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Effect of amino acid alphabet reduction on structure and function of dephosphocoenzyme A kinase
Makarov, Mikhail ; Hlouchová, Klára (advisor) ; Dračínská, Helena (referee)
It is well-known that the large diversity of protein functions and structures derives from the broad spectrum of physicochemical properties of the 20 canonical amino acids that constitute modern proteins. According to the generally accepted coevolution theory of the genetic code, evolution of protein structures and functions was continuously associated with enrichment of the genetic code, with aromatic amino acids being considered the latest addition to the genetic code to increase structural stability of proteins and diversification of their catalytic functions. The main objective of this master thesis was to test whether enzymatic catalysis could precede the appearance of aromatic amino acids in the standard genetic code. For that purpose, the effect of amino acid alphabet reduction on structure and function of dephosphocoenzyme A kinase (DPCK) was studied. Dephosphocoenzyme A kinase catalyses the final step in the biosynthesis of coenzyme A, a very conserved cofactor. Two aromatic amino acid-lacking mutants of DPCK from a thermophilic bacterium, Aquifex aeolicus, were designed by substituting aromatic amino acid residues by (i) leucines and (ii) various non-aromatic amino acids to best preserve the structural stability of the protein. Wild type protein and the two mutants were cloned and...
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Biophysical characterization of protein libraries composed of different amino acid repertoires
Neuwirthová, Tereza ; Hlouchová, Klára (advisor) ; Ptáček, Jakub (referee)
This study is part of a project which aims to understand evolution of genetic code together with structural and functional analysis of prebiotic proteins. The repertoire of amino acids in the first proteins was probably developing in time and it influenced the development of structure and function of today's proteins. First amino acid alphabet was apparently only half of the size of present alphabet, which contains twenty amino acids. These ten amino acids were probably prebiotically available from endogenous and exogenous sources. This work includes cell-free expression and purification of two randomized protein libraries (containing approximately 1011 variants) with various amino acid composition and following comparison of their propensity to form secondary (using circular dichroism) and tertiary (using proteolytical analysis of sequences) structures. First library contains only ten probably prebiotically available amino acids; second library contains all twenty amino acids in today's genetic code. This project could help us understand benefits of genetic code expansion in terms of developing structure in protein sequences. The whole research could theoretically contribute a few basic questions not only in the fields of protein evolution but also in areas of synthetic biology or protein...
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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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Inhibitors of rhomboid proteases as tools for cell biology
Kuzmík, Ján ; Stříšovský, Kvido (advisor) ; Hlouchová, Klára (referee)
Rhomboid intramembrane serine proteases cleave polypeptide chains within lipid bilayer. Rhomboid proteases were originally discovered in Drosophila melanogaster where they regulate ontogenesis of the fly, but they are present in all domains of life. Nowadays, various diseases, such as malaria, amoebiasis, Parkinson's disease, various tumour malignancies, and diabetes, have been linked with rhomboid proteases. However, natural substrates and function of most rhomboids remain elusive. Cell biology tools are needed for unravelling functions of rhomboids, as well as for potential pharmacological applications, and this together fuels the effort to develop specific rhomboid inhibitors. The inhibitors known to date always bear an electrophilic warhead attacking the nucleophilic serine of the atypical serine-histidine catalytic dyad of rhomboid. From the various developed inhibitors, peptidyl -ketoamides substituted at the ketoamide nitrogen by hydrophobic groups, discovered in our laboratory, hold the biggest potential. They are potent, reversible, selective, tunable, and are built around a pharmacophore already approved for medical use. Here, I set out to improve peptidyl -ketoamides by exploring the chemical space in the active site of rhomboid and testing substituents of the ketoamide nitrogen of increasing...
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Preparation of receptor AICL in fusion with Fc fragment of human IgG
Runová, Alžbeta ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer cells (NK cells) are one of the basic elements of innate immunity. They play a key role in immune response against virus-infected, cancerous or otherwise stressed cells. NK cells express surface activating and inhibitory receptors. Activating receptors trigger cytotoxic mechanisms that lead to the target cell's apoptosis. Inhibitory receptors provide cellular tolerance. The balance between these receptor signals determines the resultant NK cell response to the target cell. C-type lectin-like receptors include the activating receptor NKp80 and its ligand AICL. AICL is a myeloid-specific activating receptor expressed on tumor cells. The NKp80:AICL complex that assists in the cytolysis of malignant myeloid cells is being studied in the context of cancer immunotherapy. This bachelor thesis describes the preparation of vectors containing genes encoding AICL expression constructs, and the subsequent production of proteins in the human embryonic renal cell line (HEK293S GnTI- ). The expression constructs contain the extracellular domain of AICL, TEV protease site and Fc fragment. Two different constructs were prepared - one containing the native AICL sequence and the other carrying C87S mutation.
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Preparation of a library of methionine sulfoxide reductase for applications in synthesis of chiral sulfoxides
Havelka, Václav ; Míšek, Jiří (advisor) ; Hlouchová, Klára (referee)
Chiral sulfoxides are important compounds in the pharmaceutical and chemical industries, however, their enantioselective synthesis providing only one desired enantiomer is not fully mastered. Some natural enzymes can be used for the biocatalytic preparation of chiral sulfoxides. One of such enzymes is methionine sulphoxide reductase. Methionine sulphoxide reductase is an enzyme limiting the effects of reactive oxygen radicals in the organism resulting from oxygen metabolism. Its function is the reduction of methionine sulfoxide in proteins to methionine. There are two types of methionine sulphoxide reductase, methionine sulphoxide reductase A reducing only (S)-methionine sulphoxide and methionine sulphoxide reductase B reducing (R)-methionine sulphoxide. Methionesulfoxide reductase B is suitable for the preparation of (S)-sulfoxides, however its catalytic activity is not sufficient for practical use. Using the recombinant DNA and mutagenic PCR techniques, a methionine sulphoxide reductase B mutant library was prepared, and the extent and nature of mutation introduced was determined. This library will serve as a starting point for the controlled evolution of the enzyme to obtain clones with increased activity and reduced substrate specificity.
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Structural characterization of selected random protein sequences with high disorder content
Ptáčková, Barbora ; Hlouchová, Klára (advisor) ; Šilhán, Jan (referee)
An infinitesimal fraction of the practically infinite sequence space has achieved enormous functional diversity of proteins during evolution. Intrinsically disordered proteins (IDPs) which lack a fully defined three-dimensional structure are the most likely precursors to today's proteins because of their flexible conformation and functional diversity. But how have these proteins evolved into often rigid and highly specialized protein structures? This evolutionary trajectory has the greatest support in the theory of induced fold whereby the development of the structure was mediated by the interaction and coevolution of primordial unstructured proteins with different cofactors or RNA molecules. Although some random sequences from the sequence space which is not used by nature are also able to form folded proteins the more suitable candidates for evolution of structure and function appear to be random sequences with a high content of disordered which have low aggregation propensity. The selected random protein sequences with high disorder content have been structurally characterized in this work for their further use in evolutionary studies. Three artificial proteins were selected from a random-sequence library based on previous study in our laboratory. In the present work they were purified and...
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Regulation of purine nucleotide metabolism as a pharmacological target
Brinsa, Vítězslav ; Maloy Řezáčová, Pavlína (advisor) ; Hlouchová, Klára (referee)
Purine nucleotides are essential basic building blocks for DNA and RNA synthesis. They can also serve as energy storage and transfer unit and play an important role in cell signalling and regulation of variety of biochemical processes. It is crucial for the cells to maintain a sufficient supply of purine nucleotides in order to secure its survival and cell division. Level of purine nucleotide pool in the human body is regulated via purine nucleotide metabolism, which consists of three coordinated processes: de novo synthesis pathway, salvage pathway and degradation pathway of purine nucleotides. Regulation of those three pathways is under control of various mechanisms including regulation on the level of enzyme expression, allosteric regulation of enzyme activity or forming a multienzime complexes, i. e. purinosomes in the de novo synthesis pathway. Phosphoribosyl pyrophosphate synthetase I (PRS-I) and cytosolic purine 5'-nucleotidase (cN-II) play an important role in purine nucleotide metabolism. These enzymes contribute significantly to the purine nucleotide pool regulation by means of their allostericaly regulated activity. Malfunctions of their catalytic activity are connected with various pathologies such as gout, hyperuricosuria, neurological dysfunctions and acute lymphoblastic leukaemia...
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Recombinant preparation of proliferating cell nuclear antigen (PCNA)
Herynek, Štěpán ; Vaněk, Ondřej (advisor) ; Hlouchová, Klára (referee)
Natural killer (NK) cells are cells of native immunity. Their task is to recognize and destroy tumor cells or, for example, viruses infected cells. NK cells use a number of surface receptors to recognize affected cells. These receptors may be activating, i.e., activate the cytotoxic response of NK cells leading to the induction of apoptosis in the target cell, or inhibitory ones, which, on the other hand, inhibit NK cell response. Whether a cytotoxic response occurs depends on both types of NK receptors. The NKp44 receptor belongs to the family of immunoglobulin receptors and it is one of the activating receptors. Its properties are very unique among other receptors. Other activating receptors of the immunoglobulin receptor family are NKp46 and NKp30. Recently, the interaction between the NKp44 receptor and the proliferating cell nuclear antigen (PCNA) has been described. PCNA is a clamp protein that plays a key role in DNA replication and repair as it anchors replication proteins. This work focuses on the production and optimization of a production of PCNA and on the preparation of expression vectors for the later production of NKp44 and NKp46. Interaction of NKp44 and PCNA will be investigated later. For the same reason, the expression vector for the NKp46 protein is also prepared in this work....
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