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The effect of Ca2+ ion on the enzymatic activity of human glutamate carboxypeptidase II
Nedvědová, Jana ; Bařinka, Cyril (advisor) ; Hlouchová, Klára (referee)
Human glutamate carboxypeptidase II (GCPII, EC 3.4.17.24) is a homodimeric membrane glycoprotein. GCPII has been studied as a marker of prostate carcinoma and a therapeutic target of neurodegenerative disorders. The extracellular region of the protein is composed of three domains, apical, protease and C-terminal. There are two zinc ions in the active site that are essential for the enzymatic activity. A calcium ion is located between apical and protease domains near the dimeric interface approximately 20 Å away from the active site. Consequently, the Ca2+ ion in unlikely to participate in substrate hydrolysis. The aim of this thesis is to elucidate the function of Ca2+ in GCPII using a combination of molecular-biological, biochemical and biophysical approaches. To this end we prepared series of GCPII variants with mutations in calcium-coordinating amino acids. The mutant constructs were expressed in insect S2 cells and purified by combination of affinity and size exclusion chromatography. Enzymatic activity and thermostability of the mutants were decreased significantly. Furthermore, mutated proteins were aggregation prone and formed a monomeric GCPII species. Our results thus show that Ca2+ ion plays an essential role in proper GCPII folding as well as the formation of a homodimer molecule that is...
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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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Molecular mechanism of the neutral trehalase Nth1 regulation
Kopecká, Miroslava ; Obšilová, Veronika (advisor) ; Bařinka, Cyril (referee) ; Pompach, Petr (referee)
The yeast enzyme neutral trehalase (Nth1, EC 3.2.1.28) from the Saccharomyces cerevisiae helps these organisms to survive adverse living conditions. Nth1 hydrolyses a storage and protective disaccharide trehalose into two molecules of glucose. The activity of this enzyme is regulated by PKA phosphorylation, Ca2+ binding and the yeast 14-3-3 protein (Bmh1) binding. Ca2+ binds to the Ca-binding domain located within N-terminus of Nth1 and contains so called EF-hand motif (D114 TDKNYQITIED125 ) which is highly conserved among many Ca-binding proteins. The main aim of this project was to reveal the structural basis of the Bmh1- and calcium-dependent activation of Nth1. Other goals were to solve the structure of Nth1 itself and the structure of its complex with Bmh1. To reveal how the calcium regulates the Nth1 activity we prepared twelve mutant forms of Nth1 using site directed mutagenesis. These mutations were located within the region of EF-hand motif and its close vicinity. We estimated the enzymatic activity of all these mutants in the presence of Bmh1 and/or Ca2+ . The ability of Nth1 to form stable complexes with Bmh1 was verified using the native polyacrylamide gel electrophoresis and analytical ultracentrifugation. The impact of mutations on the structure and properties of Nth1 was tested using...
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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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Antibody derivatives for the detection of human glutamate carboxypeptidase II
Bělousová, Nikola ; Bařinka, Cyril (advisor) ; Černý, Jan (referee)
Prostate cancer is the most common neoplasia in men. The therapy of progressed tumor is usually not efficient and early diagnosis is therefore crucial for successful treatment. Glutamate carboxypeptidase II (GCPII) is an established marker for prostate cancer imaging and therapy as the neoplastic transformation of prostate tissue is accompanied by the substantial increase of GCPII expression levels. Currently used GCPII-specific diagnostic and therapeutic reagents can be broadly categorized as small- molecule ligands or macromolecules. Antibodies are preferred macromolecules used in clinic. At the same time, however, protein engineering is regularly applied to modify natural antibodies to enhance their utility in biomedicine applications. This thesis summarizes the current knowledge about the GCPII structure and function and its role in diagnosis and therapy of prostate carcinoma. Powered by TCPDF (www.tcpdf.org)
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Impact of the glycine-rich loop on the function of processing peptidases of the mitochondrial type
Kučera, Tomáš ; Janata, Jiří (advisor) ; Bařinka, Cyril (referee) ; Ettrich, Rüdiger (referee)
The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...
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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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Role of the 14-3-3 protein in the regulation of G-protein signaling
Řežábková, Lenka ; Obšil, Tomáš (advisor) ; Konvalinka, Jan (referee) ; Bařinka, Cyril (referee)
Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Fyzikální chemie Mgr. Lenka Řežábková Studium úlohy proteinů 14-3-3 v regulaci G-proteinové signalizace Role of the 14-3-3 proteins in the regulation of G-protein signaling Disertační práce Školitel: doc. RNDr. Tomáš Obšil, Ph.D. Konzultanti: doc. RNDr. Petr Heřman, CSc. doc. RNDr. Jaroslav Večeř, CSc. Praha, 2012 Abstract The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of their binding partners in various signaling pathways that control diverse physiological and pathological processes such as signal transduction, metabolic pathways, cell cycle and apoptosis. More than 300 different cellular proteins from diverse eukaryotic organisms have been described as binding partners for the 14-3-3 proteins. During my Ph.D., I was particularly interested in the role of 14-3-3 proteins in the regulation of G protein signaling pathway. The 14-3-3 proteins affect the G protein signaling via the interaction with negative regulators of G protein cascade - the RGS proteins and phosducin. I employed both biochemical and biophysical approaches to understand how the activity and function of RGS3/14-3-3 and phosducin/14-3-3 complexes are regulated. I solved the low-resolution solution structure of...
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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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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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