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Discovery of General Controů Nondereprssible 5 protein interactors using proximity labeling
Potapenkova, Uliana
Usually, proteins don’t act as isolated species, but they perform their function in complexes with other proteins. Identification of protein-protein interactions (PPIs) brings insights into regulation of plant developmental processes and their response to the environmental conditions. In recent years, several methods to study PPIs were developed, since they represent the key objectives of plant system biology research. This thesis is focused on the Proximity Labeling (PL) assay in order to study GCN5 protein interactors. GCN5 is a histone acetyltransferase that plays a crucial role in plants’ defense responses to various abiotic stresses. Studies of its interactors provide valuable information on the histone acetylation mechanisms and the way plants cope with adverse conditions. Here, the advantages and disadvantages of PL assay are summarized and compared with other methods of PPIs studies. In the experimental part of this work, GCN5 was cloned and plasmid for further biotin-based proximity labeling was prepared.
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Interaction of protein subunits SEC10 and SEC15 of the exocyst tethering complex
Bartáková, Anna ; Ryšlavá, Helena (advisor) ; Čermáková, Michaela (referee)
The exocyst is an evolutionarily conserved tethering complex involved in the regulation of the secretory pathway in eukaryotic cells. As an effector of Small GTPases, the exocyst contributes to efficient targeting of secretory vesicles to the sites of intense exocytosis at the plasmatic membrane through interactions with specific membrane phospholipids. The exocyst complex consists of eight subunits: SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84. Each subunit binds at least two other subunits, among them the interaction of SEC10 and SEC15 is crucial for the exocyst function. The question remains how this particular interaction is evolutionarily conserved across different plant taxonomic groups. SEC10 and SEC15 genes from the three evolutionary groups of plants (green algae - Klebsormidium nitens, mosses - Marchantia polymorpha, angiosperms - Arabidopsis thaliana) were cloned into yeast two-hybrid vectors for studying protein interactions. Testing the interaction of SEC10 and SEC15 exocyst subunits from different plant species in the yeast-two hybrid system showed that despite partially different protein sequences of the tested subunits, their interaction across different evolutionary groups of plants remains very well conserved.
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Characterization of Cbf11 and Mga2 interactions in the fission yeast
Grulyová, Michaela ; Převorovský, Martin (advisor) ; Čáp, Michal (referee)
Transcription factor Cbf11 belongs to the CSL protein family. The CSL protein family is well known for its function in Notch signalling pathway, however representatives in Notch- less fission yeast were discovered. Mga2 protein is a transcription regulator of triacylglycerol and glycerophospholipid metabolism. A crosstalk between Cbf11 and Mga2 was found. Cbf11 and Mga2 share target genes, and both are required for mitotic fidelity. This thesis aims to validate and characterize relationship between these transcription regulators. We show here that protein levels of Cbf11 and Mga2 change in response to presence of the other protein, as well as in response of nitrogen source. We also determine phylogenetic distribution of Cbf11 and Mga2 among Fungi, pointing to its connection. Using proteomic analysis of mga2 and cbf11 deletion strains we found that there is an overlap between proteins up/downregulated in these strains. Together, these results acknowledge crosstalk between Cbf11 and Mga2 proteins, bringing a novel connection between CSL protein family member and a functional analogue of mammalian SREBP-1 protein Mga2.
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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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Physical interactions of the splicing factor Prp45
Kratochvílová, Eliška ; Folk, Petr (advisor) ; Doubravská, Lenka (referee)
It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.
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Interaction of Cytochromes P450 with Flavodoxin: a theoretical study
Culka, Martin ; Martínek, Václav (advisor) ; Chmelík, Josef (referee)
Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...
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Identification of crystal packing contacts using a method for detection of protein-protein interaction sites
Mitková, Natália ; Hoksza, David (advisor) ; Jakubec, Dávid (referee)
One of the fundamental issues when obtaining the protein quaternary structures is to be able distinguish biological interfaces from crystal packing contacts. The existence of these false contacts is the consequence of the crystallographic method which is used for obtaining the tertiary and quaternary structure. Gaining knowledge of how proteins interact in 3D space is key to understanding their functions and offers the opportunity to apply the knowledge to inhibitor impact analysis, drug design, immunotherapy, or de novo protein design. Furthermore, this knowledge is an essential requirement when applying in silico approaches. The bachelor thesis is divided into two parts. The first part contains literature retrieval of existing methods addressing the problem of crystallographic contact detection together with relevant datasets for an objective evaluation. The second practical part is focused on the comparison of evaluations of the previously developed method for the protein-protein interface prediction, INSPiRE, and the EPPIC classifier. Key words: bioinformatics, crystal packing contact, crystallography, protein structure, protein- protein interactions
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Applications of graph theory in protein function prediction
Kalábová, Nikola ; Hartman, David (advisor) ; Kratochvíl, Miroslav (referee)
The rapid development of the whole-genome sequencing methods and their reducing cost resulted in a huge number of sequenced genomes. Developing reliable methods for in- silico annotation of the expeditiously growing number of sequenced genomes is the next challenge of modern biology. We described a graph-theoretical approach for function prediction from the protein-protein interaction networks and outlined its strengths and weaknesses. We illustrate the principles of this approach on selected algorithms based on different ideas and provide their comparison and evaluation. 1
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Major capsid protein of polyomaviruses and its interactions with nuclear lamins
Žáčková, Sandra ; Horníková, Lenka (advisor) ; Šroller, Vojtěch (referee)
In this study, we focused on interactions of structural proteins of mouse polyomavirus (MPyV) and BK virus (BKV) with the nuclear lamina. Our goal was to examine whether and how can the virus, hence viral structural proteins, interact with the nuclear lamina and how would these interactions affect its properties. We supposed, that the expression of viral proteins would induce disintegration of the structure of nuclear lamina, thus enabling nuclear egress of virions in the late phase of infection. Viral structural proteins were expressed transiently in cells transfected with an expression vector pMPyV LATE. In these cells, VP1 was localized in a likewise manner as it shows in infected cells - mostly in a perinuclear area. Concurrently, defects in staining of nuclear lamina were observed in these cells, similarly to infected cells. Also, another expression vector was used in our experiments, the pMPyV mut3 VP1 encoding for a mutated protein VP1. When transiently expressed in cells, the mutated VP1 protein showed mostly diffuse nuclear localization. However, we observed significant morphological deformations and defective staining of the nuclear lamina. These observations imply an important role of VP1 in mechanical and biochemical properties alterations of the nuclear lamina in transfected and...
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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