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Parasitic protease SmCB2 as a target for the treatment of schistosomiasis
Bakardjieva, Marina ; Mareš, Michael (advisor) ; Mikeš, Libor (referee)
Blood flukes of the genus Schistosoma are parasitic trematodes that cause schistosomiasis, a serious disease afflicting more than 240 million people. The proteolytic system of schistosomes is essential for their viability: it participates in important processes during host-parasite interactions such as food digestion, invasion and tissue migration. Thus, schistosomal proteases are promising molecular targets for therapeutic intervention in schistosomiasis treatment. The thesis focuses on the protease cathepsin B2 from S. mansoni (SmCB2) which has not been studied in detail so far in terms of biochemical properties and biological function. Recombinant SmCB2 was prepared using yeast and bacterial expression systems and was chromatographically purified. Using an in vitro activity assay, the first effective inhibitors of SmCB2 were identified which inhibited its proteolytic activity in submicromolar concentrations. Specific polyclonal antibodies against SmCB2 were prepared and used for immunomicroscopic localization of this protease on the surface of the parasite. ELISA analysis demonstrated that SmCB2 is a parasite antigen recognized by the host immune system in the mouse model of schistosomiasis. The thesis provides valuable information about SmCB2 as a potential target molecule for synthetic...
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Cathepsins L of Diplostomum pseudospathaceum cercariae
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Hartmann, David (referee)
This study is focused on cercarial cysteine peptidases of the trematode Diplostomum pseudospathaceum. It follows previous research which confirmed the presence of a 24kDa cysteine peptidase in cercariae biochemically and by mass spectrometry. It was postulated, that the function of this peptidase is histolytic, when cercariae penetrate the tissues. During an attempt to purify this peptidase and characterize its peptidolytic activity, it was found out that the cercarial homogenate containsmore different peptidases varying in their pI. Tests of peptidolytic activity and inhibition have shown that these peptidases are cathepsin L-like. They are active over a broad spectrum of pH with optima of activities in weakly acidicor neutral pH. Using degenerate primers based on conserved motifs of cysteine pepridases, partial sequences of three genes for cathepsin L of D. pseudospataceum (DpCL1, 2 a 3) were obtained. Then the complete sequences of DpCL2 and 3 genes and partial sequence (without 5'end) of DpCL1 were obtained by RACE PCR. To confirm function of these peptidases we tried to immunolocalize them. We assumed that they are localized in penetration glands. Preliminary results suggested that some of the cathepsins could be also localized in the gut of cercariae. For more detailed biochemical...
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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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Structural and functional analysis of cathepsin B1 from the blood fluke, Schistosoma mansoni
Jílková, Adéla ; Mareš, Michael (advisor) ; Obšil, Tomáš (referee) ; Mikeš, Libor (referee)
Schistosomiasis is a serious infectious disease that afflicts over 200 million people in tropical and subtropical regions. It is caused by Schistosoma blood flukes that live in human blood vessels and obtain nutrients from host hemoglobin, which is degraded by digestive proteases. Current therapy relies on a single drug and concern over resistance necessitates new drug development. In Schistosoma mansoni, cathepsin B1 (SmCB1) is a critical digestive protease that is a target molecule for therapeutic interventions. This thesis provides a comprehensive characterization of SmCB1 focused on structure-activity relationships and inhibitory regulation based on six crystal structures solved for SmCB1 molecular forms and complexes. SmCB1 is biosynthesized as an inactive zymogen in which the N-terminal propeptide operates as a natural intra-molecular inhibitor by blocking the active site. Detailed biochemical and structural analyses have identified a new and, so far, unique mechanism of SmCB1 zymogen activation through which the propeptide is proteolytically removed and the regulatory role of glycosaminoglycans in this process has been described. A study of SmCB1 proteolytic activity has revealed that the enzyme acts in two modes, as endopeptidase and exopeptidase, which makes it an efficient tool for host...
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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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Production of recombinant cathepsin C from human blood fluke
Illichová, Hana ; Konvalinka, Jan (advisor) ; Martínková, Markéta (referee)
Blood flukes of the genus Schistosoma cause schistosomiasis, a serious parasitic disease occurring in tropical and subtropical areas. Cathepsin C (EC 3.4.14.1) is a digestive enzyme of the blood flukes which participates in the degradation of hemoglobin through its dipeptidyl aminopeptidase activity. This enzyme is critical for metabolism of the parasite and represents a potential target for the development of antischistosomal drugs. Cathepsin C has not yet been studied in detail. This bachelor thesis is focused on the development of expression systems for production of recombinant cathepsin C of Schistosoma mansoni (SmCC). The yeast Pichia pastoris system was used for the expression of an inactive SmCC precursor (zymogen) whose proteolytic stability was analysed. Furthermore, the expression system for SmCC in the protozoan Leishmania tarentolae was employed, and four different SmCC constructs were prepared to optimize production.
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Mitochondrial proteases and their role in biogenesis of mitochondria
Krump, Ondřej ; Stibůrek, Lukáš (advisor) ; Pecina, Petr (referee)
Mitochondria are organelles of endosymbiotic origin responsible for many cellular functions, including bioenergetics, biosynthesis and apoptosis. Regulated protein turnover is crucial for proper mitochondrial function. It is controlled by cellular proteolytic system, especially by its mitochondrial part. This mitochondrial proteolytic system is comprised of severeal groups of proteases. The best characterized AAA+ proteases constitute hollow oligomeric complexes, in which the proteolytic domains are localized. Access to these domains is dependent on unfolding - an energy-consuming process driven by ATP hydrolysis mediated by ATPase domains of AAA+ protein. The main function of AAA proteases is proteolytic degradation of proteins, a part of quality control system of mitochondrial proteins. AAA proteases are localized freely in mitochondrial matrix (Lon and ClpXP), or anchorred in the inner membrane (i-AAA and m-AA). Processing peptidases cleave off the mitochondrial targetting sequences of nuclearly encoded mitochondrial proteins. Oligopeptidases cleave peptides produced by processing and proteolytical degradation to single amino acids. Incorrect function of various components of mitochondrial proteolytical system is implicated in several diseases, including certain forms of hereditary spastic paraplegia...
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Cathepsin L by parasites - occurrence, features, functions
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Cathepsines L are lysosomal cysteine endopeptidases with an universal function in protein catabolism. This work discusses present knowledge about their characteristics in the context of their specific function in parasites. Features and function differences are described in detail on molecular level. The emphasis is on the biochemical properties with resultant use of these enzymes. Cathepsines L of kinetoplastida, aplikomplexa, entamoeba and helmints (focused on Fasciola spp and Schistosoma spp) are each discussed in appropriate chapters. Key words: hydrolase, protease, cysteine peptidase, cathepsin L, lysosome, parasite
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Cathepsin C of the blood fluke Schistosoma mansoni
Oupicová, Irena ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Blood flukes of the genus Schistosoma cause schistosomiasis, one of the most serious parasitic diseases. Cathepsin C (EC 3.4.14.1) is a digestive enzyme of the blood flukes and a potential drug target. This cysteine protease has not been studied in detail yet. In this thesis, cathepsin C activity was detected in the extract of adult blood flukes S. mansoni and S. japonicum. Inhibitory specificity of these enzymes was determined using of set of selective inhibitors of mammalian cathepsins. Cathepsins C were visualized on SDS-PAGE gels with fluorescent proteomic probe. Furthermore, recombinant cathepsin C of S. mansoni (SmCC) was prepared by homologous expression in the yeast Pichia pastoris. (In Czech)
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The study of enzyme activities in compost
Pernicová, Iva ; doc.Mgr.Pavlína Pelcová, Ph.D. (referee) ; Voběrková, Stanislava (advisor)
This thesis deals with the determination of enzyme activities in compost for consideration their changes during the composting. The changes in the activity of particular enzymes serve as the indicators of the activity of microorganisms, which are found in the compost. In the practical part of the work the changes in activity of proteases, cellulases, lipases, dehydrogenases and ureases within 21 days of composting under laboratory conditions were determined. The change in pH was observed as well. The changes in the pH-values and all enzyme activities except ureases present in the first week the same trend associated with the use of available substrates. The changes in the activities of the key enzymes in compost under laboratory conditions were compared with the changes in activities in compost under real conditions, which is the indicator of the different composting process under the given circumstances.
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