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Digestive proteases of termites
Čermáková, Markéta ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)
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Structural characterization of interaction between transcription factors and DNA
Filandrová, Růžena ; Novák, Petr (advisor) ; Vondrášek, Jiří (referee) ; Wimmerová, Michaela (referee)
Structural characterization of interaction between transcription factors and DNA Mgr. Růžena Filandrová Abstract Transcription factors are proteins that mediate gene expression regulation through interactions with DNA and other factors. They allow a cell to respond to various stimuli and play a crucial role in many biological processes such as control of cell cycle progression, differentiation of cells during development or immune response. To understand these processes, the knowledge of the transcription factors 3D structure together with the mechanism of their interaction with DNA is essential. However, some of the typical features of transcription factors, such as is for example the presence of intrinsically unstructured regions, make the 3D structure determination by the commonly used high resolution methods challenging. Therefore, utilization of complementary methods like structural mass spectrometry (MS), which was used in this thesis, might prove to be beneficial to explore the structural basis of the transcription factor-DNA interaction. In first part of this work, a set of structural mass spectrometry methods with the main focus on hydrogen/deuterium exchange mass spectrometry (HDX-MS) was optimized and tested on two transcription factor-DNA complexes and their DNA binding motifs and proved to be...
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Study of enzymes secreted by oomycete Pythium oligandrum
Hrdinová, Karolína ; Ryšlavá, Helena (advisor) ; Kubíčková, Božena (referee)
Pythium oligandrum is one of the non-pathogenic microorganisms of Pythium species which found its use as biological control agent. The main modes of action of this oomycete lie in inducing the plant immunity and in mycoparasitism of pathogenic fungi and fungus-like organisms. The oomycete attacks the pathogens by secretion of hydrolytic enzymes into the environment. In the first part of this bachelor's thesis, activity of hydrolytic enzymes endo-1,3-β-glucanase, cellulase, chitinase and proteases was observed in a commercial product based on Pythium oligandrum, called Polyversum-Biogarden. It was conclu- ded that the direct hydrolysis of phytopathogens is probably not the main mechanism of this product because the activity of glycosidases increased only after six hour-long incubation of the Polyversum-Biogarden in water and the proteolytic activity was not detected. In the second part of the bachelor's thesis, properties of proteases secreted by orga- nism Pythium oligandrum were studied. The highest proteolytic activity was observed at pH 6,5. Ovomucoid acted as an inhibitor of secreted proteases. The stability of pro- teases was lowered by SDS, detergents present in liquid soaps and by a solution of a solid soap or NaOH. Only the highest concentrations of urea lowered the activity of proteases....
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Modulation of HIV-1 Protease Activity
Pokorná, Jana ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Ruml, Tomáš (referee)
HIV-1 protease plays a crucial role in the late state of the life cycle of HIV virus when it cleaves the viral polyprotein precursors into the structural and functional proteins. If it is effectively inhibited, HIV particles remain immature and noninfectious. The application of highly active antiretroviral therapy (HAART) including protease inhibitors can reduce plasma HIV-1 levels below the detection limit in adherent patients and thus dramatically change their life expectancy. The clinical utility of the first inhibitors was limited by severe side effects, low bioavailability, high pill burdens, and rapid development of viral resistance under the selection pressure of HIV antiretrovirals. To overcome these difficulties, second-generation inhibitors were developed. Despite an indisputable improvement they brought to antiretroviral therapy, the development of new highly active HIV-1 protease inhibitors with optimal pharmacokinetic properties, higher metabolic stability, little off-target activity, and particularly, more favorable resistance profiles is still of high importance. This thesis provides an overview of anti-HIV- drugs including development of substituted metallacarboranes, a new class of potent, unusual, nonpeptidic HIV protease inhibitors with therapeutic potential. Next, the impact of...
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Digestive proteases of termites
Čermáková, Markéta ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)
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Prolyl endopeptidase of the blood fluke Schistosoma mansoni
Fajtová, Pavla ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Prolyl endopeptidase SmPEP from the blood fluke Schistosoma mansoni is investigated here for the first time. This enzyme is potentially interesting as a drug target for the treatment of schistosomiasis. SmPEP was detected in the extract of adult worms by enzyme activity and immunoreactivity. Enzymatically active SmPEP was produced in the E. coli expression system and was chromatographically purified. The pH optimum of recombinant SmPEP was about 8. Substrate specificity analysis revealed that SmPEP cleaved peptide substrates by endopeptidase activity, however, macromolecular substrates were not fragmented. The residue preferences in the positions P3 to P1' were determined using synthetic fluorogenic peptide substrates. SmPEP was found to be highly sensitive to the inhibition by Z-Ala-Pro-CMK and Z-Arg-Pro-CHO. Primary screening of crystallization conditions for recombinant SmPEP was performed. " (In Czech)"
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