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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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Preparation of mutated forms of fosfatidylinositol kinase IIα
Gregor, Jiří ; Šulc, Miroslav (advisor) ; Košek, Dalibor (referee)
Phosphatidylinositol 4-kinases (PI4K) are enzymes that form phosphatidylinositol-4- phosphate, which is an important regulatory molecule and precursor for the synthesis of other regulatory molecules. PI4K are interesting from medical aspect, because of connection between their function and viral, malignant, metabolic and neurodegenerative diseases. PI4K type II are inhibited by calcium cations, whereas PI4K type III aren't. The goal of this thesis was to elucidate the relationship between structure and regulation of function of PI4K IIα, specifically regulation by calcium cations. We failed in preparation of mutated forms of PI4K IIα that wouldn't be inhibited by calcium cations. However, the results obtained suggest, how aminoacids N313, D346 and E193 affect the kinase activity of PI4K IIα. (In Czech)
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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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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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Preparation of mutated forms of fosfatidylinositol kinase IIα
Gregor, Jiří ; Šulc, Miroslav (advisor) ; Košek, Dalibor (referee)
Phosphatidylinositol 4-kinases (PI4K) are enzymes that form phosphatidylinositol-4- phosphate, which is an important regulatory molecule and precursor for the synthesis of other regulatory molecules. PI4K are interesting from medical aspect, because of connection between their function and viral, malignant, metabolic and neurodegenerative diseases. PI4K type II are inhibited by calcium cations, whereas PI4K type III aren't. The goal of this thesis was to elucidate the relationship between structure and regulation of function of PI4K IIα, specifically regulation by calcium cations. We failed in preparation of mutated forms of PI4K IIα that wouldn't be inhibited by calcium cations. However, the results obtained suggest, how aminoacids N313, D346 and E193 affect the kinase activity of PI4K IIα. (In Czech)
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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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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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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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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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