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Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography
Vaněk, Ondřej ; Bezouška, Karel (advisor) ; Hrabal, Richard (referee) ; Bařinka, Cyril (referee)
Department of Biochemistry, Faculty of Science, Charles University in Prague 2010 Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography Abstract of Ph.D. thesis Ondřej Vaněk Supervisor: Prof. RNDr. Karel Bezouška, DSc. Natural killer cells (NK cells) were found out for their ability to spontaneously kill certain allogeneic tumour cell lines, without any previous sensitization. NK cells are part of non- adaptive immune response with very short reaction time against pathogens such as viruses, intracellular bacteria, parasites, and they are responsible for elimination of certain tumour cells and thus they are able to fight against malignancy and formation of metastasis. Activity of NK cells is regulated by the balance between activation and inhibitory signals mediated by the NK cell surface receptors. From the structural point of view, the majority of NK cell surface receptors could be classified as the C-type lectin or immunoglobulin-like receptors. One of many C-type lectin subgroups are type II lymphocyte receptors that are expressed on the NK cell surface. This study had two main aims. The first one was to find suitable expression and purification systems for selected C-type lectin receptors of NK cells and the other one was to perform their...
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Structure and interactions of selected forkhead transcription factors
Kohoutová, Klára ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee)
This diploma thesis is a part of a project aiming to develop and study specific inhibitors of FOXO3 transcriptional activity. FOXO3 belongs together with FOXO1, FOXO4 and FOXO6 to FOXO subfamily of forkhead family transcription factors. FOXO transcription factors are evolutionary conserved proteins playing important roles in numerous cellular processes, such as apoptosis, cell cycle regulation and metabolism. Due to their ability to induce apoptosis and to block the cell cycle they are considered tumor suppresors. However, it has been shown that increased activity of FOXO proteins is connected with many kinds of cancer. In such cases FOXO proteins function to maintain cell homeostasis. They promote tumor resistance against chemotherapy as well as they speed up its growth. The aim of this project is to develop specific inhibitors able to bind to FOXO3 DNA-binding domain (DBD, residues 156-269) and to block its interaction with target DNA. Development of specific inhibitors of FOXO3 transcriptional programme requires knowledge of solution structure of all FOXO DBDs and detailed insight into their interaction with target DNA. So far crystal structures of complexes of FOXO1, FOXO3 and FOXO4 with target DNA and solution NMR structures of apo DBDs of FOXO3 and FOXO4 have been solved. One of the goals of...
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Detailed characterization of the interaction between LEDGF/p75 and MeCP2
Naušová, Karolína ; Veverka, Václav (advisor) ; Hrabal, Richard (referee)
Epigenetics investigates heritable phenotype changes that are not caused by alternations in DNA sequence. Major epigenetic mechanisms include covalent DNA modifications (particularly methylation), histone and chromatin modifications and RNA interference. These mechanisms are involved in number of processes from transcription to translation. Lens epithelium-derived growth factor (LEDGF/p75) is ubiquitously expressed in human body and it is considered to be a transcriptional coactivator upregulated upon stress conditions. LEDGF/p75 consists of several domains. The N-terminal PWWP domain plays very important role from epigenetic point of view as it is able to bind di- and trimethylated lysine 36 of histone 3, which is considered as an epigenetic marker of transcriptionally active chromatin. LEDGF/p75 interaction partners include e.g. HIV integrase, MLL1-MENIN complex or MeCP2. A shorter isoform of LEDGF/p75 called LEDGF/p52 shares with LEDGF/p75 its N- terminal part that is responsible for interaction with DNA and chromatin. Methyl-CpG-binding protein 2 (MeCP2) is present everywhere in human body with the highest abundance in brain. MeCP2 is a transcriptional modulator remodelling chromatin, therefore its function is to activate or repress gene depending on the molecular and cellular context. Among...
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Structural NMR studies of protein complexes
Hexnerová, Rozálie ; Veverka, Václav (advisor) ; Hrabal, Richard (referee) ; Hlouchová, Klára (referee)
Protein-protein interactions are involved in various biological processes and detailed characterization of their structural basis by the means of structural biology is often instrumental for rigorous understanding of underlying molecular mechanisms. This information is important not only for fundamental biology but also plays an important role in search for sites amenable for therapeutic intervention. Nuclear magnetic resonance spectroscopy is alongside X-ray crystallography and single-particle cryo-electron microscopy one of the key high-resolution techniques in structural biology. Although its applicability to larger systems has a well-known physical limit, it offers unique capabilities in addressing highly dynamic or inherently heterogeneous systems. In this doctoral thesis, the solution-based NMR approach was used for detailed structural characterization of selected biologically important proteins and their complexes that provided important insights into their biological roles. In three distinct projects, I (i) studied the relationship between the structural effects of particular modifications in the insulin-like growth factor II (IGF-II) and their selectivity to the insulin axis receptors; (ii) the specific binding mechanism of the SH3 domain from the Crk-associated substrate (CAS); (iii) and...
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Structural studies of selected signaling protein complexes.
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee) ; Maloy Řezáčová, Pavlína (referee)
The ability of proteins to bind other molecules in response to various stimuli in their microenvironment serves as a platform for extensive regulatory networks coordinating downstream cell actions. The correct function of these signaling pathways depends mostly on noncovalent interactions often affecting the structure of proteins and protein complexes. Understanding the molecular mechanism of a protein function in cell signaling therefore often depends on our knowledge of a three-dimensional structure. In this doctoral thesis, I present the work that led to the understanding of several protein-protein and protein-ligand interactions implicated in cell signaling at the molecular level. I applied nuclear magnetic resonance spectroscopy, small angle X-ray scattering and other biophysical methods to determine the molecular basis of inhibition of four signaling proteins: Calcium/Calmodulin (Ca2+ /CaM)-dependent protein kinase kinase 2 (CaMKK2); protease Caspase-2; Forkhead transcription factor FOXO3, and Apoptosis signal-regulating protein kinase 1 (ASK1). In particular, I investigated the distinct roles of 14-3-3 and Ca2+ /CaM in the regulation of CaMKK2 activity. I also studied in detail the mechanism how 14-3-3 interferes with the caspase-2 oligomerization and its nuclear localization as well as...
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Interactions in solutions and gels of stimuli-responsive polymer systems investigated by NMR spectroscopy
Konefał, Rafał ; Spěváček, Jiří (advisor) ; Hrabal, Richard (referee) ; Štěpánek, Miroslav (referee)
Stimuli-responsive (stimuli-sensitive, intelligent, or smart) polymers are polymer materials which, after small external stimuli, evidently change their physical or chemical properties. Smart polymers can be classified according stimuli they respond to such as: temperature changes, mechanical stress, light irradiation, ultrasonic treatment, application of external magnetic as well as electric field, changes of pH, ionic strength, addition of the chemical agents and presence of biomolecules and bioactive molecules. Stimuli-responsive synthetic polymer systems has attracted considerable attention due to wide range of applications, i.e. controlled drug delivery and release systems, diagnostics, tissue engineering and 'smart' optical systems, as well as biosensors, microelectromechanical systems, coatings, and textiles. Among the types of stimuli for this dissertation temperature, pH and reactive oxygen species (ROS) responsive polymer systems were studied. In case of thermoresponsive polymers, when polymer chains are molecularly dissolved in a good solvent, changes (increasing or decreasing) of temperature result in insolubility (globular nanoparticles formation) of polymer chains, called temperature induced phase-separation. pH responsive polymers change properties such as: solubility, volume (gels),...
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Struktura a vlastnosti modifikovaných složek nukleových kyselin
Procházková, Eliška ; Dračínský, Martin (advisor) ; Lyčka, Antonín (referee) ; Hrabal, Richard (referee)
Modified nucleic acid components (nucleotides, nucleosides, nucleobases) display a wide range of biological activities such as antiviral, cytostatic, antimicrobial or antioxidative. Many of them are successfully used in clinical practice, for instance as anti-HIV drugs or in hepatitis B treatment. The first precondition for understanding biological effects is to evaluate the chemical structure as well as the conformation of the studied compounds correctly. For this purpose, solution-state nuclear magnetic resonance (NMR) spectroscopy in combination with mass spectrometry is the most frequently used method. Not only the structure, but also physico- chemical properties can be investigated using NMR spectroscopy, for instance, conformational changes, tautomeric forms, protonation sites, acido-basic properties, non-covalent interactions, isotopic exchanges or interactions with biomolecules. Similarly to solutions, NMR spectroscopy can be employed in studies of nucleic acid components (NACs) in solid state. This technique found assertion after computational-chemistry methods development during the last decades. Different solid-state structures (polymorphic forms) or dynamics of these compounds can be established by solid-state NMR spectroscopy in combination with quantum-chemical calculations. In this...
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Understanding the interaction of antibodies and transcription factors with their ligands through structural biology
Škerlová, Jana ; Maloy Řezáčová, Pavlína (advisor) ; Hrabal, Richard (referee) ; Obšil, Tomáš (referee)
Understanding protein function highly benefits from the knowledge of its three-dimensional structure, especially in the case of protein-ligand complexes. Structural biology methods such as X-ray crystallography, SAXS and NMR are therefore widely used for structural studies of protein-ligand interaction. In this work, these methods were used to understand two biological processes involving protein interactions: X-ray structural analysis was used to study binding of effector molecule to a prokaryotic transcription factor. NMR and SAXS techniques were used to study interaction of a monoclonal antibody with its protein antigen. Transcriptional regulator DeoR negatively regulates the expression of catabolic genes for the utilization of deoxyribonucleosides and deoxyribose in Bacillus subtilis. DeoR comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain (C-DeoR), and its function is regulated by binding of a small-molecular effector deoxyribose-5-phosphate. We determined crystal structures of C-DeoR both in the free form and in complex with deoxyribose-5-phosphate. Structural analysis revealed unique covalent binding of effector molecule through a reversible Schiff-base double bond with an effector-binding-site lysine residue. The physiological nature of this binding mode was...
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Synthesis and characterization of the new insulin analogues with the aim to clarify the interaction of insulin with its receptor
Kletvíková, Emília ; Jiráček, Jiří (advisor) ; Obšil, Tomáš (referee) ; Hrabal, Richard (referee)
The objective of this thesis is to characterize insulin analogues modified at the C-terminus of the B-chain with the aim to observe the impact of the inserted modifications on the insulin-insulin receptor (IR) interaction and the ability of the analogues to dimerize. Therefore, a series of analogues with modifications at B24-B26 positions was prepared. Using the synthetic and semisynthetic methods we inserted coded and non-coded amino acids to this part of B-chain. We studied full-length analogues and analogues truncated by three to four amino acids. Binding affinity of all analogues to the insulin receptor was determined by competition of analogue with radioactive (125I) human insulin. Dissociation constant in the dimer dissociation process of selected analogues (especially of those with N- methylation of B23-B24, B24-B25 and B25-B26 peptide bonds) was determined by isothermal titration microcalorimetry. The crystal structures of several analogues were resolved by X-ray crystallography and nuclear magnetic resonance. The structural results showed the consequences of inserted modifications to the insulin molecule. We characterized analogues with higher, equipotent and lower binding affinity to the IR. The results...
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