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Proteolytic enzymes of the blood fluke Schistosoma mansoni: pathobiochemistry and their use in biomedicine
Leontovyč, Adrian ; Mareš, Michael (advisor) ; Kašný, Martin (referee) ; Mikeš, Libor (referee)
Blood flukes of the genus Schistosoma are causative agents of the disease schistosomiasis, which affects more than 250 million people worldwide and together with malaria represents the most important parasitic infection. There is a high risk of resistance development against the only drug in use, therefore novel therapeutic approaches for schistosomiasis are intensively researched. Proteolytic enzymes of schistosomes are crucial for their survival in the host and thus are promising drug and vaccine targets. This thesis is focused on two proteases of the human blood fluke Schistosoma mansoni, which were produced as recombinant proteins and functionally characterized. The first one is serine protease SmSP2, which is localized at the surface of the adult worms and secreted into the blood of the host. It was identified as a vasodilatory and fibrinolytic agent, and its modulatory role in host-parasite interactions was proposed. The second one is cysteine cathepsin SmCL3, which is involved in the digestion of host blood proteins serving schistosomes as nutrients. Potent peptidomimetic inhibitors of SmCL3 were identified, and their antischistosomal activity was demonstrated in an assay with live parasites. The thesis provides new important information about S. mansoni proteases, their pathobiochemistry...
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Proteolytic systems of the blood fluke (Schistosoma mansoni).
Fajtová, Pavla ; Horn, Martin (advisor) ; Bařinka, Cyril (referee) ; Sojka, Daniel (referee)
Schistosomiasis is a serious parasitic disease caused by blood flukes of the genus Schistosoma. It is a global health problem with more than 200 million people infected and 750 million people at risk. Current therapy relies on a single drug, praziquantel, for which there are concerns of emerging drug resistance. Proteases of schistosoma are promising target molecules for the development of new therapeutic strategies against schistosomiasis. This work focuses on the comprehensive characterization of proteolytic systems of Schistosoma mansoni and determination of their role in the interaction with the human host. First, the major proteolytic activities secreted by individual developmental stages of schistosoma that parasitize the human body were classified using functional proteomics. This analysis demonstrated their complex and specific distribution with predominant serine and cysteine proteases and metalloproteases. Second, tegumental and digestive proteases, namely prolyl oligopeptidase and cathepsins B, C and D, were identified by chemical genomics as suitable target molecules for therapeutic intervention. Prolyl oligopeptidase was biochemically characterized using a recombinant protein, its effective inhibitors were developed as templates for antischistosomal drugs, and a biological role of the...
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Proteolytic system of blood flukes of the genus Schistosoma
Bakardjieva, Marina ; Mareš, Michael (advisor) ; Dvořák, Jan (referee)
Blood flukes of the genus Schistosoma are parasitic trematodes causing a disease called schistosomiasis, which afflicts more than 200 million people in the tropics and subtropics. Adult schistosomes live in human blood vessels and feed on blood. Critical nutrients required for growth, development and reproduction of schistosomes are obtained from the major blood protein haemoglobin. Its digestion is mediated by the proteolytic arsenal of the schistosome digestive tract, which includes enzymes with complementary specificity belonging to the classes of cysteine and aspartic proteases, and metalloproteases. Proteolytic enzymes also play an important role in other processes, such as host penetration, tissue migration, immune evasion and modulation of inflammation. Here, serine and cysteine proteases importantly participate. The proteolytic system is essential for the viability of schistosomes and is a current topic of intense research focused on the development of new vaccines and chemotherapeutics for the treatment of schistosomiasis. Powered by TCPDF (www.tcpdf.org)
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Serine protease SmSP2 of Schistosoma mansoni
Leontovyč, Adrian ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Blood fluke Schistosoma mansoni is one of the most important human parasites. Proteolytic system of schistosoma is crucial for parasite - host interactions. Therefore some of the proteases became potential therapeutic targets. This work is focused on not yet characterized serine protease SmSP2. SmSP2 is newly discovered protease of S. mansoni, whose biological role is unknown. This protease is highly expressed in developmental stages parasitizing humans. SmSP2 was recombinantly expressed in prokaryotic and eukaryotic expression system (E. coli a P. pastoris) and purified using chromatographic methods. Recombinant SmSP2 was used for polyclonal antibody production. Conditions for refolding were optimized. Basic biochemical properties of the protease were detected and substrate amino acid preferences for P1 - P4 sites for single aminoacids were identified using synthetic fluorogenic peptides for positional scanning substrate combinatorial library (PS-SCL). (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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