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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
Characterisation of recombinant cathepsins B of the bird schistosome Trichobilharzia regenti
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
This study focuses on the recombinant cysteine peptidases - cathepsin B originating in the bird schistosome Trichobilharzia regenti that is unique across the whole family for its ability to migrate through the nerve tissue to the final localization. For invasion, migration, degradation of nutritional proteins and/or evasion of host immune responses, schistosome employs peptidases. This study follows the research done by researchers of Department of parasitology, Faculty of Natural Sciences, Charles University. The main goal of this study was to deepen the characteristics of recombinant cathepsins B originating in T. regenti. In T. regenti, two cysteine peptidases - cathepsins B1 (TrCB1) and B2 (TrCB2) - have been previously characterized. TrCB1 is located in the gut of schistosomula and involved in digestion. TrCB2 occurs in post-acetabular penetration glands of cercariae and probably facilitates penetration. The recombinant pro-cathepsin B (isoforms TrCB1.1, TrCB1.4 and also TrCB2) were expressed in Pichia pastoris yeast system. An attempt was made to produce in P. pastoris the recombinant isoform TrCB1.6, in which the active site cysteine is substituted by glycine. While TrCB2 underwent self-processing in the expression medium, TrCB1.1 and TrC1.4 zymogens were effectively activated only after the...
Peptidases of Trematodes
Kašný, Martin ; Mikeš, Libor (advisor) ; Kopáček, Petr (referee) ; Haas, Wilfried (referee)
90 3. SUMMARY The text above refers about the majority of characterized trematode peptidases; the fundamental enzymes for trematode existence, which are integrated in many physiological processes like pathogenesis, tissue invasion/migration, nutrition, immune evasion and host-parasite interactions. In the history (until 1996), the peptidase catalytic activities in trematode extracts have been monitored. During 1980s and 1990s, the information of first cloned trematode peptidase genes were published and during last three decades cca 90 trematode peptidase sequences belonging to 19 peptidase families of 5 clans have been identified. The most studied trematode peptidases have been of Schistosoma mansoni origin: the serine peptidase - cercarial elastase (of cercariae), cysteine peptidases - cathepsins B, L, F, C plus the asparaginyl endopeptidase SmAE and the aspartic peptidase - cathepsin D (of adult worms and some other life stages). The recent computational cluster analysis revealed that the sequence S. mansoni elastase (the main cercarial penetration enzyme) is quite divergent from other serine peptidases of the S1 family. Cercarial elastase gene was proved in S. mansoni, S. haematobium and Schistosomatium douthitti, but not in the related S. japonicum. Mass spectrometry analysis confirmed cercarial...
Acid peptidases of schistosomes and haematophagous monogeneans
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Horn, Martin (referee) ; Sojka, Daniel (referee)
Blood is a complex nutrient-rich mixture. No wonder that haematophagy has been adopted as a feeding strategy by many invertebrates, including many parasitic helminths. In general, processing of haemoglobin (and other blood proteins) in blood-feeding helminths relies on an evolutionary conserved network of cysteine and aspartic peptidases (e.g., cathepsins L, B and D). However, some helminth taxa have been neglected from this point of view - very little information has been available about the occurrence of these enzymes in haematophagous monogeneans. Therefore, the presented thesis focuses on the molecular and biochemical characteristics of peptidases that maybe potentially involved in blood processing by the monogenean Eudiplozoon nipponicum (Heteronchoinea, Diplozoidae), an ectoparasite inhabiting gills of common carp. We show that the most abundant haemoglobinolytic endopeptidase activities in soluble protein extracts and excretory/secretory products of E. nipponicum belong to the cysteine and aspartic classes, with cathepsin L-like activity predominating over cathepsin B-like activity and supplemented with cathepsin D-like activity (paper 1). Additionally, we found that E. nipponicum adults express a variety of cathepsins L with different structural characteristics and probably different...
Morphological and functional variability of secretory glands in cercariae of chosen trematode groups
Krčmářová, Veronika ; Bulantová, Jana (advisor) ; Mikeš, Libor (referee)
Trematodes are characterized by their complex life cycles that include definitive hosts and variable number of intermediate hosts. Transfer of the parasite from the first intermediate host to the other is usually realized by larval stage called cercaria. After finishing of their development within the first intermediate host, morphology and fate of these larval stages vary according to the way of infection of the next host. Some cercariae actively penetrate directly to their definitive hosts trough their body surface, other encystate in the outer environment where they wait in a form of metacercariae to be ingested by definitive host. Both of these ways can be combinated and cercariae encystate inside second intermediate host after they actively penetrate them. Exceptionally, cercariae do not leave the sporocyst in which they were developing inside the first intermediate host. They encyst there waiting for ingestion by the definitive host. Various types of secretory glands have been developed in cercariae for successful direct infection of next hosts, survival of parasite in adverse conditions of outer environment or for transformation of one larval stage to subsequent one. Variability in morphology and function of these secretory glands in cercariae is closely connected with differences in life...
Interactions between Schistosoma spp. and their hosts at the metabolome level
Kurečka, Martin ; Kameník, Zdeněk (advisor) ; Mikeš, Libor (referee)
The blood flukes of the genus Schistosoma are important parasites that cause serious chronic diseases in mammals, including humans, in tropical and subtropical countries. Treatment of these diseases is challenging; therefore, new molecular targets are still being sought for the development of vaccines and more effective drugs. To achieve this, better understanding of interactions between the parasite and the host at the molecular level is an important prerequisite. These processes can be studied by quantitative and qualitative determination of metabolite differences in healthy and infected individuals using metabolomics. The work represents a review of low molecular weight substances in tissues and body fluids of schistosome hosts, in which a change in concentration of metabolites putatively related to the infection was observed. Only metabolites with a hypothetical or known mechanism of these changes in the context of infection are covered. The thesis also includes a brief overview of basic methods of analytical chemistry, which are used in studies based on metabolomics. Key words: Schistosoma spp.; intermediate host; definitive host; spectrometry; metabolomics; low molecular compounds
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...
Biologically active compounds of selected model trematodes
Kurečka, Martin ; Kameník, Zdeněk (advisor) ; Mikeš, Libor (referee)
Trematoda are parasites known for their ability to manipulate their host for survival and reproduction. They have complex life cycles with the intermediate host represented by mollusks and the definitive host, represented by vertebrates. This work focuses on three medically important genera of trematodes: Schistosoma, Fasciola and Opisthorchis. The aim of this work is to summarize biologically active low molecular weight substances that parasites modulate or produce in order to manipulate their host. The result of the work is a literature research of a comparative change in the concentration of metabolites of infected and uninfected trematode hosts with a focus on the analytical method used. Metabolomics deals with a comprehensive analysis of the metabolism of biological samples. It uses spectrometric analytical methods such as nuclear magnetic resonance and mass spectrometry combined with gas or liquid chromatography. Part of the work is also a summary of the importance, development and perspectives of metabolic profiling in parasitology. Current research in this area focuses mainly on vertebrate hosts. In addition, for vertebrate hosts, the sum of studied substances is still much broader than that in intermediate hosts. In intermediate host studies also focus on different types of substances,...
Kunitz-type inhibitors in Eudiplozoon nipponicum
Černíková, Markéta ; Mikeš, Libor (advisor) ; Jedličková, Lucie (referee)
Proteins containing Kunitz domain are mostly inhibitors of serine proteases. Their general characteristic is the presence of three disulfide bonds and small sizes around 6-10 kDa, although sometimes they consist of several Kunitz domains or they are part of more complex proteins. Their function is usually related to the regulation of physiological and proteolytic processes, but also to an interaction with pathogens or other defense mechanisms, such as being part of the sea anemone mucus or the venom of snakes and other invertebrates. We focused on Kunitz proteins in Eudiplozoon nipponicum, a helminth of the class Monogenea parasiting on gills of common carp (Cyprinus carpio). In the transcriptome of this parasite, several sequences with Kunitz domain have been identified based on similarities with the one already described Kunitz protein, EnKT1, suggesting that this parasite, like other bloodfeeding parasites, uses a whole set of these serine protease inhibitors with other specific functions. Several sequences with the Kunitz domain found in the transcriptome were verified by PCR and optionally supplemented by RACE-PCR. One protein, called EnKC1, was subsequently produced by recombinant expression in E. coli cells of SHuffleTM and Rosetta Gami B strains. Recombinant protein with the Kunitz domain...
New inhibition mechanisms of regulation of cathepsin D activity
Hánová, Iva ; Mareš, Michael (advisor) ; Heidingsfeld, Olga (referee) ; Mikeš, Libor (referee)
The aspartic protease cathepsin D (CatD) is associated with numerous pathologies, and therefore the molecular mechanisms of its activation are studied for their potential uses in biomedicine. This dissertation thesis is focused on new, natural endogenous inhibitors of CatD, the analysis of their interaction, and the development of synthetic inhibitory biomimetics. Two groups of inhibitors of CatD, which are the first specific endogenous regulators of this enzyme, have been identified. (1) Sphingolipids are complex modulators of human CatD, depending on their structure. While sphingosines and ceramides are inhibitors of CatD, their phosphorylated derivates act as activators of CatD. A correlation was found between the action of these sphingolipids on CatD and their modulatory effect on cancer cells. (2) Using the analysis of a CatD of parasitic origin, a new mechanism of inhibition was identified, which is conserved in aspartic proteases of the pepsin family. A peptide fragment is released autocatalytically from the zymogen of CatD, which then acts as an allosteric inhibitor, binding to an exosite on the surface of the catalytically active enzyme. Furthermore, synthetic macrocyclic inhibitors of CatD were prepared, which mimic the binding conformation of the bacterial inhibitor pepstatin in the...

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