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Changes in plant metabolism due to phytoremediation of pharmaceuticals
Polívková, Linda ; Bělonožníková, Kateřina (advisor) ; Chmelík, Josef (referee)
As a result of human activity, the rate and amount at which various pollutants enter the environment are increasing. Among them, pharmaceuticals and their metabolites, whose fate in nature has not yet been fully understood, have a key role to play. Phytoremediation, as an alternative method to the traditionally established methods of remediation of pollutant- contaminated wastewater and soils, has an increasing potential to become an important and ecologically friendly tool for environmental restoration and conservation. In this bachelor thesis, the effect of the pharmaceuticals buspirone, dextromethorphan, ibuprofen, the combination of dextromethorphan and ibuprofen, paroxetine and sulpiride on the antioxidant system of maize (Zea mays L., DKC 3969) cultivated in vitro in hydroponics under sterile conditions was investigated. The activity and isoenzyme composition of peroxidases, which are among the key components of the antioxidant system, were determined in maize plants. The amount of total soluble proteins in plants was reduced in most cases due to the influence of the aforementioned pharmaceuticals. In contrast, the specific activity of total peroxidases, ascorbate peroxidase and guaiacol peroxidase was increased in most cases, most notably by buspirone, sulpiride and the combination of...
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Preparation and characterization of protein constructs for structural analysis of acylate domain of adenylate cyclase toxin from bacteria B. pertussis
Ryvola, Vojtěch ; Ječmen, Tomáš (advisor) ; Chmelík, Josef (referee)
Whooping cough or pertussis is a contagious bacterial airways diasease caused by B. pertussis. Despite the high vaccination coverage of the population in developed countries, whooping cough is one of the most common diseases that can be prevented by vaccination. Bacteria B. pertussis produces a variety of toxins that facilitate the colonization and proliferation of bacterial cells in the host ciliary epithelium of the upper respiratory tract. One of these toxins is adenylate cyclase toxin, which is released from bacterial cells by a type I secretory apparatus and, after it's calcium-induced packaging, is able to transport its N-terminal adenylate cyclase domain across the cytoplasmic membrane of host cells. This domain able to catalyse conversion of adenosine triphosphate (ATP) to a cyclic adenosine monophosphate (cAMP) after the interaction with cytoplasmatic calmodulin. High levels of cAMP subsequently lead to impaired cell signaling and to a fundamental reduction in the bactericidal functions of phagocytic cells. The aim of this bachelor thesis was to prepare and characterize 'hybrid' protein constructs, which consist of a C-terminal part of the acylation domain and an N-terminal part of the RTX domain (at positions 881-1038 and 881-1047) linked to the C-terminal part of the RTX domain (at...
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Interaction of Cytochromes P450 with Flavodoxin: a theoretical study
Culka, Martin ; Martínek, Václav (advisor) ; Chmelík, Josef (referee)
Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...
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Backbone Resonance Assignment of the mouse protein Nkrp1c
Nelic, Dominik ; Chmelík, Josef (advisor) ; Kavan, Daniel (referee)
The protein Nkrp1c is a receptor on the surface of murine natural killer cells belonging to the C-lectin receptor family. These cells create congenital immunity against tumors or pathogens before the formation of antibodies. Determining the 3D protein structure is often the key to understanding the function of the protein at the molecular level. One way to determine the structure of proteins at the atomic level is nuclear magnetic resonance. The aim of this bachelor thesis was to evaluate several already measured spectra and to assign the resonance frequencies of the peptide backbone atoms needed to obtain data for the secondary structure prediction for Nkrp1c protein, which was prepared by recombinant expression. The Sparky program was used to evaluate the measured spectra. The prediction of the secondary structure of the Nkrp1c protein itself was performed by programs Talos+ and PSIPRED. The obtained results were compared with the already published homologous model of the Nkrp1c protein receptor. Keywords: protein NMR, Nkrp1c
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Protein chemistry and mass spectrometry in biochemical research
Pompach, Petr ; Bezouška, Karel (advisor) ; Chmelík, Josef (referee) ; Kovářová, Hana (referee)
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NMR study of the extracellular part of the mouse Nkr-p1b receptor from natural killer cells
Skála, Kristián ; Chmelík, Josef (advisor) ; Martínek, Václav (referee)
Protein Nkr-p1b is a surface receptor of cytotoxic NK cells, that mediates inhibitory signal toward the body's own cells. In this study, the ligand binding domain of the mouse protein receptor Nkr-p1b (mNkr-p1b LBD) was prepared by recombinant expression in E. coli cells. Isolated protein was subsequently used for NMR structural analysis. Prediction of protein secondary structures ratio was carried out using three different methods (CD, PSIPRED and TALOS). Results correlate well with the structure of CTLD domain, that plays a key role in ligand binding and thus to function of Nkr-p1b receptor. We managed to prepare this protein in a form suitable for NMR experiments. Based on the data obtained by NMR spectra analysis, a preliminary model of the mNkr-p1b LBD protein structure was created. However, for more precise learning of the 3D structure accurate positions of individual atoms need to be determined by other NMR spectra evaluation in the next phase. Explaining the structure of the ligand binding domain of mNkr-p1b protein could help to better understand the complex mechanism of activation of NK cell cytotoxic activity, thereby contributing to its controlled use as a therapeutic against some viral and tumor diseases.
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N-oxides of morphinan alkaloids
Palušák, Martin ; Dračínská, Helena (advisor) ; Chmelík, Josef (referee)
Morphinan alkaloids belong to important class of compounds acting as painkillers. Understanding of the mechanism of their action is important for improved therapy using these alkaloids, for example, through the synthesis of new analogues with improved effect. The goal of this work is to summarize existing knowledge of these compounds including information about their biosynthesis in the plants and overall effect on the organism. The goal of the practical part of the work is a determination of the relative configuration of morphinan N-oxides produced by in situ oxidation of parent alkaloids using NMR spectroscopy and molecular modelling. Relative configurations of both oxidized and non-oxidized forms of morphine, codeine and thebaine were analyzed using NMR spectroscopy. NMR spectra were analyzed to detect ratio of different N-oxide products in situ. These values were compared with results of DFT quantum chemical computation. Using correlation analysis, reliability of computational method was verified. (in Czech) Key words: NMR spectroscopy, morphine, codeine, thebaine, N-oxides, metabolism of alkaloids
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Preparation of the soluble form of a mouse NKR-P1A protein for the NMR study
Skála, Kristián ; Chmelík, Josef (advisor) ; Adámková, Lyubina (referee)
Natural killer cells (NK cells) are a type of lymphocyte. According to their function they are defined as cytotoxic cells which cause cell death without prior sensitization. NKR-P1 is one of the families of NK surface receptors. This family belong to C-type lectin like with inhibitory or activatory function. In this work we concern of soluble form of mouse protein Nkr-p1a, that is isoform of activatory receptor Nkr-p1a. This receptor is expected to be intracellular due to lack of major part of its transmembrane domain. We focus on the optimization of Nkr-p1a production parameters. As production system we used bacterial strain E. coli BL21(DE3) Gold, in which the target protein is produced and subsequently isolated in the form of inclusion bodies. Obtained recombinant protein was refolded and purified. As purification step we used high-performance liquid chromatography. We optimized concentration of inductor of expression, production time and temperature. The objective is to set up protocol for preparation of isotopically labeled protein for nuclear magnetic resonance structure characterization. (in czech)
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Co-operativity of cytochrome P450 system and its impact on drug and carcinogen metabolism
Holý, Petr ; Hodek, Petr (advisor) ; Chmelík, Josef (referee)
The system of mixed-function oxidases (MFO system) has a significant role in metabolism of many endogenous compounds, as well as xenobiotics (for ex. karcinogens, drugs). Membrane-bound haemoproteins called cytochromes P450 are a vital part of that system. Reactions catalyzed by cytochromes P450 are influenced by another protein of the MFO system, cytochrome b5. The mechanism of this cyt b5 agency has not yet been fully described. One of methods used for study of this protein-protein interaction is covalent cross- linking. By replacing one of three methionines in the cyt b5 structure by a photo-reactive analogue (photo-methionine), an analogue of cyt b5 (photo-cyt b5) can be obtained. When activated by UV radiation, the protein covalently bonds cytochrome P450 in a membrane environment. This paper focuses on expression and isolation of a recombinant cyt b5 analogue with only one methionine position (96) in the protein structure and substitution by photo-methionine. Protein was purified in a yiedl of 6 mg from 1 liter of baterial suspension. Analysis by mass spectrometry (MALDI-TOF/TOF) showed methionine to have been substituted by the photo-reactive analogue in approx. 30 %. Photo-cyt b5 was used to fixate transient protein-protein interactions with cytochrome P450 2B4 (CYP2B4). Photo-cyt b5 was...
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Interaction of Cytochromes P450 with Flavodoxin: a theoretical study
Culka, Martin ; Martínek, Václav (advisor) ; Chmelík, Josef (referee)
Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...
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