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Preparation and characterization of cyanide hydratase from Aspergillus niger and nitrilase from Arthroderma benhamiae
Hradilová, Iveta ; Vaněk, Ondřej (advisor) ; Martínek, Václav (referee)
Nitrilases are well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used. Keywords: nitrilase, cyanide hydratase, Aspergillus niger, Arthroderma benhamiae, liquid chromatography (In Czech)
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Expression and characterisation of homologs of human glutamate carboxypeptidase II
Bäumlová, Adriana ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane bound glycoprotein that belongs to the metallopeptidase M28 family. Two physiological substrates were found for GCPII. The first one, N-acetyl-aspartylglutamate (NAAG), serves as a neurotransmiter in the brain and GCPII hydrolyzes it to yield free glutamate in the synaptic cleft. Excess glutamate might be cytotoxic and eventually lead to excitoxic nerve cells death. Inhibition of NAAG hydrolyzing activity has been shown to be neuroprotective. Therefore, GCPII inhibition was suggested as a therapeutic target in treatment of neurological disorders where excess glutamate is involved. The second substrate, polyglutamyl folate, is a precursor of folic acid which is required for cell growth and development. GCPII cleaves off glutamate from dietary folates and thus facilitates their absorption in small intestine. Although GCPII biological relevance is known only in the brain and the small intestine, its role in the prostate is also important. GCPII has been described as a prostate cancer marker as it is expressed on the membrane of prostate cancer cells. Since GCPII is type II transmembrane protein, it is enzymatically active and undergoes internalization, it has been suggested as a promising tool for specific anticancer-drug...
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LacZ-alpha complementation peptide as a tool for molecular evolution studies
Ptáčková, Barbora ; Hlouchová, Klára (advisor) ; Vaněk, Ondřej (referee)
Proteins are the key structural and functional molecules of living organisms. Although the last decades have brought a lot of knowledge about their structural and functional characteristics, science still lacks very basic answers about how these properties evolved. Current predominant opinions suggest that early genetic code contained only a subset of today's canonical amino acids. Both exogenous and endogenous sources of prebiotic amino acids imply that even though the prebiotic amino acid repertoire was very broad, only about half of the proteinogenic amino acids were present. It follows that the ''evolutionary new'' amino acids were added to the genetic coding system only after the evolution of their biosynthetic pathways. From the current scientific knowledge it is unclear whether proteins composed of "evolutionary old" amino acids could serve basic metabolic functions and if today's proteins could be "reversely-evolved" to be composed of only such a subset of amino acids while maintaining their structural and functional integrity. These questions lie at the core of this study. This thesis aims to test a starting methodology that would randomize "new" amino acid positions by "old" amino acids in the sequence of LacZ-alpha peptide. This peptide was selected as a target model protein because it...
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Biomolecule conjugates with nanodiamonds: preparation and application
Šlegerová, Jitka ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
ENGLISH ABSTRACT Fluorescent nanodiamonds are a perspective material for the preparation of fluorescent labels for bioimaging due to their stable fluorescence. Contrary to other compounds, nanodiamonds do not photobleach or photoblink. Furthermore, nanodiamonds emit fluorescence in the red part of the spectrum which is well separated from auto-fluorescence. However, before the employment of nanodiamonds as fluorescent labels in biological systems, several properties have to be improved. Primarily, their colloidal stability in biological media has to be ensured. Preventing of non-specific interactions of nanodiamonds with proteins is next crucial step. Finally, it is necessary to enable their high-yield further modifications with various molecules. Hydrophilic polymer coating surrounding a nanodiamond particle was introduced as a successful modification. Coated nanodiamonds were subsequently modified with transferrin or the inhibitor of glutamate carboxypeptidase II. These molecules bind specifically to receptors on cell membranes and enable cellular internalization. Nanodiamond conjugates were successfully prepared and the ability of binding to respective receptors was verified for the nanodiamond conjugate with the inhibitor of glutamate carboxypeptidase II. However, the effect of transferrin or the...
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Cloning, expression and biochemical characterisation of mouse glutamate carboxypeptidase II.
Knedlík, Tomáš ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
English Abstract Glutamate carboxypeptidase II (GCPII) is a membrane metallopeptidase expressed in many human tissues, predominantly in prostate, brain and small intestine. In brain it cleaves the most abundant peptide neurotransmitter N-acetyl-L-aspartyl-α-L-glutamate into N-acetyl-L-aspartate and free L-glutamate. Thus, GCPII participates in glutamate excitotoxicity through the release of free glutamate into the synaptic cleft. Inhibition of this activity has been shown to be neuroprotective in rats. In the human jejunal brush border, GCPII cleaves off terminal glutamate moieties from poly-γ-glutamylated folates, which can be then transported across the intestinal mucosa. The function of GCPII in human prostate is unknown but it is overexpressed in prostate cancer. Therefore, GCPII is an important marker of prostate cancer and its progression.Moreover, it could become a perspective target for treatment of prostate cancer as well as neuronal disorders associated with glutamate excitotoxicity. For the development and testing of novel drugs and therapeutics it is necessary to have an appropriate animal model. Mouse (Mus musculus) is such a model and it is widely used by many experimentators. However, no detailed comparison of mouse and human GCPII orthologs regarding their enzymatic activity, inhibition...
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The influence of heterodimezation of splicing variants of metobotropic glutamate receptor 1a and 1b on the intracellular distribution of receptor complexes
Dvořáková, Michaela ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
L-glutamate is a major excitatory neurotransmitter in vertebrate central nervous system. L-glutamate enables synaptic transmission through ionotropic and metabotropic glutamate receptors. These receptors are indispensable in the brain. The main role of metabotropic glutamate receptors is to mediate slow excitatory and inhibitory responses by activation of intracellular messengers and to regulate cationic channels. Metabotropic glutamate receptors are involved in synaptic plasticity, different types of memory, learning, motoric coordination and neural development. On the other hand excitotoxicity of glutamate is often associated with neurodegenerative processes such as Alzheimer, Huntington and Parkinson disease. Metabotropic glutamate receptors are promising therapeutic targets for a treatment of psychiatric and neurological diseases. Targeted trafficking of metabotropic glutamate receptors to distinct parts of neurons is influenced by neuronal polarity and thus regulates sensing and transmission of extracellular signals. Newly detected heterodimeric receptors might be trafficked in a different way than homodimers and therefore our knowledge of molecular pathways of these complexes could help us with subsequent drug targeting. This work confirms heterodimerization of metabotropic glutamate receptor 1 into...
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Study of interaction between ASK1 kinase and thioredoxin.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
MAP kinase signaling cascade plays an important role in the cellular response to various stress stimuli from the external environment. This signaling cascade is divided into three levels: MAP kinase kinase kinases (MAP3K) phosphorylate and thus activate MAP kinase kinases (MAP2K) and those subsequently phosphorylate and thus activate MAP kinase (MAPK) pathway, which regulates many cellular functions such as apoptosis, cell differentiation and morphogenesis. One of the important MAP3K is protein kinase ASK1 (Apoptosis signal-regulating kinase 1), which is an important regulator of cellular immune and stress responses. Given that the increased activity of ASK1 is related to the development of serious diseases such as cancer, cardiovascular and neurodegenerative diseases, ASK1 is an interesting target in the pharmacy in the development of new drugs. Human ASK1 consists of 1374 amino acids and is divided into three domains: a central Ser/Thr catalytic domain and two coiled-coil domains, of which the first is located at the N- and the second at the C-terminus of the molecule of this protein kinase. ASK1 is regulated by its binding partners, which include a small cellular redox protein thioredoxin (Trx-1), which binds to the N-terminal part of ASK1. Trx-1 is a potent antioxidant and so it protects cells...
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Preparation of expression constructs of phosducine binding partners.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
Key words: SUG1, phosducin, G protein signaling, expression, pGEX-4T-1, pST39 Phosducin is an acidic protein found in cytosol and is involved in G protein signaling within rods of photoreceptors. Phosducin also influences stress-dependent hypertension. One of phosducin's important binding partners is SUG1, a 26S proteasome subunit, which belongs to a family of proteins that have an ATPase activity. This bachelor thesis takes part in a broader research, which tries to clarify physiological functions of the interaction of SUG1 with phosducin. Main goal of this study was to prepare constructs of SUG1 (human isoform 1) suitable for the recombinant protein production in an expression system E. coli BL21 (Rosetta). Gene that encodes SUG1 was inserted into two different plasmid DNAs that allow expression of SUG1 as two different fusion proteins in a prokaryotic expression system. Nucleotide sequences of these constructs were verified using sequencing. Next, expression tests revealed sufficient production of both recombinant fusion proteins. The main result of this bachelor thesis is the successful expression of human SUG1 in E. coli BL21 (Rosetta) cells. This result will enable the development of the purification protocol for the large scale expression of both recombinant fusion proteins as well as the...
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Study of structure and interaction of human lymphocyte receptors
Bláha, Jan ; Vaněk, Ondřej (advisor) ; Šulc, Miroslav (referee) ; Obšil, Tomáš (referee)
Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to...
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