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Parasite cystatins as inhibitors of cysteine proteases: structural aspects of functional specificity and their evolution
Buša, Michal ; Mareš, Michael (advisor) ; Hudeček, Jiří (referee) ; Kukačka, Zdeněk (referee)
Members of the cystatin family are important inhibitors of cathepsin-type cysteine proteases and are involved in a number of pathologies. Parasite cystatins are attractive target molecules for parasite control, but our knowledge about them is still limited. This work is focused on cystatins of two blood-feeding parasites: the common tick (Ixodes ricinus) as the main vector of Lyme disease and tick-borne encephalitis, and the liver fluke (Fasciola hepatica), the causative agent of fasciolosis. Four novel cystatins were functionally and structurally characterized to determine the structural determinants of their inhibitory specificity and describe them in the context of evolution and physiological role of cystatins. The cystatin FhCyLS-2 from F. hepatica has broad inhibitory specificity and is suggested to play a dual role in the regulation of proteolytic systems in host tissue and the parasite gut. FhCyLS-2 combines the characteristics of two cystatin subfamilies in a unique way and is a model representative of a novel evolutionary group of cystatins identified in several orders of parasitic flukes. Ricistatin and iristatin are salivary cystatins of I. ricinus with immunomodulatory effects on the host caused by an exceptionally narrow inhibitory specificity. It was explained by structural modifications of...
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Production of cysteine cathepsins and structural characterization of their interaction with peptidomimetic inhibitors
Wichterle, Filip ; Mareš, Michael (advisor) ; Knejzlík, Zdeněk (referee)
Cysteine cathepsins participate in many pathological processes such as cancer and neurodegen- erative, cardiovascular, or autoimmune diseases. This work is focused on cathepsins B, L, and V (CatB, CatL, CatV), which represent attractive targets for the development of inhibitors as potential chemotherapeutics and diagnostic tools. The aim of this study was to prepare these cathepsins and structurally characterize their complexes with selected synthetic peptidomimetic inhibitors. Recombinant CatB and CatL were prepared in the yeast Pichia pastoris, and the expression conditions were optimized for the production of cathepsin zymogens. A chromato- graphic purification protocol was designed for CatB and CatL, while CatV was purified using a previously developed procedure. The obtained enzymes were used to prepare complexes with six peptidomimetic inhibitors equipped with a carbamate, vinyl sulfone, or azanitrile warhead, which selectively target CatB, CatL, and CatV, respectively. Their inhibition parameters were determined in a kinetic assay and initial crystallization conditions were identified. After opti- mizing the crystallization conditions for CatB with three carbamate inhibitors, crystals suitable for X-ray crystallography were obtained. Based on the crystal structures of these complexes, the...
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Regulation of the activity of aspartic and serine proteases by selective natural inhibitors
Srp, Jaroslav ; Mareš, Michael (advisor) ; Novák, Petr (referee) ; Bařinka, Cyril (referee)
Proteases are involved in many physiological processes and their dysregulation is associated with various pathologies. Protease activity is effectively controlled by natural inhibitors. This PhD thesis is focused on the inhibitors of aspartic and serine proteases of animal and plant origin and provides the identification, biochemical characterization and structural description of their inhibition mechanisms. Plant Kunitz inhibitors are produced as defensive proteins, and they are able to block activities of a broad spectrum of proteases. In this thesis, the digestive proteolytic system of the Colorado potato beetle, a herbivore pest of potato plants, was described with the help of functional proteomics. It was shown that aspartic and serine proteases from this herbivore are effectively blocked by two potato Kunitz inhibitors (namely PCDI, PSPI). Using structural analysis, novel types of reactive centers were identified on PCDI and PSPI molecules for the inhibition of aspartic protease cathepsin D and the serine proteases trypsin and chymotrypsin. The analysis of the reactive center on a PCDI with the crystal structure of digestive cathepsin D from the Colorado potato beetle explained the mechanism of their interaction. Sphingolipids were identified as the first endogenous inhibitors of human...
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Media
Mareš, Michael ; Zamazal, Ondřej (advisor) ; Kraus, Jiří (referee)
Diploma thesis "Media as an Agent of National Self-Confidence: Analysis of the Victory of the Czechoslovak Ice Hockey Players at the 1947 World Championship" deals with the reflection of the major sport event in the Czechoslovak media and how this reflection inspired the audience. The goal of this diploma thesis is to describe the dependance between the media coverage and the national self-confidence, which results from the national identity. Therefore, I focused on the chosen newspaper titles to analyze the content they produced during the 1947 Ice Hockey World Championship. I defined a hypothesis, based on observation and scholarly literature, that the media used the coverage to strengthen the national self-confidence and redefine the national identity. My second hypothesis was the idea, that this kind of coverage led to an active response among the audience. Before I started analyzing the three chosen titles (Lidova demokracie, Rude Pravo, Prace), I delivered complex structure of theoretical concepts, describing the influence media have towards the audience and also reasons why the members of the audience use media in their everyday life. I also focused on the historical and political context, as well as the media landscape that shaped the content of that time. I conducted the analysis and...
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Regulation of cathepsin K activity by reactive inhibitory molecules applicable in biomedicine
Benýšek, Jakub ; Mareš, Michael (advisor) ; Obšilová, Veronika (referee) ; Kutil, Zsófia (referee)
3 Abstract Human cathepsin K (KatK) is a lysosomal cysteine protease expressed predominantly in osteoclasts. It is the most effective enzyme for collagen breakdown and its physiological function lies in the degradation of the extracellular bone matrix. Increased enzymatic activity of KatK is associated with osteoporosis, rheumatoid arthritis, and osteoarthritis. This makes KatK a target for the treatment of pathologies, and chemotherapeutics based on protease inhibitors are being developed for its regulation. This work deals with reactive peptidomimetic and low molecular weight inhibitors of KatK namely thiadiazoles, vinyl ketones, and cyanohydrazides. It focuses mainly on the determination of the binding mode of selective inhibitors and characterization of their key interactions with the active site of KatK. Crystallographic structural analysis was combined with approaches of computational chemistry, enzymological analysis, and cell-based assays to elucidate the relationship between structure and biochemical activity of the investigated inhibitors. The obtained results provide important information for the design and optimization of new highly effective and selective KatK inhibitors as potential drugs and diagnostic probes. Keywords: cathepsin K, protease, inhibitor, 3D structure, osteoporosis
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Regulation of Cathepsin D Activity and Activation
Máša, Martin ; Mareš, Michael (advisor) ; Jonáková, Věra (referee) ; Holada, Karel (referee)
PhD Thesis ABSTRACT REGULATION OF CATHEPSIN D ACTIVITY AND ACTIVATION Martin Máša supervisor: Michael Mareš Department of Biochemistry Faculty of Science Charles University Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic PRAGUE 2009 Introduction Cathepsin D (CD) is an aspartic peptidase located in the lysosomes of all mammalian cells, its main role is catabolic degradation of proteins. More over CD is known to participate in a range of physiological processes such as apoptosis and tissue homeostasis, as well as in the regulation of angiogenesis and the production of peptidic antigens. The role of CD in pathophysiology is associated with several diseases such as Alzheimer's disease and cancer. Alzheimer's disease is a neurodegenerative disorder, which is generally considered to be the most common form of dementia. Progression of this disease is accompanied with the deposition of amyloid plaques (AP) in the brain, which leads to neurodegeneration. The AP is a fragment released from amyloid precursor protein (APP) cleaved by secretases1 . High levels of CD were found in cerebrospinal fluid of the Alzheimer's patients2 . It was demonstrated that CD is able to cleave APP and produce the pathogenic AP. A genetic polymorphism in the CD gene was reported, which changes...
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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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