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The two-component signal transduction system in bacteria utilizing heme analogues
Bečková, Alexandra ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
6 Abstract Hemoproteins are essential components of both prokaryotic and eukaryotic organisms, where they play a role in fulfilling many significant life functions. According to their function, hemoproteins can be divided into several types. One specific type of hemoproteins, which was also the object of interest in this bachelor thesis, is the heme sensor proteins. The function of part of these sensor hemoproteins is the ability to detect diatomic gas molecules and subsequently bind them to the heme molecule in the protein. This process leads to changing the properties of the sensor hemoprotein and the signal then becomes part of the signalling pathways. An example of sensor hemoprotein is the histidine kinase, with a globin structure of the sensor domain, from Anaeromyxobacter sp. Fw 109 (AfGcHK), which was studied during the experimental part of the bachelor thesis. First, a transformation of E. coli BL-21 (DE3) cells with the pET21c(+)/AfGcHK plasmid was performed, followed by cell cultivation and expression of the AfGcHK protein in the cells. The expressed AfGcHK protein was isolated in two forms. One form represented AfGcHK protein containing heme, that is, with iron incorporated in its structure. The second form represented AfGcHK proteins containing a heme analogue, where nickel was incorporated...
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Heme sensor proteins as potential biomarkers of cellular and oxidative stress processes induced by ionizing radiation
Vávra, Jakub ; Martínková, Markéta (advisor) ; Souček, Pavel (referee) ; Tichý, Aleš (referee)
[IN CZECH] Ionizing radiation is a potential inducer of the oxidative stress processes in cells. As a result, reactive radicals are formed in the intracellular space modifying the essential biomolecules. Ionizing radiation has either direct (radiation sickness) or indirect (malignant processes) effect on the organism. Therefore, a fast determination of the dose is required when suspected irradiation of the organism occurs. However, a method routinely applicable, fast enough and at the same time suitable for dose estimation based on biomarkers has not been developed so far. The aim of this thesis is to describe new properties of the selected heme sensor proteins and discuss their potential importance for the cellular adaptation to oxidative stress conditions. Specifically, the thesis is focused on two eukaryotic proteins, heme regulated inhibitor (HRI) and transcription factor p53. The study of functional regulation as well as the conformational changes of these proteins induced by heme is greatly emphasized. Besides, the optimization of the key experimental methods was conducted. Specifically, Phos-tag electrophoresis was applied for the kinetics study of HRI wild type and its Gly202Ser mutant form, which is a characteristics of lung cancer development. Unsurprisingly, for both HRI forms studied,...
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Detail enzymatic characterization of a model heme-containing oxygen sensor
Vojáčková, Lukrécie Sophie ; Martínková, Markéta (advisor) ; Čermáková, Michaela (referee)
This thesis focuses on heme-based gas sensors, particularly phosphodiesterase from the bacterium Escherichia coli, referred to as EcDOS. The enzyme catalyzes the degradation of c-di-GMP, an important signaling molecule in bacteria that affects cellular processes, such as bacterial motility or biofilm formation. The thesis deals with detailed enzyme kinetics of protein forms in different redox and ligand states of the heme iron ion [Fe(III), Fe(II) and Fe(II)-O2], as well as a mutant form of the enzyme (EcDOS H77A) which does not bind heme. Results confirmed that the EcDOS WT Fe(II)-O2 form has higher kcat values than the EcDOS form with the ferrous ion of heme in the reduced state. Other significant result was that the enzyme activity is affected not only by the state of the heme iron ion but also by the presence and concentrations of divalent metal cations. The presence of the metal cation is essential for enzyme function, and suitable metal ions that stimulate enzyme activity are Mg2+ , Mn2+ and Zn2+ or their mixtures, which act synergistically on enzyme activity under chosen conditions. Analysis by ICP-MS also showed that Zn2+ cations are natural components of the enzyme. Thus, for further kinetic studies, it would be appropriate to use Zn2+ or mixtures of metal ions that are physiological for...
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Structure and function relationships of model hemoproteins
Lengálová, Alžběta ; Martínková, Markéta (advisor) ; Hudeček, Jiří (referee) ; Muchová, Lucie (referee)
Heme is one of the most important and most studied cofactors that are essential for proper function of many proteins. Heme-containing proteins comprise of a large group of biologically important molecules that are involved in many physiological processes. The presented dissertation is focused on two groups of heme sensor proteins, namely prokaryotic heme-based gas sensors and eukaryotic heme-responsive sensors. Heme-based gas sensors play an important role in regulation of many bacterial processes and consist usually of two domains, a sensor domain and a functional domain. The dissertation thesis aims at the study of two model bacterial heme-based gas sensors, histidine kinase AfGcHK and diguanylate cyclase YddV, in order to elucidate their mechanism of interdomain signal transduction. Using X-ray crystallography and hydrogen-deuterium exchange coupled to mass spectrometry approaches, significant differences in the structure of the AfGcHK protein between the active and inactive forms were described. The signal detection by the AfGcHK sensor domain affects the structural properties of the protein, and these conformational changes then have indirect impact on the enzyme activity of the functional domain. Further, the dissertation pays more attention to the effect of a sensor domain dimerization...
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Study of the effect of heme analogues on the structural-functional characteristics of a model representative of heme sensor proteins
Ďatko, Peter ; Martínková, Markéta (advisor) ; Vávra, Jakub (referee)
Heme sensor proteins allow bacteria to react to changes of concentration of certain molecules in their environment. This reaction depends on the coordination of the ligand to the heme iron atom. Model representative of this signaling system is a histidine kinase containing a sensor domain with a globin structure, AfGcHK. The aim of this bachelor thesis was to prepare and characterize a modified form of AfGcHK containing manganese within its protoporphyrine complex. To express the protein, E. coli BL-21 (DE3) cells were transformed using a plasmid pET21c(+)/AfGcHK. The protein was isolated and purified using affinity chromatography and gel chromatography. To determine its enyzmatic activity, polyacrylamid gel electrophoresis in the presence of sodium dodecyl sulfate with Phos-Tag was used. It was determined, that this novel form of AfGcHK is enzymatically active. Spectroscopic analysis has shown, that the modified form of AfGcHK containing manganese within its protoporphyrine complex is susceptible to reduction by sodium dithionate. Key words: heme, heme sensor proteins, oxygen sensors, signal transduction [IN CZECH]
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Biochemical characterization of a model heme sensor protein containing a heme analog
Hlubučková, Darina ; Martínková, Markéta (advisor) ; Prošková, Veronika (referee)
An integral part of the life of cells is cell signaling, which is ensured, among other things, by heme proteins, specifically by their members called heme sensor proteins. Heme sensor proteins are divided into two groups, heme sensor proteins detecting heme, which are found more in eukaryotic cells, and heme sensor proteins detecting gaseous molecules, which are more typical of prokaryotic cells. The gas molecules in this case can be CO, NO and O2. A family of oxygen-detecting heme sensor proteins is crucial for bacteria that must adapt to changing oxygen concentrations in the environment. One of these bacteria is the soil bacterium Anaeromyxobacter sp., strain Fw 109-5, which contains a heme sensor protein with the globin structure of the sensor domain and the histidine kinase activity of the functional domain (AfGcHK), enabling the bacteria, among other things, to form a biofilm. As part of the theoretical part of this thesis, the current knowledge about heme sensor proteins was summarized, with a focus on the AfGcHK protein. In the practical part of this bachelor's thesis, bacterial cells E. coli BL-21 (DE3) were transformed with the plasmid pET21(+)/AfGcHK containing the gene encoding AfGcHK, from which two forms of AfGcHK were subsequently isolated, the natural form and the form containing a...
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ALAS2 gene sequencing
HOLOUBKOVÁ, Renata
In my bachelor thesis I dealt with the detection of mutations in the ALAS2. In the theoretical part, I dealt with the introduction to genetic concepts and the field of genetics itself. The work begins from the cell itself, to the chromosomes, through mutations. I also dealt directly with the ALAS2 gene. In the following chapters, I focused on heme, because the ALAS2 gene encodes an enzyme that is responsible for heme production. Last but not least, I have described the disease erythropoietic protoporphyria and sideroblastic anemia, which arise precisely due to mutations in this gene. In the practical part, I examined the ALAS2 gene of exon 11. I examined 25 anonymized samples, from which I isolated DNA. I then amplified the desired section of the gene by PCR. I prepared the samples for the sequencing method, which took place at GenSeq s.r.o. In the last part of my bachelor's thesis, I dealt with the evaluation of sequences and processing of results, but also with the evaluation of sequences from children from Karviná and České Budějovice, which I received. No mutations were found from the whole examined group and the group to be evaluated. For evaluation from a larger data set, the results of the mutations found in the research were added.
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Light-harvesting like domain of the cyanobacterial ferrochelatase
PAZDERNÍK, Marek
This thesis is focused on elucidating the function of the C-terminal transmembrane lightharvesting complex like (LHC) domain of the cyanobacterial ferrochelatase (FeCh). Using the model cyanobacterium Synechocystis PCC 6803, I show that the FeCh LHC domain can bind chlorophyll (Chl) and carotenoids; however, this pigment binding occurs only when the biosynthesis of heme and Chl in the cell is misbalanced. Further, I found that point mutation, which prevents the pigment binding to FeCh LHC domain results in a misregulated ratio between heme and Chl during stress conditions due to low heme accumulation. My data also show that the FeCh LHC domain interacts with CurT protein most likely to localize the FeCh into a specialized membrane domain, where the synthesis of photosystem II is proposed to occur. Based on my data I propose that the role of the FeCh LHC domain is to monitor the availability of Chl during photosystem biogenesis and to coordinate Chl availability with the synthesis of heme.
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Specific heme interaction modulates the conformational dynamics and function of p53
Sergunin, Artur ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Tumor suppressor p53 is one of the most studied proteins in terms of cancer and the mechanism of its formation. The general function of p53 is based on the transcriptional regulation of various genes, which can differently influence numerous cellular processes. Recent studies revealed a relationship between p53 and iron homeostasis within the cell. In particular, p53 was shown to interact with a molecule of heme, and this interaction ultimately disrupts the DNA-binding ability of p53 and promotes its proteasomal degra- dation. This work focuses on a detailed description of heme binding to the p53. For this purpose, we isolated two forms of p53, heme-free and heme-bound. We discovered that conformational dynamics of heme-free and heme-bound p53 differ, with the latter exhibi- ting a higher degree of flexibility. We also confirmed previous reports that heme indeed interacts with a cysteine residue in a specific manner. However, heme binding does not disrupt the oligomeric state of p53 or its native zinc binding ability. Finally, we showed that heme-bound p53 exhibits severely impaired DNA-binding ability as opposed to the heme-free form. Keywords: heme, sensor proteins, p53 protein, transcription factor, intrinsically disor- dered proteins
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The role of iron in the metabolism of the amoeba Naegleria gruberi
Ženíšková, Kateřina ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
Iron is a biogenic trace element that is vital for all organisms on the planet Earth. This element occurs in biological systems in the form of Fe3+ and Fe2+. These two forms are often incorporated in heme structures or iron-sulfur clusters. Proteins containing iron ions have a wide range of functions in organisms. The main functions include the transport of electrons in the respiratory chain (Rieske's proteins, cytochromes), DNA synthesis (ribonucleotide reductase) and the participation in the Krebs' cycle (aconitase, succinate dehydrogenase). Naegleria gruberi is a nonpathogenic amoeba known for its pathogenic relative Naegleria fowleri. This organism causes the primary amoebic meningoencephalitis. An interesting fact about Naegleria gruberi genome is that it contains genes for both aerobic and anaerobic metabolisms. The purpose of my bachelor work was to investigate the effect of availability of iron ions on metabolism in Naegleria gruberi. Changes in the activities of enzymes from different metabolic pathways were studied including lactate dehydrogenase, isocitrate dehydrogenase, Fe- hydrogenase, aconitase and fumarase. The most significant changes were observed in the activities of alcohol dehydrogenase and Fe-hydrogenase. Key words: Iron, heme, iron-sulfur clusters, availability of iron ions,...
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