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Detail enzymatic characterization of a model heme-containing oxygen sensor
Vojáčková, Lukrécie Sophie ; Martínková, Markéta (advisor) ; Čermáková, Michaela (referee)
This thesis focuses on heme-based gas sensors, particularly phosphodiesterase from the bacterium Escherichia coli, referred to as EcDOS. The enzyme catalyzes the degradation of c-di-GMP, an important signaling molecule in bacteria that affects cellular processes, such as bacterial motility or biofilm formation. The thesis deals with detailed enzyme kinetics of protein forms in different redox and ligand states of the heme iron ion [Fe(III), Fe(II) and Fe(II)-O2], as well as a mutant form of the enzyme (EcDOS H77A) which does not bind heme. Results confirmed that the EcDOS WT Fe(II)-O2 form has higher kcat values than the EcDOS form with the ferrous ion of heme in the reduced state. Other significant result was that the enzyme activity is affected not only by the state of the heme iron ion but also by the presence and concentrations of divalent metal cations. The presence of the metal cation is essential for enzyme function, and suitable metal ions that stimulate enzyme activity are Mg2+ , Mn2+ and Zn2+ or their mixtures, which act synergistically on enzyme activity under chosen conditions. Analysis by ICP-MS also showed that Zn2+ cations are natural components of the enzyme. Thus, for further kinetic studies, it would be appropriate to use Zn2+ or mixtures of metal ions that are physiological for...
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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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Characterisation of recombinant mouse glutamate carboxypeptidase III
Janoušková, Karolína ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Glutamate carboxypeptidase II (GCPII, PSMA, NAALADase) is transmembrane metalopeptidase and due to cleavage of substrates β-citryl-L-glutamate (BCG), N-acetyl-L-aspartyl-L-glutamate (NAAG) and polyglutamylated folates (Pte-Glun) is being studied as potential therapeutic target. Enzymes, which could compensate for enzyme activity and functions of GCPII, are thus relevant targets of enzymology as well. One of GCPII's homologs with similar enzyme activity is mouse glutamate carboxypeptidase III (GCPIII, NAALADase II). Enzymatic cleavage has not been determined using recombinant mouse GCPIII yet. It is important to kinetically characterize mouse GCPIII so that we can compare enzyme activity with human ortolog. Then we can find out whether mouse model is comparable with human. Recombinant mouse GCPIII was kinetically characterized. Kinetic parameters (KM, kcat) for recombinant mouse GCPIII were measured for substrates NAAG and BCG using radioactive assay. Experiments with the substrate Pte-Glu2 were analyzed using HPLC method. Although human GCPIII is more effective than mouse ortolog at clearage of NAAG, both enzymes are comparable during hydrolysis of BCG. Those results can contribute to better understanding of the role of GCPIII in the most commonly used animal model.
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Separation and determination of possible products of enzymatic cleavage of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside using capillary electrophoresis
Velvarská, Romana ; Křížek, Tomáš (advisor) ; Kalíková, Květa (referee)
This work deals with the development and optimization of conditions of a method that can be used to compare the activity of the enzyme β-N-acetylhexosaminidase in hydrolysis of a natural substrate and a chromogenic substrate, which is often used in the study of enzyme kinetics. As a substrate, 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside was selected for cleavage. This oligosaccharide contains bond, which the enzyme cleaves in the natural substrate, and the bond that occurs in the chromogenic substrate. To determine the products arising from enzymatic hydrolysis of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside, capillary zone electrophoresis was used. First, it was necessary to find the optimal composition of the electrolyte, its pH and concentration. The optimal background electrolyte was a solution of sodium tetraborate at a concentration of 25 mmol/l and a pH of 10.25. Subsequently, repeatability, calibration curves and linearity, limit of detection and limit of quantification were investigated. Repeatability of migration times ranged up to 0.6%, the repeatability of peak areas between 2.5 and 6.3%. Limits of detection were ranging from 0.005 to 0.120 mmol/l. Finally, the optimized method was successfully used to monitor the actual enzyme cleavage.
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Preparation and characterization of the catalytic domain of human protein kinase ASK1.
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Protein kinase ASK1 (apoptosis signal-regulating kinase 1) is a member of the mitogen- activated protein kinase kinase kinase (MAP3K) family and plays a crucial role in immune and stress responses. Since the increased activity of ASK1 has been linked to the development of several diseases including cancer, cardiovascular and neurodegenerative diseases, this enzyme is a promising target for therapeutical intervention in these pathologies. The molecule of ASK1 consists of 1374 amino acid residues, but catalytic activity possesses only a kinase domain located approximately in the middle of the molecule. The activity of ASK1 is regulated by interactions with various proteins including the 14-3-3 protein. This protein recognizes a phosphorylated motif around Ser966 at the C-terminus of the catalytic domain of ASK1. This binding interaction inhibits ASK1 through unknown mechanism. ASK1 under stress conditions, such as oxidative stress, is dephosphorylated at Ser966 and the 14-3-3 protein dissociates. This dissociation is then one of the factors that lead to the activation of ASK1. The aim of this diploma thesis was to prepare a complex of the catalytic domain of ASK1 with the 14-3-3 protein for subsequent structural studies. Both proteins were expressed in E. coli cells and successfully purified. In...
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Analysis of the mechanism of action of metallacarborane inhibitors of HIV PR
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
English Abstract Shortly after the identification of HIV as a causative agent of AIDS, an aspartic protease was identified in the viral genetic information. The very same time protease has become one of the dominant therapeutical targets in AIDS therapy. The introduction of protease inhibitors into the antiretroviral therapy has led to a significant improvement in the quality and length of life of HIV patients. However, the virus is still able to effectively prevent the impact of an inhibitor via generating inhibitor-resistant mutated protease variants. Thus, there is a constant need for novel types of inhibitors that would be capable of effectively blocking these resistant variants and simultaneously not supporting the development of novel resistant viral strains. One way to identify such inhibitors could be searching for compounds interacting with the enzyme at different sites than the active cavity, via the mechanisms of noncompetitive or uncompetitive inhibition. The group of compounds called metallacarboranes - inorganic compounds consisting of carbon, boron, hydrogen and metall ion - were shown to exhibit such an activity against HIV-1 protease. However, for further optimization of these inhibitors, detailed biophysical investigation of the enzyme-inhibitor complex is needed. This work focuses on the...
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Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases
Hympánová, Michaela ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...
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Structure-assisted development of a continuous carboxypeptidase assay
Rakhimbekova, Anastasia ; Bařinka, Cyril (advisor) ; Bouřa, Evžen (referee)
Glutamate carboxypeptidase II (GCPII) is a zinc-dependent carboxypeptidase with high expression levels in prostate carcinoma. As the enzyme represents a validated target for cancer therapy and imaging, the development of new GCPII-specific ligands is still a focus of an active academic and industrial research. However, existing assays to screen inhibitor libraries and determine inhibitor efficacy are suboptimal at best. This thesis is aimed at the development of small internally quenched probes that could be used for continuous measurement of the GCPII enzymatic activity. These probes are derived from natural GCPII substrates and consist of a fluorophore/quencher pair connected by a GCPII-hydrolysable linker. I first characterized biophysical properties of the probes and then determined kinetic parameters of their hydrolysis by GCPII. The optimized activity assay was then used to determine inhibition constants of several GCPII-specific inhibitors. Finally, complexes between the inactive enzyme and several probes were co-crystallized and one of the complexes refined and analyzed. Our data show that the probes are involved in non-covalent interactions with the same amino acid residues of the enzyme's active site as natural substrates. The developed assay could be optimized for high-throughput...
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Inhibitors of mouse serine racemase
Vorlová, Barbora
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
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Protease Inhibitors as a Research Tool: Design, Synthesis and Evaluation of HIV PR and GCPII Inhibitors
Schimer, Jiří
This dissertation thesis focuses on creating tools for the analysis and potential therapeutic intervention in the biological processes regulated by proteolysis. I focus on two important proteolytic enzymes: HIV-1 protease, which is indispensable for the polyprotein processing of the nascent virus and thus for the development of infectious viral particle, and glutamate carboxypeptidase II, a tumor marker and a neuropeptidase from the prostate and central nervous system. Rational design of inhibitors of these therapeutically relevant enzymes serves two purposes: firstly, protease inhibitors were shown to be powerful drugs (HIV protease is in fact the example of successful drug development driven by structural biology). Secondly, and in the context of this thesis perhaps more importantly, inhibitors of medicinally relevant proteases might serve as tools for the elucidation of basic biological questions concerning regulation, timing and spatiotemporal control of such key processes as virus maturation or cancer development. The experimental work described in this thesis summarizes my results in both these areas. Human Immunodeficiency Virus Protease Human immunodeficiency virus (HIV), a causative agent of AIDS, has been estimated to kill close to 40 million people during the past four decades with 1.5...
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