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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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Effect of heme analogs on structure and conformational stability of cytochrome b5
Maroušková, Růžena ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Cytochrome b5 is a component of the system of mixed function oxidases (MFO system) participating in metabolism of many endogenous and exogenous substances. Its main function in MFO system is reduction of cytochrome P450. The first aim of the thesis was to employ the pulse proteolysis to evaluate the influence of heme analogs on structure and conformational stability of cytochrome b5. This method utilizes the cleavage of protein by nonspecific protease (thermolysin) what is active even in the medium with high concentration of urea. Four forms of cytochrome b5, reconstituted with protoporphyrine IX containing Fe3+ , Mn3+ , Cr3+ or Co3+ ions, were prepared using titration of apocytochrome b5 with hemin or its analogs. The stability of individual forms of cytochrome b5 was than compared with apocytochrome b5 using pulse proteolysis. Finally, the relative amounts of residual cytochrome b5 were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Key words: electrophoresis, proteolysis, cytochrome b5, stability. (In Czech)
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Analysis of substrate specificity and mechanism of GlpG, an intramembrane protease of the rhomboid family.
Peclinovská, Lucie ; Stříšovský, Kvido (advisor) ; Konvalinka, Jan (referee)
Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...
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Effect of heme analogs on structure and conformational stability of cytochrome b5
Maroušková, Růžena ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Cytochrome b5 is a component of the system of mixed function oxidases (MFO system) participating in metabolism of many endogenous and exogenous substances. Its main function in MFO system is reduction of cytochrome P450. The first aim of the thesis was to employ the pulse proteolysis to evaluate the influence of heme analogs on structure and conformational stability of cytochrome b5. This method utilizes the cleavage of protein by nonspecific protease (thermolysin) what is active even in the medium with high concentration of urea. Four forms of cytochrome b5, reconstituted with protoporphyrine IX containing Fe3+ , Mn3+ , Cr3+ or Co3+ ions, were prepared using titration of apocytochrome b5 with hemin or its analogs. The stability of individual forms of cytochrome b5 was than compared with apocytochrome b5 using pulse proteolysis. Finally, the relative amounts of residual cytochrome b5 were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Key words: electrophoresis, proteolysis, cytochrome b5, stability. (In Czech)
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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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Kinetic behavior of the NAD(P)H:Quinone oxidoreductase WrbA from Escherichia coli.
KISHKO, Iryna
This Ph.D. thesis addresses the structure-function relationship of the multimeric oxidoreductase WrbA with the principal aim being the explanation of the unusual kinetics of this enzyme in molecular terms, and thus getting an insight about its physiological role in bacteria. WrbA is a multimeric enzyme with FMN as a co-factor, catalyzing the oxidation of NADH by a two electrons transfer. Structure and function analysis of WrbA places this enzyme between bacterial flavodoxins and eukaryotic oxidoreductases in terms of its evolutionary relationship. The kinetic activity of WrbA was studied under varying conditions such as temperature, pH etc, and its kinetic mechanism was evaluated from parameters KM and Vmax and confirmed by product inhibition pattern experiments. Crystallization and proteolytic experiments also underpin the functional importance of the multimeric nature of WrbA and aid the understanding of the physiological role of this enzyme in molecular terms.
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