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Structural characterization of biotechnologically and medicinally important proteins.
Filandr, František ; Man, Petr (advisor) ; Šebela, Marek (referee) ; Škultéty, Ľudovít (referee)
A large number of biological processes depends on dynamics of protein structure and specific protein-protein and protein-ligand interactions occurring under specific native conditions in or outside of cells. Standard methods for protein structure analysis like x-ray crystallography, nuclear magnetic resonance or cryo-EM are able to obtain important atomic or near- atomic resolution protein structures, however these are usually a static snapshot of protein locked in a specific conformation and mostly in non-native conditions. Structural mass spectrometry on the other hand, allows to describe protein structure dynamics, protein-protein and protein-ligand interactions and obtain inter- and intraprotein distance constraints between amino acid residues, all while working with proteins in their native conditions and needing only a fraction of sample. In this work, hydrogen/deuterium exchange mass spectrometry (HDX-MS) and classical proteomic approaches were used together with other methods to analyse biotechnologically important proteins of fungal cellulolytic system lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH) as well as plant-derived photosensitizer protein LOV2 with potential use in biologically targeted photodynamic therapy. These methods allowed us to follow...
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Regulation of C-MYC oncoprotein by natural drugs.
Filandr, František ; Novák, Petr (advisor) ; Flieger, Miroslav (referee)
Sesqiterpene lactones, a group of plant secondary metabolites which include Cnicin from Cnicus benedictus plant, have an anti-proliferative and anti-tumor effect on mammalian cells by activating specific signaling pathways while also generating oxidative stress. These factors combined drive tumor cell apoptosis. A few of these compounds have reached clinical trials and seem to be a promising chemotherapeutics. The focus of this work is to elucidate the effect of cnicin on C-MYC transcription factor and oncoprotein which is overexpressed in majority of tumor tissues, the effect of cnicin on DEAD-box RNAhelicase DDX3 and on the expression levels of several metabolic genes is also studied. Through the use of western blotting, immunodetection and qPCR it was found out, that cnicin is regulating the expression of C-MYC oncoprotein on both transcriptional and translational levels, while also lowering C-MYC protein stability probably through the effect on PIM-2 kinase. Cnicin is not affecting the total amount of DDX3 protein in cells, but it seems it is lowering its degradation rate. The possible transcriptional regulation of DDX3 by cnicin is still not clear and requires further research. With the use of LC-MS quantitative analysis and qPCR, it was found out that cnicin does not affect the metabolism of...
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Mechanism of anti-cancer activity induced by sesquiterpene lactones.
Filandr, František ; Novák, Petr (advisor) ; Grobárová, Valéria (referee)
One way to stop tumor growth in organism is to induce differentiation, apoptosis or necrosis of tumor cells. A class of chemicals known to induce apoptosis and inhibit proliferation in leukemia cells are sesquiterpene lactones. One of these lactones is cnicin, a bitter tonic used in liqueurs, found in the plant Cnicus benedictus. The mechanism of this inhibition, is not fully understood but certain signaling pathways are suspected, mainly the recently discovered Hippo signaling pathway which controls the organ size and apoptosis in mammalian cells. The core kinase of this signaling pathway is MST1/2 protein and its activation by sesquiterpene lactone cnicin resulting in cleavage of its active N-terminal domain is observed in this work. Also, the effect of cnicin on down-regulating main oncoprotein deregulated in leukemia cells C-MYC is studied. In addition the results of q-PCR also show significant down-regulation of anti-apoptotic BCL2 and MCL1 genes and cMYC oncogene. (In Czech)
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