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Preparation of constructs for protein isolation and its testing
Osadchuk, Olha ; Kostovová, Iveta (referee) ; Brázda, Václav (advisor)
This study is focused on describing of recombinant protein production. Protein p53 was chosen, as one of the most important tumor suppressor proteins, for studying this issue. The p53 protein is responsible for the gene regulation, control of cell cycle and DNA replication. P53 is the most mutated gene in human cancer. Several point mutations of p53 protein was chosen for work with. The theoretical part describes main properties of protein, expression systems, Gateway cloning system and methods of protein purification. In the experimental part are described the procedures of preparing of the expression vectors by Gateway technology, cell transformation and DNA plasmid isolation. Using cloning technology were prepared three expression clones, they were transformed into competent cells and after was done DNA isolation.
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P53 protein isoforms production and purification in the bacterial expression system
Vadovičová, Natália ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.
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Prediction of p53 Protein Binding Sites
Radakovič, Jozef ; Vogel, Ivan (referee) ; Martínek, Tomáš (advisor)
Protein p53 which is encoded by gene TP53 plays crucial role in cell cycle as a regulator of transcription of genes in cases when cell is under stress. Therefore p53 acts like tumor suppressor. Understanding the pathway of p53 regulation as well as predicting its binding sites on p53 regulated genes is one of the major concerns of modern research in genetics and bioinformatics. In first part of this project we aim to introduce basics from molecular biology to better understand the p53 protein pathway in gene transcription and introduction to analysis of prediction of p53 binding sites. Second part is about implementation and testing of tool which would be able to predict transcription factor binding sites for protein p53.
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Preparation and expression of p53 protein isoforms using the GATEWAY expression system
Wikarská, Monika ; Hrstka, Miroslav (referee) ; Brázda, Václav (advisor)
The TP53 gene can express protein p53 and 11 another isoform proteins N- and/or C-terminally truncated by using two promoters and alternative splicing. The p53 isoforms are found in both healthy and tumorous tissues, and are intensively studied in relation to cancer diagnosis, prognosis and treatment. In this work, the p53 isoforms were subcloned into expression vectors by LR reaction adapted from Gateway cloning system. The expression vectors were designed for protein production by bacteria E. coli strain BL-21. The constructs containing p53 isoforms were encoded together with two fusion proteins, glutathione-S-transferase and polyhistidine tag under the control of the same promotor for the affinity chromatography protein isolation. All the clones underwent Sanger sequencing for verification after homologous recombination. Sequencing confirmed the accuracy of the subcloned isoforms p53, 133p53, 160p53, p53 and 160p53 into an expression vector pDEST15-N6xHis-GST-GW-DEST. Protein 160p53 was expressed in BL-21 and isolated using both HIS and GST tag interacion. Isolation using HIS tag yielded in a higher protein concentration then the isolation mediated by the interaction of the glutathione-S-transferase.
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Infuence of natural and synthetic G4-ligands to p53 transactivatio
Perná, Kristýna ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
Secondary DNA structures, such as G-quadruplexes, occur in the promoters of human genes. Dysfunctions of these quadruplex structures have been observed in several cases of cancer, and therefore these structures are the subject of research for the design of new anticancer drugs. The p53 protein is an important regulatory protein in the process of cell cycle control and DNA repair. Mutations in the gene encoding this protein occur in more than 50 % of cancer cases. In this thesis, the influence of natural ligands binding to G-quadruplexes on the binding and activity of the p53 protein was investigated. The theoretical part of the thesis describes the structure and binding properties of p53 protein, the structure and role of G-quadruplexes and describes selected G4-ligands occurring in food - curcumin, quercetin, berberine and ellagic acid. The aim of the experimental part of this thesis was to determine the effect of these substances on the transactivation activity of the p53 protein in vivo based on their interaction with G-quadruplexes using yeast isogenic systems. The interaction between selected G-quadruplex structures and ligands was first verified in vitro using the ThT assay and circular dichroism.
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Optimization of p53 mutant protein isolation and its DNA binding properties
Osadchuk, Olha ; Zemanová, Jana (referee) ; Brázda, Václav (advisor)
Protein p53 je jednou z nejdůležitějších molekul v lidském těle. P53 reguluje celou řadu procesů v buňce, jako je například oprava DNA, buněčný cyklus nebo indukce apoptózy. Protein p53 je známý i jako „strážce genomu“. DNA vazebné schopnosti proteinu p53 jsou důležité pro normální vývoj a růst buňky. Mutace genu pro p53 mohou vést ke ztrátě jeho DNA vazebných vlastností a funkce nádorového supresoru, což muže způsobit rozvoj rakoviny. Teoretická část této diplomové práce je zaměřena na popis vlastností, funkce a mechanismus aktivace proteinu p53 a popis lokálních sekundárních struktur DNA. Hlavním cílem experimentální části byla produkce čtyř mutantních forem proteinů p53 a wild-type p53 proteinu a studium jejich vazebných vlastnosti s různými lokálními sekundárními strukturami DNA. Pomoci Gateway klonovacího systému byly připraveny čtyři expresní vektory, které byly použity pro produkci proteinů v bakteriálním expresním systému. Celkem byly úspěšně připraveny čtyři mutantní formy a wild-type p53 protein. Jejich vazebné vlastnosti byly studovány gelovou retardační analýzu. Výsledky naznačují různé DNA-vazebné vlastnosti wild-type p53 a studovaných mutantních forem tohoto proteinu. Všechny mutantní proteiny ztratily schopnost sekvenčně specificky vázat DNA, zatímco nespecifická interakce s DNA byla pozorována u tří ze čtyř mutantních forem. Jeden ze studovaných mutantních proteinů se vázal jenom na superhelikální formu DNA.
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Eucaryotic cells systems and their biotechnologycal applications
Porubiaková, Otília ; Obruča, Stanislav (referee) ; Vorlíčková, Michaela (referee) ; Brázda, Václav (advisor)
The submitted dissertation is divided into several parts. The first part deals with the interactions of the p53 protein and its isoforms with different potential DNA substrates under different experimental conditions. These are predominantly DNA and its non-canonical structural motifs, such as G-quadruplexes or cruciform structures, whose interactions have been studied in yeast isogenic systems or by in vitro methods. The seconds part deals with the bioinformatic analysis of the mentioned secondary DNA structures in different organismal groups, and the result is a set of publications that show their non-random distribution in the genome and their relationship with regulation. The last part of the work contains unpublished results, including the results of testing the effect of natural and synthetic substances on aging in model human cells.
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Analyses of inverted repeats localization in bacterial genomes
Šedý, Michal ; Zemanová, Jana (referee) ; Brázda, Václav (advisor)
Inverted repeats (IR) are common part of DNA of all living prokaryotic and eukaryotic organisms. Inverted repeats plays an important role in the regulation of basics cells processes. They are responsible for formation of cruciform structures. Inverted repeats also cause genomic instability and can be a source of numerous mutations. Cruciform structures can be recognized by DNA-binding proteins and can also act as a transcriptional regulators. Using the Palindrome Analyser tool, the frequency of IR and localization of inverted repeats in bacterial genomes was analyzed. The frequency of IR across the bacterial genome is variable. The frequency of short inverted repeats shows an approximately quadratic dependence on the %GC content in the genome with a minimum of about 50% of GC content. The localization of inverted repeats with respect to “annotated features” show a non-random distribution. The frequency of IR for most features is higher “outside” than “inside”.
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Effect of natural substances from tea on G-quadruplexes and binding and transactivation properties of p53 protein
Foltanová, Klára ; Řeháková, Veronika (referee) ; Brázda, Václav (advisor)
The tumor suppressor protein p53 belongs to the most important regulator of the cell cycle in case of DNA damage. Apart from stopping the cell cycle and inducing apoptosis, protein p53 interacts with other proteins and DNA. Mutation of the TP53 gene encoding the protein p53 are a common feature of human cancer. This Bachelor thesis investigates natural substances from tea which could play a positive role in the activation of protein p53 in cancer and be used to support the treatment of these diseases. In the theoretical part of the Bachelor thesis, the secondary structures of DNA were described, specifically the structure and properties of G-quadruplexes, as well as protein p53 and its binding activity to the secondary structure of DNA. Selected natural substances found in tea and food were described – gallic acid and apigenin. The aim of the experimental part of this work was to verify the ability of these substances to interact with G-quadruplexes in vitro and thus to stabilize them in the yeast model and to assess the subsequent effect on measuring the effect of protein p53 transactivation. The interaction of quaternary structures with G4 ligands was verified in vitro by using the ThT fluorescence assay and the luciferase reporter assay. It was found that G4 ligands at 30 M concentration after 20 hours of incubation did not show a significant effect on the tested yeast cultures. At a 60 M concentration of G4 ligands, an increase in protein p53 production was observed due to cellular stress caused by the presence of G4 ligands.
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Specific heme interaction modulates the conformational dynamics and function of p53
Sergunin, Artur ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Tumor suppressor p53 is one of the most studied proteins in terms of cancer and the mechanism of its formation. The general function of p53 is based on the transcriptional regulation of various genes, which can differently influence numerous cellular processes. Recent studies revealed a relationship between p53 and iron homeostasis within the cell. In particular, p53 was shown to interact with a molecule of heme, and this interaction ultimately disrupts the DNA-binding ability of p53 and promotes its proteasomal degra- dation. This work focuses on a detailed description of heme binding to the p53. For this purpose, we isolated two forms of p53, heme-free and heme-bound. We discovered that conformational dynamics of heme-free and heme-bound p53 differ, with the latter exhibi- ting a higher degree of flexibility. We also confirmed previous reports that heme indeed interacts with a cysteine residue in a specific manner. However, heme binding does not disrupt the oligomeric state of p53 or its native zinc binding ability. Finally, we showed that heme-bound p53 exhibits severely impaired DNA-binding ability as opposed to the heme-free form. Keywords: heme, sensor proteins, p53 protein, transcription factor, intrinsically disor- dered proteins
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