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The photolabile protein nanoprobes expression optimization of human structural dental enamel proteins
Štrohalmová, Jana ; Šulc, Miroslav (advisor) ; Kubíčková, Božena (referee)
Dental enamel is the hardest tissue of the body. It is formed by an evolutionarily highly conserved biomineralization process that is controlled by proteins of extracellular matrix. Amelogenin (AMEL) and ameloblastin (AMBN) are key element of the correct enamel formation. Simultaneously the proteins serve as a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts (the cells involved in dental enamel formation). AMEL and AMBN belong to the family of intrinsically disordered proteins (IDPs) therefore it is very difficult to find a methodology for studying the structure and action of molecular mechanism. This bachelor's thesis is aimed at optimization of preparation process of photolabile protein "nanoprobes" of ameloblastin and amelogenin using recombinant expression in E. coli by incorporation of the photolabile analogs of amino acids (methionine, leucine). The prepared protein " nanoprobes " will be used to study protein-protein interactions in solution and to elucidate the structure-function relationships of human dental enamel proteins (ameloblastin, amelogenin). (In Czech) Keywords: Dental enamel Ameloblastin Amelogenin Photolabile protein "nanoprobes" Mass Spectrometry
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Structural basis for interspecies differences in the TRPA1 receptor activation
Synytsya, Viktor ; Šulc, Miroslav (advisor) ; Hudeček, Jiří (referee)
Ankyrin transient receptor potential channel TRPA1 is an excitatory ion channel that transduces nociceptive information on primary aferent sensory nerves of mammals and other organisms. Structure function studies on TRPA1 are valuable for understanding the mechanisms of channel activation and for specific drug discovery efforts, however, significant interspecies differences hamper direct transfer of findings in animals to human. On the other hand, the interspecies differences may prompt identification of many important functional domains. The aim of this bachelor thesis is to give an overview of recent evidence regarding the functional and structural properties of human TRPA1 ion channels from the point of view of the comparison of the most important interspecies differences among TRPA1 orthologs. The experimental part is focused on the comparison of activation properties of human TRPA1 channel with a chimera in which the fifth transmembrane region was replaced by that from Drosophila melanogaster. The presented results obtained by electrophysiological technique patch-clamp demonstrate that outward membrane currents induced by depolarizing voltage are significantly reduced in chimera, which indicates an important role of the fifth transmembrane domain in TRPA1 channel gating (In Czech). Key words:...
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Proteomic approaches in cancer biology
Lorková, Lucie ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kovářová, Hana (referee)
Proteomics as a modern comprehensive approach to the analysis of proteomes was applied in three projects aimed at diagnosis and therapy of cancer. The aim of the first the project was to find a new diagnostic biomarker for ovarian cancer. Two different comparative proteomic approaches were used for comparative analysis of sera from patients diagnosed with ovarian cancer and from healthy age-matched women. We identified -1-antitrypsin with increased concentration in patien sera, and apolipoprotein A4 and retinol-binding protein 4 (RBP4) with significantly decreased concentration in patients. The significantly decerased concentration of RBP4 in patients is a new observation. We propose that RBP4 is either decreased in ovarian cancer patients as a result of its reduced production by ovary or it may reflect less specific systemic changes, for instance early onset of cancer cachexia. The second project was focused on gaining insight into the molecular mechanism of cytarabine resistance in mantle cell lymphoma (MCL). Proteomic and transcriptomic analyses of cytarabine-resistant cells revealed marked downregulation of deoxycytidine kinase (DCK) - a protein essential to intracellular activation of purine and pyrimidine nucleosides and their analogues including cytarabine. The cytarabine-resistant MCL...
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Preparation of DNA-binding domain of Forkhead transcription factor FOXO3
Dolejš, Vojtěch ; Šulc, Miroslav (advisor) ; Ptáček, Jakub (referee)
This bachelor thesis is part of a project aiming for the development of low molecular compounds which would be capable to inhibit the interaction between human transcription factor FOXO3 and DNA. Main goal of this thesis is preparation of 15 N-labelled DNA-binding domain of FOXO3 protein (FOXO3-DBD) and verification of its native structure using 1 H- 15 N HSQC NMR experiment. FOXO transcription factors are important and evolutionary conserved regulatory proteins, which are involved in many crucial cellular processes. The activity of FOXO proteins is regulated by posttranslational modifications, out of which the most important are phosphorylation, acetylation and ubiquitination. Forkhead transcription factors participate in a variety of different cellular functions, although its expression is limited to specific tissues. They contain approximately 100 amino acids long DNA-binding domain composed of several parts. Among its main functions belong the regulation of cell cycle and apoptosis, proliferation and cell differentiation, metabolism control and stress-response regulation. Some types of tumor cells have developed resistance against chemotherapy by increasing activity of FOXO3 transcription factors. For this reason, it is necessary to look for means to specifically suppress the function of this...
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Proteomická a bioinformatická charakterizace N-terminálních sekvencí proteinů modifikovaných po importu do hydrogenosomu Trichomonas vaginalis.
Zákoucká, Eva ; Man, Petr (advisor) ; Šulc, Miroslav (referee)
Trichomonas vaginalis is a human pathogen causing trichomoniasis, one of the most common non-viral sexually transmitted diseases in both men and women. Trichomoniasis is currently treated with metronidazole, but the pathogen is known to develop resistance against this drug. However as the pathogen is eukaryotic, the targets for the pathogen elimination without seriously affecting the host are limited. Throughout the evolution Trichomonas vaginalis adapted to anaerobic environments by developing an alternative metabolism resulting in a reduced form of mitochondria named hydrogenosome. Hydrogenosomes lack genetic information, therefore all its proteins are nucleus-encoded and need to be transported inside the hydrogenosome using a targeting N-terminal presequence. The peptidase recognizing and cleaving those presequences at the entrance of the organelle, the hydrogenosomal processing peptidase (HPP), is unique for hydrogenosomes and therefore represents a potential drug target against the pathogen. In this work the HPP's substrate specificity towards the targeting presequences was investigated. To do so a proteomic analysis of the proteome of Trichomonas vaginalis hydrogenosomes was performed using a novel optimized protocol for N-terminal peptide sequencing. N-terminal peptides were captured using a...
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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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Structure and interaction of human 14-3-3 regulatory protein using in vitro photoaffinity labelling in combination of protein nano-probes and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Dračínská, Helena (referee)
This thesis is focused on the study of the structure and mechanism of human 14- 3-3 protein, which is one of the important regulatory proteins present in all eukaryotic cells. Nowadays it is known seven isoforms of this protein in mammals. Although their crystal structure shows a high similarity, their mutual comparison reveals some changes. The aim of this work is to prepare experimental tools for verification whether the differences in the crystal structure of the ζ isoform are present in solution and how the structure-functional mechanism of this isoform is affected. The otimization of 14-3- 3zeta recombinant protein expression with incorporated a photo-labile analog of leucine in the protein sequence was performed using limiting medium with prokaryotic expression system of E. coli BL-21 DE3 Gold or system of auxotrophic E. coli K-12 with non-functional leucine biosynthesis.
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Cytochrome P-450: Study of structure and interactions using chemical modification, photo-initiated cross-linking and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor) ; Petrák, Jiří (referee) ; Šebela, Marek (referee)
ABSTRACT Mixed function oxygenase system participates in biosynthesis of endogenous and metabolism of exogenous substances (e.g. drugs or chemical procarcinogens) in an organism. Substrates are biotransformed by terminal oxygenases - cytochromes P450 (P450). Catalytic properties of certain P450s (e.g. studied isoform 2B4) are altered in the presence of a redox partner - cytochrome b5 (cyb5). Both cytochromes are anchored by hydrophobic domains in a lipid membrane of endoplasmic reticulum whereas their catalytic domains are exposed to cytosol. Two zero-length cross-linking approaches were employed to extend present knowledge of P450 2B4 and cyb5 protein structure and protein-protein interactions: (1) interlinking of carboxylate and primary amine groups of amino acids by water soluble 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and (2) photo-initiated cross-linking by photo-labile methionine analog (pMet), which links to any amino acid after activation by UV-irradiation, either in hydrophilic or hydrophobic environment. pMet was incorporated to methionine site(s) of cyb5 during recombinant expression in E. coli, which was carried out in limit medium supplemented with amino acid analog. Optimization of experimental conditions led to ~20-30% substitution of the natural amino acid. Covalent...
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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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Proteome analysis of anti-cancer drug effects and characterisation of drug resistance
Hrabáková, Rita ; Kovářová, Hana (advisor) ; Hernychová, Lenka (referee) ; Šulc, Miroslav (referee)
Despite significant progress in the development of anti-cancer drugs, there is still a need for novel therapeutic strategies that would improve the outcome of cancer patients. Using proteomic technologies and cell lines with different phenotype of p53 tumour suppressor, we monitored cancer cell response to anti-cancer treatment with focus on the development of drug resistance. The different levels of metabolic proteins were identified in our study which may help to explain different anti-cancer activity of drugs with only a subtle difference in structure. More importantly, proteins associated with the development of drug resistance were identified and such expression changes have become a focus of interest. Our findings demonstrate a higher protein level of serine hydroxymethyltransferase, serpin B5 and calretinin in cancer cells resistant to Aurora kinase inhibitors. Such proteins promote the tumour growth with no apparent impact of p53 phenotype whilst voltage-dependent anion-selective channel protein 2 contributes to the development of resistance only in cells with functional p53 which is accompanied by the decreased level of elongation factor 2. On the other hand, cancer cells with loss of p53 appear to amplify alternative mechanisms such as protection against oxidative stress. The results...
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