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Regulatory mechanism of the cyclin-dependent kinase 16 through the cyclin Y/14-3-3 complex
Janáčková, Zuzana ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
CDK16 is a cell-cycle-related cyclin-dependent kinase that functions primarily in neurite growth processes, axonal transport regulation and sperm development. It also plays a role in the progression of X-linked intellectual disability and, together with its activating partner cyclin Y, in the development and progression of various types of cancer. Consequently, CDK16 and cyclin Y could represent potential new therapeutic targets. Both CDK16 and cyclin Y are structurally atypical among similar proteins, and the mechanism of activation of CDK16 by cyclin Y is unclear. However, it is clear from recent studies that the interaction between CDK16 and cyclin Y requires the prior interaction of cyclin Y with 14-3-3 proteins. In addition, for cyclin Y to interact with 14-3-3 proteins, its phosphorylation on the S100 and S326 binding sites of 14-3- 3 is required. To investigate the mechanism of activation of CDK16 by cyclin Y, we decided to characterize the interaction between CDK16, cyclin Y and 14-3-3 proteins in detail. The interaction between CDK16, cyclin Y and 14-3-3 proteins was characterized using biophysical methods including fluorescence anisotropy measurements, native electrophoresis, size exclusion chromatography and protein crystallography. The for- mation of binary pCCNY-14-3-3 and ternary...
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Heat-shock protein 90 (HSP90) in cell physiology.
Karmazin, Alina ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Heat-shock protein 90 (HSP90) is a molecular chaperone that represents one of the most important proteins for cellular homeostasis in all life domains. Chaperones are proteins that assist other proteins in proper folding and refolding. First discovered as a protein of a heat-shock response, HSP90 eventually emerged as a hub connecting multiple cellular functions, such as transcription, translation, DNA repair, immune response, cell signaling, etc. Unsurprisingly, HSP90 also plays a role in the pathogenesis of human diseases: various cancers, and neurodegenerative and respiratory diseases. For that reason, it became a target of medical research. HSP90 is a homodimer consisting of two protomers, each of which is composed of three domains: N-terminal domain, middle domain, and C-terminal domain. To fulfill its functions, HSP90 goes through an ATP-dependent conformational cycle, tightly regulated by a large group of assisting proteins-co-chaperones, and several post-translational modifications, such as phosphorylation and acetylation. Acetylation is known to affect HSP90 binding to nucleotides, clients, and co-chaperones, and thus it is suggested as a control mechanism of HSP90 function. Potentially, HSP90 acetylation can be utilized in the treatment of hormone-dependent cancers. Therefore, regulators of HSP90...
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Characterization of protein-protein interactions between Forkhead box O (FOXO) and p53 transcription factors
Mandal, Raju ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee) ; Pavlíček, Jiří (referee)
The transcription factor p53 plays a key role in cell cycle arrest, DNA repair, apoptosis, tumor suppression, and maintaining cellular homeostasis. Under cellular stress, p53 directly interacts with the Forkhead box O (FOXO) 4 transcription factor, thereby upregulating the expression of the p21 gene, resulting in the induction of cellular senescence. However, the detailed molecular mechanism behind FOXO4-p53 interaction remains unclear due to the unavailability of structural data. Therefore, main goal of this doctoral thesis was the characterization of the interaction between FOXO4 and p53 using several biophysical techniques including sedimentation velocity analytical ultracentrifugation (SV AUC), nuclear magnetic resonance (NMR) spectroscopy and chemical cross-linking coupled to mass spectrometry. Furthermore, we also investigated the DNA binding properties of both proteins with their respective consensus DNA sequences in the presence or absence of their binding partners by fluorescence anisotropy measurements along with the comparison of p53-binding surfaces of the Forkhead domain of three different FOXO proteins by NMR spectroscopy. In addition, we also optimized small molecule compounds for the inhibition of FOXO3-DNA interaction. Our results revealed that the p53 interacts with FOXO4 through...
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The effect of 14-3-3 protein on intradomain interaction of ubiquitin ligase Nedd4-2
Pohl, Pavel ; Obšilová, Veronika (advisor) ; Žáková, Lenka (referee) ; Pavlíček, Jiří (referee)
EN The human ubiquitin ligase Nedd4-2 (NEDD4L) ubiquitinates a wide range of membrane proteins and receptors, playing a key role in maintaining homeostasis. This enzyme is regulated by phosphorylation and subsequent interaction with 14-3-3 proteins, which primarily affects its ability to interact with various substrates. However, very little is known about the molecular basis of this protein-protein interaction. In this work, we focused on biophysical characterization of the role of individual phosphorylation sites and also on mapping the structural changes in the Nedd4- 2 protein induced by 14-3-3 protein binding. Our experiments using analytical ultracentrifugation methods revealed that two phosphorylation sites Ser342 and Ser448 are primarily required for stable binding of Nedd4-2 to 14-3-3 proteins. The crystal structure of the 14-3-3ηΔC:Nedd4-2335-455 T367A complex than revealed the simultaneous binding of both phosphorylated residues to the binding groove of 14-3-3 protein. Subsequent modeling based on small-angle X-ray scattering and chemical cross-linking data combined with mass spectrometry indicated extensive structural changes in the individual domains of the Nedd4-2 protein. Binding of 14-3-3η protein blocks the WW3 domain of Nedd4-2 in the central channel of 14-3-3 protein, while...
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Characterization of the binding interface between transcription factors FOXO4 and p53
Brzezina, Adam ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
This work deals with the study of human transcription factors FOXO4 and p53. FOXO4 is a member of the "O" subfamily of FOX transcription factors. Genes encoding FOXO proteins are evolutionarily conserved across species. FOXO transcription factors regulate the expression of genes involved in the control of metabolism, cell cycle and cell proliferation, cell survival and stress resistance. They are considered tumour suppressors because of their ability to arrest the cell cycle and induce apoptosis. However, their function in tumorigenesis appears to be more complicated, as recent studies indicate a poorer prognosis for the development of tumours that express higher levels of FOXO4. The p53 protein is a thoroughly studied naturally occurring tumour suppressor. The cellular response after its activation is somewhat similar to that of FOXO4, it can also block cell cycle progression or induce apoptosis depending on the cell type and severity/type of cellular stress. Both FOXO4 and p53 appear to be key molecules affecting aging. Under stress conditions, p53 and FOXO4 interact with each other and together increase the expression of p21 protein, thereby inducing the transition of cells to a senescent state. The accumulation of senescent cells is recognised as one of the main causes of ageing and the...
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Advanced methods of assaying lectin-carbohydrate interaction
Červený, Jakub ; Bosáková, Zuzana (advisor) ; Pavlíček, Jiří (referee)
A reliable and reproducible quantification and detailed characterization of the interactions between a biomedically relevant protein and its synthetic carbohydrate ligand can provide new information for the design of better diagnostic and therapeutic tools. A high sensitivity and specificity are essential requirements for the feasibility of an analytical method. These parameters can be difficult to achieve for highly complex systems such as lectin-saccharide complexes. On the other hand, carbohydrate-based diagnostics and therapeutics offer the great advantage of detecting biologically active lectin receptors, which cannot be achieved with antibodies. In this work we demonstrate the use of the BLI technique to monitor lectin- carbohydrate interactions for a multivalent system. Using functionalization of a novel construct of galectin-1 with an in vivo biotinylated AVI tag, suitable immobilization on a biosensor was achieved without the loss of its lectin activity. Due to the high sensitivity of this technique, kinetic parameters of interaction with prepared multivalent neoglycoproteins based on LacNAc epitopes were obtained in the micro- to nanolar KD range. The complementarity of this method was demonstrated by parallel ITC and competitive ELISA measurements. The neoglycoprotein with a lower LacNAc...
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Eukaryotic expression of acid proteases.
Vodolánová, Lucie ; Man, Petr (advisor) ; Pavlíček, Jiří (referee)
Nepenthesin I and neprosin are acid proteases which are present in the pitcher fluid of carnivorous plants genus Nepenthes. Both enzymes are produced as zymogens which are activated in acidic conditions. Due to their properties and specific preferences, they are suitable for protein mass spectrometry or as a supplement in gluten-free diet of celiac patients. Quantity of the enzymes in the pitcher fluid is very low and the currently developer expression protocols do not show satisfactory results, we tried to find more suitable expression system. We would like to produce high quantitites of enzymes, which will have the same stability as natural ones. The aim of this work is preparation of expression vectors with nepenthesin I and neprosin genes for transfection into eukaryotic expression systems of S2 and HEK 293 cells. In Czech. Keywords: acid proteases, nepenthesin I, neprosin, expression vector
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Modulation of DNA Binding Affinity of Transcription Factors FOXO and p53 Through Protein-protein Interactions
Hofmanová, Adéla ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract The forkhead box "O" (FOXO) proteins are a subclass of the Forkhead family of transcription factors that play a critical role in a variety of cellular processes such as response to cellular stress, gluconeogenesis, cell cycle control, apoptosis, senescence, and repair of DNA damage. They are generally considered to be tumor suppressors. However, it has been shown that they can promote tumorigenesis and induce resistance to the chemotherapeutic agents. Despite many years of research into the biological role of FOXO proteins, a number of questions remain to be answered. For example, whether the slight structural differences observed in the otherwise highly homologous DNA-binding domains of individual FOXO transcription factors affect their DNA binding affinity. Furthermore, it is unclear how protein-protein interactions affect DNA binding affinity of FOXO proteins. Recent study has described the interaction of FOXO transcription factors with the p53 protein. Protein p53 is called the guardian of the genome due to its ability to mediate the response to acute DNA damage. The interaction of FOXO and p53 proteins appears to have a major effect on the DNA binding affinity of both these proteins. Based on this, DNA-binding domains of the human transcription factors FOXO1, FOXO3 and FOXO4 (FOXO1(144-270),...
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Implementation of the Roofing of the Stable for Cattle in Studenec
Pavlíček, Jiří ; Bříza, Lukáš (referee) ; Mohapl, Martin (advisor)
The topic of the bachelor’s thesis is the elaboration of a construction technological project for the roofing of a cattle stable in Studenec. The work contains a technical report focusing on the installation of the roof structure, the construction situation with broader relations of transport routes, technological regulation for the roofing stage, item budget with area statement, site equipment design with technical report site equipment, time schedule, machine design. Furthermore, the work addresses the control and test plan, occupational safety. The last point of the thesis deals with the implementation of roofing of buildings with a larger span.
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