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Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicus
Černý, Zbyněk ; Španová, Alena (referee) ; Pepeliaev,, Stanislav (advisor)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
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Identification of residues of acylated domain of RTX toxins involved in acyltransferase binding
Grobarčíková, Michaela ; Mašín, Jiří (advisor) ; Černý, Ondřej (referee)
Both adenylate cyclase toxin (CyaA) and α-hemolysin (HlyA) are members of Repeats in ToXins (RTX) cytolysins that play key roles in the virulence of Bordetella pertussis and Escherichia coli, respectively. Bacterial RTX toxins represent a growing group of proteins produced by gram- negative bacteria. These pore-forming RTX toxins share several notable common features: (1) they require post-translational activation by attachment of fatty acid chains to two lysine residues; (2) they contain a hydrophobic domain that forms cation-selective pores in target cell membranes; (3) they are secreted by a type I secretion system; (4) after secretion, they become biologically active by binding of Ca2+ to the nonapeptide glycine- and aspartate-rich repeats. CyaA translocates a unique AC enzyme to the cytosol of phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. CyaA and HlyA also permeabilize the cell membrane of eukaryotic cells through cation-selective pores. Both toxins preferentially bind to cells expressing β2 integrins but can also interact with a variety of cells that do not express integrins or with naked lipid membranes. Both toxins are activated from protoxin form by post- translational acylation mediated by a specific acyltransferase. CyaA is activated by...
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Role of Arginine 717 in insulin receptor respective Arginine 704 in IGF-1 receptor for the interaction with ligands
Kertisová, Anna ; Selicharová, Irena (advisor) ; Ryšlavá, Helena (referee)
Insulin and insulin-like growth factor 1 (IGF-1) are peptide hormones that are important regulators of cellular metabolism, proliferation and apoptosis. Disruptions in signalling pathways may cause a whole range of diseases from diabetes mellitus type 1 and type 2 to cancer or neurodegenerative diseases. The cellular response to these hormones is mediated by insulin (IR) and IGF-1 receptors (IGF-1R) with a tyrosin-kinase activity. Receptors are created as hetero-tetramers of two extracellular α-subunits and two intracellular β-subunits. Studies of receptor structures try to elucidate the basic principles of the interaction of receptors with their ligands. However, the role of some amino-acid residues in binding remains unclear. It was suggested that the arginine 704 of IGF-1R may interact with Glu58 IGF-1. In comparison with IGF-1R, the equivalent arginine 717 IR was not associated with an important role in insulin binding in previous studies. This thesis is focused on clarifying the role of Arg704 IGF-1R and for comparison analogically on Arg717 IR isoform A (IR-A) in ligand binding to the receptors. Therefore, mutant variants of IGF-1R in positions His697 and Arg704 and variants IR-A in positions His710 and Arg717 were created. The role of histidines 697 IGF-1R and 710 IR was already elucidated...
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Preparation of mutant variants of Kingella kingae RtxA cytotoxin for membrane topology research
Lichvárová, Michaela ; Osičková, Adriana (advisor) ; Malý, Petr (referee)
Kingella kingae is a facultative anaerobic, β-hemolytic, gram-negative bacterium. It has been shown, that K. kingae is an important cause of invasive infections in young children, especially between 6 to 36 months of age. The most common diseases caused by K. kingae are septic arthritis, osteomyelitis, bacteremia and infective endocarditis. The key virulence factor of K. kingae is the secreted RtxA toxin, which belongs to the RTX toxins family (Repeats in ToXin). These are divided into two categories, hemolysins and leukotoxins, based on the cellular specificity of their action. The broad specificity of the RtxA toxin indicates that RtxA can be classified as a cytolytic RTX hemolysin. RtxA molecules are inserted into the host cell membrane and form cation-selective membrane pores that trigger cation flux. This disrupts normal cell physiology and eventually leads to cell lysis. The aim of this bachelor thesis was to prepare mutant variants of the K. kingae RtxA cytotoxin with lysine substitutions in the pore-forming domain for future study of the membrane topology of the toxin using biotin binding to the lysine residues. In order to observe the topology of the RtxA toxin in the host cell membrane, the toxin must be able to insert to the cell membrane. Therefore, another objective was to determine...
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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Mechanism of auxin transport across plasma membrane through PIN auxin efflux carriers
Lefnar, Radek ; Petrášek, Jan (advisor) ; Nodzynski, Tomasz (referee)
Phytohormone auxin and its directional distribution plays an essential role in the regulation of numerous processes during vegetative and reproductive plant development. Regulation of the expression, localization and activity of the PIN-FORMED (PIN) proteins is important for proper polar auxin transport in plant tissues. PIN proteins have been described as the major auxin efflux carriers regulating auxin's directional flow to build up gradients that provide information for the coordination of plant development. PIN protein structure topology prediction through bioinformatic analysis is still insufficient to understand their transport mechanism. Experimental analysis of PIN protein domains can provide valuable insight into understanding their role in mediating auxin transport. In this study, the C-terminal part of PINs have been modified by gradual trimming to determine the existence of relevant functional domains, which could be important for auxin transport. Seven modified PIN proteins from Arabidopsis thaliana and Nicotiana tabacum were prepared. Transiently transformed tobacco cell line Bright Yellow-2 (BY-2) was used to monitor differences in PIN transport activity. This approach allowed indirect monitoring of intracellular auxin levels using the DR5 reporter system. Transiently expressed...
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan ; Martínek, Václav (advisor) ; Befekadu, Asfaw (referee) ; Souček, Pavel (referee)
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5 can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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