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Analysis of intermediate filament structure by chemical cross-linking
Dlabolová, Lada ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
Intermediate filament proteins create a dynamic cytoskeletal filamentous network, which due to its elastic properties, significantly contributes to the resistance of cells and tissues to mechanical stress. An important protein from the family of intermediate filaments, vimentin, is expressed mainly in cells of mesenchymal origin. Vimentin has been associated with a large number of pathophysiological conditions, and current studies consider vimentin as clinically promising target for the diagnosis, prognosis and treatment of a wide range of diseases from cancer to infectious and inflammatory diseases. Although in terms of structural characterization, vimentin belongs to one of the most studied proteins from the family of intermediate filaments, our knowledge is currently limited to the structure of the vimentin tetramer. Vimentin is capable of self-assembly into filaments formed by homo-oligomeric ULF subunits and the assembly process involves several steps of the organization of subunits. Structural characterization of the oligomeric subunits involved in the assembly of vimentin filaments is a prerequisite for elucidating the architecture of mature filaments, which can significantly contribute to understanding and connecting the mechanisms of many diseases associated with changes in vimentin...
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Characterization of protein structures using chemical cross-linking and mass spectrometry.
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Rozbeský, Daniel (referee) ; Hernychová, Lenka (referee)
Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.
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Studies on interactions between NKR-P1D and Clrb membrane receptors
Hanč, Pavel ; Novák, Petr (advisor) ; Brdička, Tomáš (referee)
Studies on interactions between NKR-P1D and Clrb membrane receptors Interaction between murine NKR-P1D and Clrb receptors was originally described as a novel type of "MHC class-I independent missing-self recognition" and was shown to confer protection from killing by natural killer cells.[1] However, further study brought conflicting results suggesting that NKR-P1D does not binds Clrb strongly if it does at all.[2] In order to address the issues arising from these conflicting results, we have recombinantly expressed the extracellular domains of both receptors in E. coli cells and refolded the proteins in vitro. The quality of refolding was confirmed both by determining the disulphide bonding pattern using FTMS and measuring 1 H/15 N-HSQC spectra. By means of size exclusion chromatography and analytical ultracentrifuge we were unable to provide convincing results for the interaction itself. However, using SPR technique, a weak, specific, pH-dependent interaction was observed. Interaction between the proteins in solution was immobilized using chemical cross-linking technique. Three cross-linking reagents, EDC, DSG and DSS were used. The reaction mixture was separated by means of SDS-PAGE and protein bands corresponding to dimers were digested in gel. Using FT-MS we were able to find peptides from both...
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Biophysical characterization of the N-terminal part of protein kinase ASK1.
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Apoptosis signal-regulating kinase 1 (ASK1) is an apical kinase of the mitogen-activated protein kinase cascade. Its activity is triggered by various stress stimuli such as reactive oxygen species (ROS), cytokines, endoplasmic reticulum (ER) stress or osmotic stress resulting in the activation of p38 and c-Jun N-terminal kinase metabolic pathways and leading to inflammation or cell death. Dysregulation of ASK1 is linked to several pathologies such as neurodegenerative and cardiovascular diseases and cancer, which makes this protein a potential target of therapeutic intervention. The activity of ASK1 is regulated through protein-protein interactions with 14-3-3 proteins and thioredoxin1 being among the most important negative regulators and tumour necrosis factor receptor-associated factors being an example of positive regulators. Apart from that, ASK1 is also tightly regulated via oligomerization. Despite continual progress being made, the precise molecular mechanism of ASK1 regulation and the role of ASK1 oligomerization in this process still remains unclear to this day owing to the lack of structural data. Interaction of the N-terminal parts of two protomers of ASK1 dimer is one of the key steps in ASK1 activation. It was shown, that the isolated ASK1 catalytic domain (ASK1-CD) forms stable...
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Studies on interactions between natural killer cell lectin receptors and their protein ligands.
Hernychová, Lucie ; Novák, Petr (advisor) ; Drbal, Karel (referee)
NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.
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Characterization of cofactor influence on protein structure using mass spectrometry
Rosůlek, Michal ; Novák, Petr (advisor) ; Vaněk, Ondřej (referee)
Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...
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Study on conformational changes in proteins using mass spectrometry.
Rosůlek, Michal ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
Some proteins and enzymes require presence of their specific ligand, cofaktor or prosthetic group for their activity. Binding of this specific molecule causes conformational changes, which permits to perform their function. In some occasions the identification of conformational changes is difficult. Using chemical cross-linking coupled with mass spectrometry perform complex tool for searching and low resolution visualization of this changes. The aim of this thesis is study of conformational changes induced by binding of calcium ion to calmodulin protein molecule. Calmodulin is a secondary intermediate messenger, which can interact with various proteins. This feature associates with wide dynamical range of calmodulin. Thus calmodulin is the suitable target for identifying conformational changes. After reaction of protein with chemical cross-linkers with different arm length (DSG and DSS) were products of reaction digested by trypsine. Formed linked peptides were separated by high-performance liquid chromatography and analysed followed mass spectrometry. Seven unique intramolecular cross-links were identified. Using isotope unlabeled cross-link reagents in the presence of Ca2+ in combination with using isotope labeled reagents in calcium free conditions we quantified formed lysine-lysine cross-links....
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Structural characterization of interaction between transcription factors and DNA
Filandrová, Růžena ; Novák, Petr (advisor) ; Vondrášek, Jiří (referee) ; Wimmerová, Michaela (referee)
Structural characterization of interaction between transcription factors and DNA Mgr. Růžena Filandrová Abstract Transcription factors are proteins that mediate gene expression regulation through interactions with DNA and other factors. They allow a cell to respond to various stimuli and play a crucial role in many biological processes such as control of cell cycle progression, differentiation of cells during development or immune response. To understand these processes, the knowledge of the transcription factors 3D structure together with the mechanism of their interaction with DNA is essential. However, some of the typical features of transcription factors, such as is for example the presence of intrinsically unstructured regions, make the 3D structure determination by the commonly used high resolution methods challenging. Therefore, utilization of complementary methods like structural mass spectrometry (MS), which was used in this thesis, might prove to be beneficial to explore the structural basis of the transcription factor-DNA interaction. In first part of this work, a set of structural mass spectrometry methods with the main focus on hydrogen/deuterium exchange mass spectrometry (HDX-MS) was optimized and tested on two transcription factor-DNA complexes and their DNA binding motifs and proved to be...
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Utilization of chemical cross-linking for studying intermediate filaments organization
Dlabolová, Lada ; Novák, Petr (advisor) ; Sabó, Ján (referee)
Intermediate filaments are cytoskeleton components formed by a large family of fibrous proteins specifically expressed in nearly all differentiated cells. Under physiological conditions, they spontaneously assemble into fibers in a process that involves several stages in the organization of subunits. These fibers provide elastic properties to the cells, allowing them to maintain their structural and mechanical integrity. While the structure of other cytoskeletal components is now well researched, detailed information on the structure of intermediate filaments at various stages of assembly is still not available. Thus, new insights into the structure of these proteins could be of great benefit in understanding of various pathological mechanisms associated with changes in their expression in cells. This thesis studies interactions of dimeric subunits in the tetrameric assembly of vimentin, class III protein of intermediate filaments. By chemical cross-linking of isotopically labeled and unlabeled tetrameric vimentin mixture, followed by proteolytic cleavage and mass spectrometry analysis, interdimeric, intradimeric and intrapeptide cross-linking products were identified. Quantification yielded information on interdimeric and intradimeric distance constraints, which allow the characterization of a...
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Biophysical characterization of the N-terminal part of protein kinase ASK1.
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Apoptosis signal-regulating kinase 1 (ASK1) is an apical kinase of the mitogen-activated protein kinase cascade. Its activity is triggered by various stress stimuli such as reactive oxygen species (ROS), cytokines, endoplasmic reticulum (ER) stress or osmotic stress resulting in the activation of p38 and c-Jun N-terminal kinase metabolic pathways and leading to inflammation or cell death. Dysregulation of ASK1 is linked to several pathologies such as neurodegenerative and cardiovascular diseases and cancer, which makes this protein a potential target of therapeutic intervention. The activity of ASK1 is regulated through protein-protein interactions with 14-3-3 proteins and thioredoxin1 being among the most important negative regulators and tumour necrosis factor receptor-associated factors being an example of positive regulators. Apart from that, ASK1 is also tightly regulated via oligomerization. Despite continual progress being made, the precise molecular mechanism of ASK1 regulation and the role of ASK1 oligomerization in this process still remains unclear to this day owing to the lack of structural data. Interaction of the N-terminal parts of two protomers of ASK1 dimer is one of the key steps in ASK1 activation. It was shown, that the isolated ASK1 catalytic domain (ASK1-CD) forms stable...
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