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The basis of the structural-functional study of selected enzymes from the 4-alkyl- L-proline pathway
Zachovalová, Veronika ; Zdvořáková, Lucie (advisor) ; Košek, Dalibor (referee)
4-alkyl-L-proline derivate (APD) is building block of some important, bioactive specialized metabolites produced by Actinobacteria. It is found, for example, in the structure of lincosamide antibiotic lincomycin, bacterial hormone hormaomycin having antimicrobial activity, the antituberculosis compound griselimycin and pyrrolo-1,4-benzodiazepins (PBD) which have anticancer effects. APDs, or their precursors, are formed from L-leucine or L-tyrosin in a unique biosynthetic pathway consisting of up to six homologous proteins named Apd1 - Apd6. The first part of this thesis lays the foundation for a crystallization study of SAM-dependent methyltransferase Apd3 from the biosynthesis of lincomycin - the protein LmbW, in order to elucidate the identity of its natural substrate, which remains up to date a subject of scientific discussions. A purification method providing LmbW protein of sufficient quantity, quality and purity for crystallization was optimized. Subsequently the crystallization conditions allowing crystal growth were defined. The second part of the thesis deals with the elucidation of molecular basis of different reaction specificity of homologous F420H2-dependent oxidoreductases, Apd6. They catalyse reduction of identical substrate with two conjugated double bonds, however, some of them...
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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 contructs for transgenic expression of DPP-IV and FAP
Košek, Dalibor ; Bezouška, Karel (advisor) ; Křepela, Evžen (referee)
Preparation of contructs for transgenic expression of DPP-IV and FAP Bc. Dalibor Košek Abstract: DASH (Dipeptidyl peptidase-IV Activity and/or Structure Homologues) protein group involves multi-funcional molecules typically bearing enzymatic activity similar to the Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with lymphocyte differentiation antigen CD26). In general, they cleave multiple regulatory as well as structural peptides and proteins, possessing proline residue on the penultimate position from the N-terminus. We focused on two members of this group: canonical DPP-IV and Fibroblast activation protein alpha (FAP-α). Both are typically type II plasma membrane proteins with specific tissue distribution. Soluble extrecellular forms have also been identified. Available knowledge suggest important roles of these proteins in oncogenesis, executed by their enzymatic activity but also by non-proteolytic interactions. To study their role in gliomagenesis we designed several experimental models exploiting astrocytoma cell lines with defined DPP-IV or FAP-α phenotype. Enzymatically inactive forms and analogues with different subcellular distribution will also be included. These models will allow to assess the impact of DPP-IV and FAP-α on the glial tumor development and the importance of their...
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Expression of recombinant form of nepenthesin I from Nepenthes gracilis.
Tretyachenko, Vyacheslav ; Man, Petr (advisor) ; Košek, Dalibor (referee)
Nepenthesin is an aspartic proteinase from pitchers of genus Nepenthes and Drosera. Native form is characterised by high thermostability and endurance in wide pH range, properties required for digestive enzymes in preparational stage of hydrogen/deuterium exchange mass spectrometry experiment. Despite of this characteristics, it was shown, that the enzyme has relatively low stability in presence of chaotropic agents (urea, guanidinium chloride). The functional analysis based on revealed structure is necessary to find the reason for this inconsistency. X-ray studies of protein were not possible, mainly in cause of protein glycosylation and low extraction yields of enzyme in pitcher plants. The aims of this project is production of an active recombinant form of nepenthesin for its common utilization in H/D based mass spectrometry analysis and for Xray crystallography based structure solution. (In Czech)
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Preparation of the transcription factor FOXK1 DNA binding domain
Procházková, Valérie ; Novák, Petr (advisor) ; Košek, Dalibor (referee)
Transcription factors are proteins that regulate gene expression in different cell types. They play an important role in many cellular processes including regulation of cell cycle and cell differentiation. They possess DNA binding domains to recognize and bind specific DNA sequences. One type of DNA binding domain is the forkhead domain, which contains a region of 100-110 amino acid residues. This sequence is referred to DBD FOX and its spatial arrangement resembles a "winged helix". Proteins of the FOX family interact with double- stranded DNA via the α-helix H3, which represents highly conserved region within the proteins of this family. Other regions of the DBD further contribute in DNA binding, but as not significantly conserved, and their different properties are responsible for variable affinities of individual FOX proteins against binding motifs. Differences in three-dimensional structure may also alter biological functions of FOX proteins in the organism. FOX proteins are divided into 19 subfamilies, including the FOXK subfamily, consisting of two members, FOXK1 and FOXK2. FOXK proteins regulate aerobic glycolysis, cell proliferation and carcinogenesis. Their increased expression has been reported in cancer cells of skeletal tissue, stomach, colon, breast, lung, ovary, etc. However, the...
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Involvement of cellular signal systems controlled by small G-proteins in neuroregenerative processes
Dušek, Jakub ; Novotný, Jiří (advisor) ; Košek, Dalibor (referee)
G-proteins repeatedly seem as significant signal mediators according to actual "cellular signal during neuroregeneration" reviews. This thesis aimed to find out how much small G-proteins correlate with neural tissue regeneration and which molecular mechanisms induce the regeneration. The thesis mentions pathologies of nervous system which are caused by traumas or neurodegenerative diseases in wider social perspective. Pivotal part of the text describes principles of functioning for each small GTPase family and its involvement in nervous tissue repair. This thesis demonstrates crucial role of small GTPases in neuroregeneration and points to considerable therapeutic potential of these GTPases. Key words: neuroregeneration; small GTPase; small G-protein; Ras; signal systems; neurodegeneration
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Role of protein-protein interactions in regulation of signalling proteins and enzymes
Košek, Dalibor ; Obšil, Tomáš (advisor) ; Karpenko, Vladimír (referee) ; Pompach, Petr (referee)
EN Protein-protein interactions have an exceptional position among other mechanisms in the regulation of signal transduction. Their systematic investigation is very important and logical step in the process of understanding to the transduction and its mechanisms at a molecular level. During my Ph.D. I was particularly interested in three important processes. ASK1 kinase is well-known initiator of the apoptosis. Under physiological conditions it is maintained in an inactive state by its two interaction partners the 14-3-3 protein and TRX1. These two proteins dissociate in the presence of reactive oxygen species by unclear mechanism and the kinase is therefore activated. The next process is an interaction between the 14-3-3 protein and phosducin and investigation of their role in the G protein signalling especially important in the biochemistry of vision. The third process is an activation of protein Nth1 through the interaction with Bmh1, yeast analog of the 14-3-3 protein, and calcium cations. I employed various biophysical method, particularly analytical ultracentrifugation, in order to explain molecular mechanisms of described processes. These techniques were used to solve the low-resolution structures of complexes TRX1 and the 14-3-3 protein with corresponding binding domains of ASK1. These...
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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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Expression of recombinant form of nepenthesin I from Nepenthes gracilis.
Tretyachenko, Vyacheslav ; Man, Petr (advisor) ; Košek, Dalibor (referee)
Nepenthesin is an aspartic proteinase from pitchers of genus Nepenthes and Drosera. Native form is characterised by high thermostability and endurance in wide pH range, properties required for digestive enzymes in preparational stage of hydrogen/deuterium exchange mass spectrometry experiment. Despite of this characteristics, it was shown, that the enzyme has relatively low stability in presence of chaotropic agents (urea, guanidinium chloride). The functional analysis based on revealed structure is necessary to find the reason for this inconsistency. X-ray studies of protein were not possible, mainly in cause of protein glycosylation and low extraction yields of enzyme in pitcher plants. The aims of this project is production of an active recombinant form of nepenthesin for its common utilization in H/D based mass spectrometry analysis and for Xray crystallography based structure solution. (In Czech)
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