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The role of N-glycosylation in homooligomerization of natural cytotoxicity receptor NKp30
Tulpa, Matouš ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
NK cells play a key role in the defence against cells that have been infected by a virus, a protozoan or have undergone malignant transformation. In addition, they also regulate the activity and quantity of other cells of the immune system. Target cells are recognized using their activating and inhibitory receptors, from which they receive activating and inhibitory signals, on which the cytotoxic response of NK cells depends. There is a dynamic balance between the signals that determines the life and death of the target cell. If activation signals prevail, the target cell will be eliminated. If inhibitory signals prevail, then a cytotoxic response will not be triggered. The NKp30 receptor, which belongs to the immunoglobulin-like receptor superfamily, is an important activating receptor that recognizes a number of ligands, including hemagglutinin of vaccinia and ectromelia virus, human cytomegalovirus pp65 protein, B7-H6, BAG-6, and galectin-3. The extracellular domain of the NKp30 receptor is capable of homooligomerization in solution under certain conditions. The first requirement is the presence of N-glycosylation, the second requirement is the presence of a 15 amino acid long "stalk" domain that connects the ligand binding domain with the transmembrane α-helix. The aim of this thesis was to...
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Dynamical Behaviour of Matrix Proteins from Mason-Pfizer Monkey Virus
Srb, Pavel ; Lang, Jan (advisor) ; Žídek, Lukáš (referee) ; Brus, Jiří (referee)
Title: Dynamical behaviour of matrix proteins from Mason-Pfizer Monkey Virus Author: Pavel Srb Department: Department of Low temperature physics Supervisor: doc. RNDr. Jan Lang, PhD. Abstract: We studied the oligomeric properties of betaretroviral nonmyristoylated ma- trix protein (MA) and its R55F mutant from the Mason-Pfizer monkey virus in solution by means of NMR spectroscopy. We have proven that the wild- type (WT) MA forms oligomers in solution. The final model of oligomeriza- tion of the WT MA was derived by concerted use of chemical shift mapping and diffusion-ordered spectroscopy measured on a set of protein samples with varying concentrations. We found that the Mason-Pfizer monkey virus WT MA exists in a monomer-dimer-trimer equilibrium in solution. Further a combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly de- tailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidence have been obtained that the oligomerization capacity of the wild-type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on a nanosecond time scale. In- creased flexibility has been observed for two regions: the loop between he- lices α2 and α3 and the C-terminal...
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Analysis of the intracellular localization of nucleophosmin: effect of C-terminal mutations
Kráčmarová, Markéta ; Brodská, Barbora (advisor) ; Čáp, Michal (referee)
C-terminal mutations of the phosphoprotein nucleophosmin (NPM) are the most frequent genetic aberration detected in adult acute myeloid leukemia (AML). I focused on characterization of type A, B and E of AML-related C-terminal mutations. The plasmids bearing fluorescently labeled wild type or mutated NPM have been constructed to characterize mutation-induced changes in the localization of NPM. Mammalian cell lines HEK293T, HeLa and NIH 3T3 were used for production of the chimeric proteins. The intracellular localization of the mutated forms of NPM was analyzed by immunofluorescence staining and fluorescence microscopy of the living cells. The localization of the mutNPM type A and B was almost identical and predominantly cytoplasmic, while mutNPM type E was detected in nucleolus and cytoplasm simultaneously. However localization of the mutated forms was greatly influenced by the used cell line. It has been demonstrated that the exogenous NPM interacts with the endogenous NPM and that they mutually affect their intracellular localization due to heterooligomer formation. Detailed analysis of the relationship between the C-terminal mutations and the localization of the mutated NPM improves understanding of specific mutation effect on the formation and progression of AML and also specifies its prognostic...
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Study of NKp30 oligomerization and its interaction with B7-H6
Pažický, Samuel ; Vaněk, Ondřej (advisor) ; Man, Petr (referee)
NK cells are important part of immune system, recognizing and eliminating tumor cells and cells infected by viruses. For the target cell recognition, binding of ligands by activating receptors plays a crucial role. Activating receptor NKp30, protein of family of natural cytotoxicity receptors, is able to bind multiple ligands either present on tumor cell surface or being part of some viruses. B7-H6 is one of the ligands of NKp30 and its specific constitutive expression on some tumor cells and cell lines makes it an interesting biological target. Although the NKp30/B7-H6 complex structure has been solved, structural basis of some important features of their binding is not explained yet. Soluble form of NKp30 receptor binding domain creates oligomers, presence of which is dependent on C-terminus length of its domain and its N-glycosylation; however, structural insight into formation of the oligomers and their significance is not known. Furthermore, binding affinity of NKp30 to its ligands is dependent on presence of its glycosylation and glycosylation type. We have already found out that NKp30 oligomerization is dependent on its glycosylation. In my work, I attempted to gain detailed functional and structural information about oligomerization of NKp30 and its binding to B7-H6 by multimethodical...
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Preparation of glycosylated form of human immunoreceptor NKp30
Kalousková, Barbora ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
NK cells (natural killer cells) play a key role in innate immunity. Their function is to recognize and kill infected, stressed or malignantly transformed cells. A range of surface receptors promotes this recognition. Cytotoxic mechanisms, lead to induction of apoptosis in the target cell. Receptor NKp30 is one of cytotoxic reaction triggers. It belongs, with NKp46 and NKp44, to NCR (natural cytotoxicity receptors) family. This work describes preparation of NKp30 receptor with natural and simple glycosylation in expression system of human embryonic kidney cell line 293 (HEK293). It was found that glycosylated receptor NKp30 forms noncovalent oligomers. Equilibrium is formed in solution between oligomers and monomers. Oligomerization depends on glycosylation, deglycosylated protein doesn't form oligomers. A recombinant endoglycosidase ENDO F1 was prepared for purposes of deglycosylation.
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The role of protein kinase StkP in regulation of the cell division in Streptococcus pneumoniae
Malíková, Eliška ; Doubravová, Linda (advisor) ; Kuthan, Martin (referee)
Protein phosphorylation by protein kinases is a key mechanizm that enables both eukaryotic and prokaryotic organizm sense and read environmental signals and convert these signals into changes in gene expression and thus proper biological response. One of the main phosphorylation systems in bacteria consists of eukaryotic-like Ser/ Thr protein kinases. The genome of human pathogen Streptococcus pneumoniae contains single Ser/ Thr protein kinase StkP. StkP regulates virulence, competence, stress resistance, gene expression and plays an important role in the regulation of cell division cycle. Analysis of phosphoproteome maps of both wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the cell division protein DivIVA (NOVÁKOVÁ et al., 2010). DivIVA in S. pneumoniae is localized at midcell and at the cell poles. It was proposed to be primarily involved in the formation and maturation of the cell poles (FADDA et al., 2007). The aim of this thesis was to investigate phosphorylation of the cell division protein DivIVA in S. pneumoniae. Gene divIVA was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of DivIVA by StkP was examined in a kinase assay. We confirmed that DivIVA is a direct...
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Dynamical Behaviour of Matrix Proteins from Mason-Pfizer Monkey Virus
Srb, Pavel ; Lang, Jan (advisor) ; Žídek, Lukáš (referee) ; Brus, Jiří (referee)
Title: Dynamical behaviour of matrix proteins from Mason-Pfizer Monkey Virus Author: Pavel Srb Department: Department of Low temperature physics Supervisor: doc. RNDr. Jan Lang, PhD. Abstract: We studied the oligomeric properties of betaretroviral nonmyristoylated ma- trix protein (MA) and its R55F mutant from the Mason-Pfizer monkey virus in solution by means of NMR spectroscopy. We have proven that the wild- type (WT) MA forms oligomers in solution. The final model of oligomeriza- tion of the WT MA was derived by concerted use of chemical shift mapping and diffusion-ordered spectroscopy measured on a set of protein samples with varying concentrations. We found that the Mason-Pfizer monkey virus WT MA exists in a monomer-dimer-trimer equilibrium in solution. Further a combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly de- tailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidence have been obtained that the oligomerization capacity of the wild-type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on a nanosecond time scale. In- creased flexibility has been observed for two regions: the loop between he- lices α2 and α3 and the C-terminal...
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