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The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan ; Honys, David (advisor) ; Paleček, Jan (referee) ; Smýkal, Petr (referee)
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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New molecular mechanisms involved in cell cycle control
Aquino, Cecilia ; Macůrek, Libor (advisor) ; Anger, Martin (referee) ; Braun, Marcus (referee)
Cecilia Aquino Perez, M. Sc. Doctoral thesis abstract In this doctoral, thesis we aimed to find and study novel mechanisms regulating cell cycle phase transitions in non-stressed conditions and in context of the cell response to various types of stress. First, we focused on studying Polo-like kinase 3 that has previously been implicated in activation of the cell cycle checkpoint after DNA damage. For this, we employed CRISPR/Cas9- mediated gene editing to knock-out PLK3 in RPE cells while in parallel performing RNA interference assays and submitting the cells to different types of stress. The main observation was that in both systems PLK3 was disposable for response to DNA damage, hypoxia and osmotic stress. Through mass spectrometry analysis of purified EGFP-PLK3 we identified PP6 and its regulatory subunits PPP6R1 and PPP6R3 as novel PLK3 interactors. We observed that PLK3 is phosphorylated in its conserved residue Thr-219 and that PP6 depletion boosted PLK3 phosphorylation status but did not affect its kinase activity. The possible regulation of PLK3 trough PP6 is interesting and its biological relevance will be addressed by future research. Next, we performed a transcriptomic analysis in human RPE-FUCCI cells aiming to identify new regulators of the cell cycle. We selected Family with sequence...
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Preparation and characterization of the catalytic domain of human protein kinase ASK1.
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Protein kinase ASK1 (apoptosis signal-regulating kinase 1) is a member of the mitogen- activated protein kinase kinase kinase (MAP3K) family and plays a crucial role in immune and stress responses. Since the increased activity of ASK1 has been linked to the development of several diseases including cancer, cardiovascular and neurodegenerative diseases, this enzyme is a promising target for therapeutical intervention in these pathologies. The molecule of ASK1 consists of 1374 amino acid residues, but catalytic activity possesses only a kinase domain located approximately in the middle of the molecule. The activity of ASK1 is regulated by interactions with various proteins including the 14-3-3 protein. This protein recognizes a phosphorylated motif around Ser966 at the C-terminus of the catalytic domain of ASK1. This binding interaction inhibits ASK1 through unknown mechanism. ASK1 under stress conditions, such as oxidative stress, is dephosphorylated at Ser966 and the 14-3-3 protein dissociates. This dissociation is then one of the factors that lead to the activation of ASK1. The aim of this diploma thesis was to prepare a complex of the catalytic domain of ASK1 with the 14-3-3 protein for subsequent structural studies. Both proteins were expressed in E. coli cells and successfully purified. In...
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Study of interaction between ASK1 kinase and thioredoxin.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
MAP kinase signaling cascade plays an important role in the cellular response to various stress stimuli from the external environment. This signaling cascade is divided into three levels: MAP kinase kinase kinases (MAP3K) phosphorylate and thus activate MAP kinase kinases (MAP2K) and those subsequently phosphorylate and thus activate MAP kinase (MAPK) pathway, which regulates many cellular functions such as apoptosis, cell differentiation and morphogenesis. One of the important MAP3K is protein kinase ASK1 (Apoptosis signal-regulating kinase 1), which is an important regulator of cellular immune and stress responses. Given that the increased activity of ASK1 is related to the development of serious diseases such as cancer, cardiovascular and neurodegenerative diseases, ASK1 is an interesting target in the pharmacy in the development of new drugs. Human ASK1 consists of 1374 amino acids and is divided into three domains: a central Ser/Thr catalytic domain and two coiled-coil domains, of which the first is located at the N- and the second at the C-terminus of the molecule of this protein kinase. ASK1 is regulated by its binding partners, which include a small cellular redox protein thioredoxin (Trx-1), which binds to the N-terminal part of ASK1. Trx-1 is a potent antioxidant and so it protects cells...
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In search of DUSP specificity
Sladeček, Stanislava ; Novotný, Marian (advisor) ; Martínková, Natália (referee)
Dual specificity phosphatases (DUSP) are enzymes that dephosphorylate both phosphoserine/threonine and phosphotyrosine residues on one substrate. Most of them specifically dephosphorylate family mitogen-activated protein kinases (MAPK). Number of DUSPs increases with complexity of organisms and in human genome there are 25 DUSPs described. Some DUSPs can dephosphorylate only one protein while other interact with wider spectrum of substrates. Except for substrate specificity DUSPs differ in expression, subcellular localization etc. Although first DUSPs were described about 20 years ago, a clear factor responsible for their substrate specificity is not known. This works uses in silico methods to discover and describe similarities and differences between DUSPs which may be important in determining DUSP specificity. Key words: phosphatase, kinase, DUSP, MAPK, substrate specificity, conservation of residues, phylogenetic tree, in silico methods
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The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
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The Role of Lck Kinase in T-cell Antigen Receptor Signaling
Němec, Dušan ; Štěpánek, Ondřej (advisor) ; Rösel, Daniel (referee)
LCK activity is crucial for the triggering of the entire T cell activation process. The primary function of LCK is to convert the signal of TCR:pMHC ligation into the intracellular environment. The outcome of the LCK-triggered pathway is T cell activation, cytokine production, differentiation, and clonal expansion. This thesis provides a summary of recent knowledge about the unique position of LCK in the T cell signaling machinery as well as an overview of molecules and interacting partners that regulate LCK activity. It describes the importance of the LCK-coreceptor association for optimal TCR signaling and physiological thymocyte development and mentions discussed adaptor role of LCK in the T cells. Keywords: LCK, T-cell, antigen, kinase, enzyme
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Counterbalances: antagonistic regulation of fission yeast growth and proliferation under favourable conditions and stress
Hohoš, Patrik ; Převorovský, Martin (advisor) ; Groušl, Tomáš (referee)
Microorganisms come across dramatically changing conditions in the environment. It is important for them to be agile for a quick and effective response. Signal transduction pathways are essential for this ability. They can sense a broad spectrum of extracellular and intracellular stimuli and regulate a great number of processes in the cell. For unicellular microorganisms, the most essential ability is to sense environmental conditions for proliferation or abnormal stress conditions. One of the most popular model microorganisms, the fission yeast Schizosaccharomyces pombe, is used for the signal transduction pathways research. Findings obtained by research on the fission yeast are applicable to other eukaryotic organisms, thanks to the high conservation of the signal transduction pathways between the fission yeast and other eukaryotic organisms. Proliferation-promoting signal transduction pathways promote cell proliferation, growth and mitotic cell cycle in fission yeast. The stress-response signal transduction pathways play an opposite role. They promote cellular defence against stress stimuli and promote the sexual differentiation process alongside meiotic cell cycle. At first sight, the whole machinery may look like a switch mechanism. There is, however, a more complex crosstalk mechanism...
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Characterization of selected properties of model heme-containing sensor proteins
Fojtík, Lukáš ; Martínková, Markéta (advisor) ; Svášková, Dagmar (referee)
Heme sensor proteins are the fourth group of hemoproteins. In this group of hemoproteins heme plays an important role in signalization. Dissociation and/or association of heme detecting proteins serves as an important physiological function in regulation of enzyme activity or gene expression. In this bachelor thesis all the actual knowledge about selected forms of eukaryotic heme sensor proteins previously published in scientific articles are summarized. The experimental part of this bachelor thesis is focused on preparation of recombinant protein heme regulated inhibitor (HRI) and its substrate eukaryotic translation initiation factor 2 alpha (eIF2α). Firstly the preparation of the plasmids with genes HRI and eIF2α was conducted. In the next step these proteins were prepared in prokaryotic system formed by E. coli BL-21(DE3). The final sample of HRI (7,7 μM in total volume 400 µl and 60 % of homogenity) and the final sample of eIF2α (51,3 μM in total volume 400 µl and 80 % of homogenity) were obtained by the purification process. The study of thermal stability of these samples provided important informations on appropiate storage and manipulation with them in further experiments. Key words: heme-base sensors, heme, kinase, tranduction of signal, isolation of plasmids, prokaryotic expresion,...
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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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