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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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Never Born Proteins: Occurence and characterization of secondary structure motifs
Treťjačenko, Vjačeslav ; Hlouchová, Klára (advisor) ; Kopecký, Vladimír (referee)
An experimental study on randomly generated protein sequences can provide important insights into the origin and mechanism of secondary structure formation and protein folding. In this study we bring biophysical characterization of five protein sequences selected from the in silico generated library of random chains. The sequences were selected on the basis of bioinformatic analysis in order to find the candidates with the maximum potential to possess secondary structure. This study shows that the random polypeptide sequences form stable secondary structures and in some show the signs of tertiary structure, such as hydrophobic core formation and distinctive oligomerization pattern. While the work presented in this thesis is work in progress on a larger study, the data already demonstrate that unevolved protein sequence space provides a lot of potential for secondary and tertiary structure formation that awaits its characterization. Powered by TCPDF (www.tcpdf.org)
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The role of iron in the metabolism of the amoeba Naegleria gruberi
Ženíšková, Kateřina ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
Iron is a biogenic trace element that is vital for all organisms on the planet Earth. This element occurs in biological systems in the form of Fe3+ and Fe2+. These two forms are often incorporated in heme structures or iron-sulfur clusters. Proteins containing iron ions have a wide range of functions in organisms. The main functions include the transport of electrons in the respiratory chain (Rieske's proteins, cytochromes), DNA synthesis (ribonucleotide reductase) and the participation in the Krebs' cycle (aconitase, succinate dehydrogenase). Naegleria gruberi is a nonpathogenic amoeba known for its pathogenic relative Naegleria fowleri. This organism causes the primary amoebic meningoencephalitis. An interesting fact about Naegleria gruberi genome is that it contains genes for both aerobic and anaerobic metabolisms. The purpose of my bachelor work was to investigate the effect of availability of iron ions on metabolism in Naegleria gruberi. Changes in the activities of enzymes from different metabolic pathways were studied including lactate dehydrogenase, isocitrate dehydrogenase, Fe- hydrogenase, aconitase and fumarase. The most significant changes were observed in the activities of alcohol dehydrogenase and Fe-hydrogenase. Key words: Iron, heme, iron-sulfur clusters, availability of iron ions,...
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The effect of amino acid repertoire on protein structure and function
Treťjačenko, Vjačeslav ; Hlouchová, Klára (advisor) ; Obšil, Tomáš (referee) ; Pačes, Jan (referee)
To understand protein structure emergence is to comprehend the evolutionary transition from messy chemistry to the first heritable molecular systems. Early proteins were probably flexible in structure, promiscuous in activity and ambiguous in sequence. Moreover, first sequences were presumably composed of prebiotically plausible amino acids from endogenous and exogenous sources which form only a subset of the extant protein alphabet. Here we investigate the effect of most recent additions to the amino acid alphabet on protein structure/function relationship and the properties of random proteins as the evolutionary point-zero for the earliest sequences as well as for proteins emerging de novo from the non-coding parts of the genome. Random or never born proteins are of a special interest for the contemporary biology as they unveil the unexposed side of the protein sequence space. We constructed an in silico library of random proteins with the natural amino acid alphabet, analyzed its structure/disorder/aggregation content and selected 45 sequences for subsequent experimental preparation and biophysical characterization. We observed that structure content in random sequence space does not differ significantly from the natural proteins. However, the analyses of the aggregation propensity showed a...
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Recombinant expression and functional characterization of plant Kunitz inhibitors
Rybáriková, Renata ; Mareš, Michael (advisor) ; Hlouchová, Klára (referee)
PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...
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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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