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Synthesis of peptides as potential ligands of aspartate protease HIV and MAY1 and confirmation of their inhibitory activity
Klikarová, Ivana ; Šácha, Pavel (advisor) ; Žáková, Lenka (referee)
The lentivirus known as the human immunodeficiency virus (HIV) is transmitted through blood and body fluids, causing the destruction of CD4 lymphocytes and leading to opportunistic infections that define acquired immunodeficiency syndrome (AIDS). The urgent need for new antiretroviral drugs stems from concerns about the long-term toxicity of existing drugs, HIV-1 variants resistant to treatment, and frequent changes in patient treatment. Drug development is focused on inhibitors of two viral enzymes, reverse transcriptase and protease. Antiretroviral therapy uses protease inhibitors in combination with nucleoside analogs to effectively suppress viral replication, prolonging the lives of HIV-infected patients and reducing morbidity. Cryptococcus neoformans and cryptococcus gattii infections primarily affect the immunocompromised population and have high morbidity and mortality rates. Resistance to commonly used antifungals has been emerging, making it more difficult to treat these infections. Protease inhibitor components used in antiretroviral therapies have shown some clinical efficacy in these opportunistic infections, particularly in Major Aspartyl peptidase 1, an aspartate protease belonging to the same family of proteases as the HIV protease. To search for low molecular weight peptide ligands,...
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Adaptation of enzymatic assays for high-throughput screening with use of ECHO-MS
Kráľová, Zuzana ; Mertlíková Kaiserová, Helena (advisor) ; Šácha, Pavel (referee)
High throughput screening for potential inhibitors of enzyme purine nucleoside phosphorylase (PNP) was developed during the work on this thesis. Products of PNP-catalysed reaction, hypoxanthine and ribose-1-phosphate, were quantified using mass spectrometry. Advantages and disadvantages of an Echo® MS device were explored and compared to the previously used radioenzymatic method. The emphasis was put on assay robustness and automatization of the protocol. Acoustic droplet ejection technique (ADE) and contactless dispensing of chemicals were employed, using Echo® 550 and Certus Flex® devices. These allowed to reduce reaction volume from original 20 µl to 5 µl and perform the whole reaction in a 384-well plate, decreasing the costs and increasing the throughput. Using a developed Echo® MS method, the time of analysis was shortened from 8 minutes per sample for radiometric analysis to 0,2 minute per sample. Furthermore, enzyme assay parameters, such as PNP concentration, composition of reaction mixture, incubation time and reaction termination were optimized, following the rules of HTS. The method developed in this work was employed for PNP inhibitors screening and their IC50 determination. Moreover, the HTS method enabled screening of the whole library of IOCB in-house synthesized compounds (~ 8000)....
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Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
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Epigenetically based chemoresistance of cancer cells
Feriančiková, Barbara ; Eckschlager, Tomáš (advisor) ; Šácha, Pavel (referee)
Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...
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Nephropathy and tumour development caused by plant alkaloids aristolochic acid
Bárta, František ; Stiborová, Marie (advisor) ; Šácha, Pavel (referee)
Aristolochic acids (AA) are alkaloids contained in plant species of the family Aristolochiaceae. These plants are used since antiquity in traditional medicine to treatment of many varied diseases. There are known anti-inflammatory effects of these compounds, however these alkaloids exhibit mutagenic and carcinogenic properties. Despite of this fact, plant extracts AA are still used in traditional medicine, e.g. in China, India, Taiwan. Aristolochic acids are proven to be the cause of disease designated Aristolochic Acid Nephropathy (AAN, theretofore known as Chinese Herbs Nephropathy (CHN). This unusual nephropathy leads to a total renal failure. The late complication of this disease is the development of tumours in urothelial tissue of patients. AA can form persistent stable covalent DNA adducts. Formation of these DNA adducts lead to AT→TA transversion, the unique mutation in tumour suppressor gene p53 responsible for tumour formation. Balkan Endemic Nephropathy (BEN) is associated with AA, too. In this instance is supported also influence of another factors, e.g. mycotoxins (ochratoxin A). However, in all probability AA contribute to a development of this disease particularly. This hypothesis is supported by finding of AA-DNA adducts in tissues of patients suffering from AAN and BEN and that of...
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Structure and Function of Glutamate Carboxypeptidase II
Šácha, Pavel ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Blahoš, Jaroslav (referee)
4 Závěr GCPII je důležitý protein, který hraje roli v mnoha fyziologických i patologických procesech. Proto bylo třeba získat větší množství enzymaticky aktivního proteinu pro jeho další biochemický výzkum. Heterologní expresí v hmyzích buňkách S2 bylo exprimováno a následně purifikováno dostatečné množství velmi čistého a aktivního enzymu. To umožnilo jeho biochemickou charakterizaci, krystalizaci a později vedlo i k vyřešení krystalové struktury. GCPII je aktivní v širokém rozmezí pH 6 - 8, s maximem kolem pH 7,5. Zjistili jsme, že kromě přirozeného substrátu Ac-Asp-Glu GCPII štěpí také acetylované dipeptidy Ac-Asp-Met, Ac-Glu-Met, Ac-Glu-Glu, Ac-Ala-Glu a Ac-Ala-Met. U těchto substrátů byly změřeny kinetické parametry štěpení. Nalezení dalších substrátů může vést k objevení dosud nepopsaných fyziologických rolí GCPII. Také byly porovnány hodnoty IC50 inhibitorů známých z literatury u námi připravené GCPII a GCPII izolované z potkaních mozků. IC50 bylo u všech méně specifických inhibitorů nižší u rekombinantní GCPII než u GCPII izolované z mozků. Rozdíly ve výsledcích vysvětlujeme vazbou méně specifických inhibitorů na jiné proteiny preparátů z mozku. Vůbec nepochybujeme o tom, že data získaná za použití čistého rekombinantního proteinu jsou přesnější než ta, ktará byla získána ze špatně definovaných...
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Optimization of recombinant protein expression in HEK293 cell line
Bláha, Jan ; Bezouška, Karel (advisor) ; Šácha, Pavel (referee)
Mammalian cells have become the dominant system for recombinant expression of pharmaceutical proteins. This system is becoming suitable also for structural biology, with the advances in methodology of transient transfection of mammalian cells. This work dealt with optimization of recombinant expression in HEK293T and HEK293-6E cell lines in various media using easily quantifiable markers - secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). Emphasis was placed on optimizing key factors behind the creation of transfection complex - the ratio of DNA to polyethyleneimine and the amount of DNA used. The positive influence of histone deacetylases inhibitor valproic acid and also of casein hydrolysate Tryptone N1 was also studied. (The thesis is written in Czech.)
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Generation and Characterization of Glutamate Carboxypeptidase II (GCPII)-Deficient Mice
Vorlová, Barbora ; Šácha, Pavel (advisor) ; Eckschlager, Tomáš (referee) ; Bařinka, Cyril (referee)
Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein, which consists of short intracellular and transmembrane domains, and a large extracellular domain possessing carboxypeptidase activity. In the human body, GCPII fulfils a neuromodulatory function in the brain and facilitates folate absorption in the small intestine. In addition to the brain and small intestine, high level of GCPII is also present in the prostate and kidney. However, GCPII function in these tissues has not been determined yet. To study the role of GCPII in detail, several research groups attempted to inactivate GCPII encoding gene Folh1 in mice. Surprisingly, the experiments led to rather conflicting results ranging from embryonic lethality to generation of viable GCPII-deficient mice without any obvious phenotype. This dissertation project aimed to dissect the discrepancy using alternative strategy for gene modification. For this purpose, we designed TALENs that specifically targeted exon 11 of Folh1 gene and manipulated mouse zygotes of C57BL/6NCrl genetic background. We analysed all genetically modified mice of F0 generation for presence of TALEN-mediated mutations and established 5 different GCPII-mutant mouse colonies from founder mice that altogether carried 2 frame-shift mutations and 3 small in-frame...
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Epigenetically based chemoresistance of cancer cells
Feriančiková, Barbara ; Eckschlager, Tomáš (advisor) ; Šácha, Pavel (referee)
Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...
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Structure and Function of Glutamate Carboxypeptidase II
Šácha, Pavel ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Blahoš, Jaroslav (referee)
4 Závěr GCPII je důležitý protein, který hraje roli v mnoha fyziologických i patologických procesech. Proto bylo třeba získat větší množství enzymaticky aktivního proteinu pro jeho další biochemický výzkum. Heterologní expresí v hmyzích buňkách S2 bylo exprimováno a následně purifikováno dostatečné množství velmi čistého a aktivního enzymu. To umožnilo jeho biochemickou charakterizaci, krystalizaci a později vedlo i k vyřešení krystalové struktury. GCPII je aktivní v širokém rozmezí pH 6 - 8, s maximem kolem pH 7,5. Zjistili jsme, že kromě přirozeného substrátu Ac-Asp-Glu GCPII štěpí také acetylované dipeptidy Ac-Asp-Met, Ac-Glu-Met, Ac-Glu-Glu, Ac-Ala-Glu a Ac-Ala-Met. U těchto substrátů byly změřeny kinetické parametry štěpení. Nalezení dalších substrátů může vést k objevení dosud nepopsaných fyziologických rolí GCPII. Také byly porovnány hodnoty IC50 inhibitorů známých z literatury u námi připravené GCPII a GCPII izolované z potkaních mozků. IC50 bylo u všech méně specifických inhibitorů nižší u rekombinantní GCPII než u GCPII izolované z mozků. Rozdíly ve výsledcích vysvětlujeme vazbou méně specifických inhibitorů na jiné proteiny preparátů z mozku. Vůbec nepochybujeme o tom, že data získaná za použití čistého rekombinantního proteinu jsou přesnější než ta, ktará byla získána ze špatně definovaných...
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