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Role of PML in ribosomal stress
Kremserová, Petra ; Vašicová, Pavla (advisor) ; Sztacho, Martin (referee)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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Effect of cytochrome b5 on enzyme kinetics of Sudan I hydroxylation catalyzed by human cytochrome P450 1A1
Netolický, Jakub ; Martínek, Václav (advisor) ; Černá, Věra (referee)
Cytochromes P450 are the major xenobiotics converting enzymes. They are classified as mixed function monooxygenases (MFO). Isoform 1A1 is a extrahepatic form found mainly in the lung and other tissues. It is strongly induced by polycyclic aromatic hydrocarbons and their derivatives via the Ah receptor. As a marker reaction for this enzyme can be used hydroxylation of Sudan I, which has previously been widely used as a azo dye in industry, but since 1980s it is banned for coloring food and cosmetics for its negative influence on the organism. NADPH:cytochrome P450 reductase is the major electron donor for cytochrome P450 catalyzed monooxygenation reactions. Another electron carrier for cytochrome P450 catalyzed reactions is cytochrome b5. It was shown that cytochrome b5 can stimulate, inhibit or have no effect on P450 catalyzed reactions. This thesis aims to evaluate the influence of the ration between NADPH:cytochrome P450 reductase and cytochrome b5 on cytochrome P450 1A1 catalyzed Sudan I hydroxylation. The main goal is to characterize the influence of electron donor and electron transfer ratios on hydroxylation of Sudan I, and to determine the kinetic parameters KM and VMAX for selected protein ratios. Partial aims of the thesis were to characterize the recombinant proteins used in this study...
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Structural studies of selected signaling protein complexes.
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee) ; Maloy Řezáčová, Pavlína (referee)
The ability of proteins to bind other molecules in response to various stimuli in their microenvironment serves as a platform for extensive regulatory networks coordinating downstream cell actions. The correct function of these signaling pathways depends mostly on noncovalent interactions often affecting the structure of proteins and protein complexes. Understanding the molecular mechanism of a protein function in cell signaling therefore often depends on our knowledge of a three-dimensional structure. In this doctoral thesis, I present the work that led to the understanding of several protein-protein and protein-ligand interactions implicated in cell signaling at the molecular level. I applied nuclear magnetic resonance spectroscopy, small angle X-ray scattering and other biophysical methods to determine the molecular basis of inhibition of four signaling proteins: Calcium/Calmodulin (Ca2+ /CaM)-dependent protein kinase kinase 2 (CaMKK2); protease Caspase-2; Forkhead transcription factor FOXO3, and Apoptosis signal-regulating protein kinase 1 (ASK1). In particular, I investigated the distinct roles of 14-3-3 and Ca2+ /CaM in the regulation of CaMKK2 activity. I also studied in detail the mechanism how 14-3-3 interferes with the caspase-2 oligomerization and its nuclear localization as well as...
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Identification of small compounds disrupting protein-protein interaction in influenza A polymerase.
Hejdánek, Jakub ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...
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Study of protein-protein interaction in bacterial pathogenesis: PIXL (photo-induced cross-linking) methodology
Žídek, Radek ; Šulc, Miroslav (advisor) ; Ječmen, Tomáš (referee)
Gramnegativní bakterie druhu Bordetella pertussis jsou původci smrtelného lidského onemocnění označovaného jako pertuse, častěji jako černý kašel. Tyto bakterie produkují adenylátcyklázový toxin (ACT), který se váže na povrch makrofágů a umožňuje vpravit do cytosolu hostitelské buňky přes cytoplazmatickou membránu adenylátcyklázovou doménu (dAC). Abychom mohli studovat kovalentní interakce mezi proteiny pomocí fotochemického zesítění, byl náš studovaný protein exprimován s foto-methioninem (kyselinou L-2-amino-5,5- azi-hexanovou) v kultivačním médiu v bakteriálním kmenu Escherichia coli B834 (DE3). Foto- methionin je netoxickým analogem L-methioninu, takže je normálně inkorporován pomocí aminoacyl-tRNA syntház do struktury adenylátcyklázové domény. Maximální míry inkorporace foto-methioninu do struktury dAC bylo dosaženo po optimalizaci celého expresního protokolu. Celková míra inkorporace foto-methioninu do struktury proteinu po optimalizaci zjištěná hmotnostně-spektrometrickou analýzou byla až 80 %. Získaný protein s inkorporovaným foto- methioninem byl izolován. Byly provedeny síťovací experimenty s kalmodulinem a vláknitým hemaglutininem. Při těchto experimentech bylo provedeno jak fotochemické, tak i chemické zesítění. Vzniklé kovalentně zesítěné produkty byly rozděleny pomocí SDS-PAGE a...
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Expression and purification of protein photo-initiated nanoprobe: tool to study clinically relevant protein-protein interactions
Knížek, Antonín ; Šulc, Miroslav (advisor) ; Koblihová, Jitka (referee)
Cytochrome b5 is a key protein in the function and regulation of the mixed function monooxygenase (MFO) system in mammalian endoplasmic reticulum and is, therefore, a clinically relevant target for biochemical studies. To study its interactions within the MFO system using photo-initiated crosslinking, we have developed cytochrome b5 mutants with methionine in several key amino acid positions within the primary amino acid sequence, such as serine 23 and leucine 41. Also, naturally presented Met in positions 96, 126 and 131 were mutated to Leu with no effect to cytochrome b5 activity. Our protein was expressed in E. coli B834 auxotrophic type with L-2-amino-5,5-azi-hexanoic acid (photo-Met) present in the cultivation medium. This methionine analogue with photolabile diazirine ring is readily incorporated in Met positions into the primary sequence of proteins by aminoacyl-tRNA synthetases. The whole expression protocol was optimized to achieve maximal percentage of photo-Met incorporation into the expressed protein sequence. Up to 93.4% incorporation of photo-Met was achieved. The expressed protein was isolated and photo-Met incorporation was established with MALDI-TOF mass spectrometry. After reconstitution with its natural interaction partners - full-length cytochrome P450 2B4 (rabbit isoform) or...
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Interaction PI4 kinase IIalpha with VAMP3 protein
Dubánková, Anna ; Šulc, Miroslav (advisor) ; Teisinger, Jan (referee)
Phosphoinositides are very important in regulation activity of many signaling proteins not just in cellular membranes. Phosphatidylinositol - 4 - kinases (PI4K) generate phosphatidylinositol - 4 - phosphate, an emerging regulatory molecule and precursor of important regulatory phosphoinositides. PI4Ks are associated with pathogenicity of several RNA viruses including Picornaviridae (poliovirus, coxsackie virus, aichi virus, enterovirus 71) and Flaviviridae (hepatitis C virus). PI4Ks also play important role in cancer. This study strives to clarify the mechanism of regulation of PI4K type II α by its potential interaction with Vesicle - associated membrane protein 3 (VAMP 3) of the SNARE protein family (Soluble N - ethylmaleimide Sensitive Fusion Attachment Protein Receptor).
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Application potential of modification approaches (chemical agents, photo-nanoprobes) and mass spectrometry to study protein structure and protein-protein interaction
Ptáčková, Renata ; Šulc, Miroslav (advisor) ; Levová, Kateřina (referee) ; Osička, Radim (referee)
A comprehensive understanding of physiological role of proteins requires knowledge of their three-dimensional structure, dynamics and protein-protein interactions. Chemical cross-linking in combination with mass spectrometry represents an alternative approach to standard methods for protein structure elucidation (X-ray crystalography, NMR spectroscopy) and enables characterization of interaction interface within protein complexes in their native states. The presented thesis is mainly focused on novel cross-linking methodology based on the in vivo incorporation of methionine analog with photo-reactive functional group (photo-Met) into the sequence of studied protein (so called protein photo-nanoprobe). Interaction between two molecules of 14-3-3zeta protein was used as a model to test and optimize the protein photo-nanoprobe production. The findings confirmed usefulness of this approach for mapping the protein-protein interactions. The photo-initiated cross-linking was used to detect the heterooligomeric membrane structures of cytochromes P450 2B4 and b5 and the molar ratio of cytochromes within individual complexes was assessed. The chemical cross-linking in combination with mass spectrometry was employed to characterize the interaction of their catalytic domains and two mutual orientations of...
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Study of interactions of PI4 kinase
Eisenreichová, Andrea ; Šulc, Miroslav (advisor) ; Obšilová, Veronika (referee)
The family of 14-3-3 proteins is one of great regulatory significance, which can be found in all the eucaryotic organisms and consists of seven isoforms in human cells. The function of 14-3-3 proteins rests in the interaction with their ligands, of which several hundreds has been identified. The key role of these partners comes to pass in many cellular processes such as signalization, regulation of a cell cycle and division, apoptosis and others. This thesis deals with the interaction of 14-3-3 protein with fosfatidylinositol 4-kinase IIIβ on a molecular level using the method of X-ray crystallography. Phosphatidylinositol 4-kinase IIIβ (PtdIns4KIIIβ) situated on a cytosol side of mostly Golgi aparatus membranes catalyses the connection of a phosphate group to the fourth carbon of an inositol circle. The activity of PtdIns4KIIIβ depends upon the phosphorylation of Ser294. Not only this phosphorylation increases the kinase activity PtdIns4KIIIβ, but is the condition of 14-3-3 proteins binding as well. This interaction provides the protection of PtdIns4KIIIβ against dephosphorylation and this way it guarantees continual synthesis of phosphatidylinositol 4-phosphate, a major signalization molecule and the precursor of other phosphate derivatives of phosphatidylinositol. (In Czech)
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