for regulation of medically important peptidases. (In Czech) Key words: peptidase inhibitors, proteolytic enzymes, activity and inhibition of enzymes, recombinant expression, protein purification, protein crystallization, equistatin
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Preparation of the human NK cell receptor KACL
Nový, Jiří ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
NK buňky neboli přirození buněční zabíječi jsou důležitou součástí imunitního systému organismů. Jejich specifickou vlastností je to, že dokážou rozeznat a zneškodnit některé nádorové buňky a buňky infikované virem bez jakéhokoliv předchozího signálu. Jejich funkce je závislá na aktivitě povrchových stimulačních a inhibičních receptorů, které se aktivují interakcí s ligandy na povrchu cílových buněk. Interakce lidského NK receptoru NKp65 a ligandu KACL je velmi specifická, což potvrzuje velmi vysoká afinita interakce, která je oproti jiným příbuzným komplexům až 400× vyšší. O tomto komplexu je zatím známo jen málo informací, ale je jisté, že se účastní imunitních procesů v kůži, neboť KACL je produkován výhradně keranocyty. Náplní této bakalářské práce byla příprava rozpustné formy receptoru KACL za pomoci rekombinantní exprese proteinů v lidských embryonálních ledvinných buňkách s homogenní glykosylací (linie HEK293S GnTI- ). Protein byl následně charakterizován gelovou chromatografií a SDS elektroforézou. Správné zapojení čtyř cysteinů do dvou disulfidických můstků bylo ověřeno hmotnostní spektrometrií. S připraveným proteinem KACL bylo zahájeno strukturní studium metodou krystalizace proteinů.
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Nové inhibitory HIV proteasy: návrh, synthesa a testování aktivity
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
More than 20 years after its discovery HIV protease still remains one of the primary targets in HIV treatment. Currently there are 9 approved protease inhibitors on the market. However, due to immense replication rate and the high error prone nature of reverse transcriptase, resistance to each of them has already been described. Therefore, the search for new protease inhibitors with different binding mode is still active. A novel type of protease inhibitors (1, 4-benzodiazepine analogs) was recently discovered in our laboratory. Even though this new class of inhibitors is highly potent (Ki' in range of 10-9 ), it also has several undesirable qualities, such as low solubility and a high number of stereogenic centers. Primary objective of this study was to try to prepare more soluble compounds with lower number of possible stereoisomers, enzymologically characterize its binding to the wild-type and mutated HIV protease and to determine its structure in the complex with the enzyme. A small library of 1, 4-benzodiazepine inhibitors of HIV protease was synthesized and fully characterized using NMR spectroscopy and mass spectroscopy. The number of stereogenic centers was successfully reduced from 4 to 2 without loosing activity of the inhibitor. The improvement in solubility was always associated with a...
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Study of receptor-ligand pair NKR-P1F and Clrg
Kotýnková, Kristýna ; Man, Petr (advisor) ; Schneider, Bohdan (referee)
Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...
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