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Antibody derivatives for the detection of human glutamatecarboxypeptidase II
Bělousová, Nikola ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Prostate cancer is one of the most common human malignancies and, consequently it is critical to develop appropriate diagnostic and therapeutic tools. Glutamate carboxypeptidase II (GCPII) is currently being considered one of the most important prostate cancer markers due to its tissue- specific expression. Whereas in healthy prostatic tissue the expression levels of GCPII are low, the transformation into the tumor is associated with the substantial increase of GCPII expression, with the highest levels observed in androgen-independent metastatic tumors. GCPII is thus considered a promising marker for early phase as well as advanced metastatic stages of prostate cancer. Current research is focused on the development of highly sensitive and specific reagents that allow detection of small amounts of GCPII, for example in early stages of cancer. Antibody derivatives are promising molecules for this purpose because they have high affinity and specificity and minimum negative side effects. Protein engineering is a prefered approach for preparation of various antibody molecules that differ in size, binding properties, stability, solubility, and production means. Different types of derivatives are being developed for medical needs such as in vitro diagnosis, therapy, and in vivo imagingSmall molecular...
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Structural and functional study of viral RNA polymerases
Dubánková, Anna ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee) ; Plevka, Pavel (referee)
Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...
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Glutamate Carboxypeptidase II - Structural and Biochemical Characterization and Structure-Assisted Drug Design
Ptáček, Jakub ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Brynda, Jiří (referee)
Glutamate carboxypeptidase II (GCPII) is a human membrane-bound metallopeptidase discovered more than 30 years ago. It has attracted attention of biomedical scientists thanks to its diverse tissue expression profile and different biological functions. GCPII is detected on the surface of astrocytes in both central and peripheral nervous systems where it is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. Glutamate, one of the hydrolytic products, is a potent excitatory neurotransmitter and its overproduction has been shown to be responsible for cell death in various neurological disorders by a so-called glutamate excitotoxicity mechanism. Together with the fact that NAAG acts neuroprotectively it has been postulated (and later confirmed) that GCPII inhibition has a therapeutic potential in such disorders. Prostate cancer (PCa) is the second most prevalent cancer in men and despite its slow progression it is prone to metastasize thus posing a life threat. GCPII has been found to be overexpressed in prostate tumor cells compared to the healthy tissue (therefore it is also termed prostate-specific membrane antigen - PSMA) thus representing an excellent biomarker of PCa validated by many publications and clinical studies....
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The use of phage display to investigate Leishmania mexicana surface antigens
Krylová, Anna ; Spitzová, Tatiana (advisor) ; Bařinka, Cyril (referee)
Leishmania is a protozoan parasite of vertebrates transmitted by the bite of infected phlebotomine sandflies. In humans, it causes a disease called leishmaniasis, which ranks as one of the most serious neglected tropical diseases. In the vectorial part of the life cycle, the crucial moment is when the flagellate forms (promastigotes) attach to the midgut epithelium of the sandfly. For most leishmania species, little is known about which types of phlebotomine receptors and leishmania surface antigens participate in the binding. Phage display was used to screen for Leishmania mexicana peptide ligands which may play a role in such binding. By affinity selection of phages incubated with promastigote cells, 16 unique peptides were identified. Fluorescent labelling of peptide-bearing phages indicated their putative binding sites on the leishmania surface. Based on the hypothesis that the identified peptides may be a part of receptors found in the phlebotomine midgut, experiments were performed where the sandflies were infected with promastigotes whose binding sites were blocked by two different peptide-bearing phages. The extent of the infection was different between the two cases. However, no statistically significant difference from the control group was observed. Despite unsuccessful attempts to identify a...
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Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Novák, Petr (referee)
Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...
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Vývoj chemických regulátorů drah mikroRNA a RNAi
Bruštíková, Kateřina ; Svoboda, Petr (advisor) ; Bařinka, Cyril (referee) ; Pospíšek, Martin (referee)
MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...
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High-throughput screening for the discovery of small molecules modulating cell fate
Ribeiro Pombinho, António José ; Bartůněk, Petr (advisor) ; Bařinka, Cyril (referee) ; Jiráček, Jiří (referee)
The discovery of chemical compounds able to modify the way cells proliferate, differentiate or die can lead not only to the formulation of new drugs for disease treatment or prevention but also to their use as biological probes in the study of the molecular pathways involved in these processes. In order to test thousands of these small molecules in cellular assays, instrument automation and assay miniaturization are necessary. In this thesis, applications of High-Throughput Screening campaigns are described. The Hypoxia and Wnt pathways involved in stem and cancer cell proliferation; the differentiation of hematopoietic, neural and mesenchymal stem cells; and the TRAIL pathway leading to selective cancer cells death were the main subjects chosen. With this approach, it was possible to test the effect of small molecules in eukaryotic cells and in unicellular organisms as exemplified by the search of compounds leading to the death of the protozoan parasite Leishmania. Several chemical compounds were identified as active in modulating cell fate. Of remark were: Monensin that inhibits the Wnt pathway and prevents the growth of tumors in a mouse model of colorectal cancer; Homoharringtonine that, only in combination with TRAIL, induces the death of cancer cells implanted in immunodeficient mice; and...
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Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography
Vaněk, Ondřej ; Bezouška, Karel (advisor) ; Hrabal, Richard (referee) ; Bařinka, Cyril (referee)
Department of Biochemistry, Faculty of Science, Charles University in Prague 2010 Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography Abstract of Ph.D. thesis Ondřej Vaněk Supervisor: Prof. RNDr. Karel Bezouška, DSc. Natural killer cells (NK cells) were found out for their ability to spontaneously kill certain allogeneic tumour cell lines, without any previous sensitization. NK cells are part of non- adaptive immune response with very short reaction time against pathogens such as viruses, intracellular bacteria, parasites, and they are responsible for elimination of certain tumour cells and thus they are able to fight against malignancy and formation of metastasis. Activity of NK cells is regulated by the balance between activation and inhibitory signals mediated by the NK cell surface receptors. From the structural point of view, the majority of NK cell surface receptors could be classified as the C-type lectin or immunoglobulin-like receptors. One of many C-type lectin subgroups are type II lymphocyte receptors that are expressed on the NK cell surface. This study had two main aims. The first one was to find suitable expression and purification systems for selected C-type lectin receptors of NK cells and the other one was to perform their...
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Study of the factors affecting the binding specificity of the 14-3-3 proteins.
Veisová, Dana ; Obšilová, Veronika (advisor) ; Bařinka, Cyril (referee) ; Krůšek, Jan (referee)
113 11. Summary The 14-3-3 proteins are dimeric molecules with a characteristic shape and molecular mass about 30 kDa found in all eukaryotes. They are playing a key role in a variety of biological processes such as signal transduction, cell differentiation and apoptosis. The C- terminal segment of human 14-3-3ζ plays an important role as an autoinhibitor which can occupy the ligand binding groove in the absence of binding partner and blocks the binding of inappropriate ligand. The C-terminal segment structure has not been identified for any of the known crystallographic structures. Unlike the helical region α1-α9, the C-terminal segment shows the highest sequence variability. It is believed that the C-terminal segment is the most flexible region and can exist in a lot of conformations. The yeast isoforms of the 14-3-3 proteins Bmh1 and Bmh2 possess a distinctly variant C-terminal segment which is longer and contains a polyglutamine stretch of unknown function. The role of this C-terminal part has been studied with many of different biophysical methods. Dynamic light scattering, sedimentation velocity, time resolved fluorescence anisotropy decay, and size exclusion chromatography measurements showed that an apparent size of the molecules Bmh1 and Bmh2 is significantly bigger compared to the 14-3-3 isoforms....
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The crystal structure of PI4 kinase
Bäumlová, Adriana ; Bouřa, Evžen (advisor) ; Obšil, Tomáš (referee) ; Bařinka, Cyril (referee)
Phosphatidylinositol 4-kinases (PI4K/PI4-kinases) catalyse the production of phosphatidylinositol 4-phosphate (PtdIns4P), the first step in the generation of higher phosphoinositides. PtdIns4P is an essential precursor in the production of second messengers, Ins(1,4,5)P3 and diacylglycerol, in a receptor activated phospholipase C signalling pathway. Moreover, PtdIns4P itself regulates conserved compartment-specific biological processes, mainly via recruiting a broad spectra of effector proteins. Because PI4-kinases have a central position in PtdIns4P synthesis on a surface of intracellular membranes, they are implicated in a wide range of PtdIns4P-induced processes such as lipid transport and metabolism, intracellular trafficking processes and cargo sorting, membrane and cytoskeleton remodelling events, signal transduction and many others. In mammals, two types of PI4-kinases were identified: type II and type III. Both types do not bear high sequence similarity to each other and, therefore, they possess diverse biochemical properties. In order to elucidate their structural relationship to other lipid kinases, structural analysis is highly demanded. The structural characterisation of individual PI4-kinases could also clarify the catalytic mechanism of PtdIns4P synthesis. Furthermore, information...
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