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Mass Spectrometry-Based Identification of a Potential Binding Partner of Glutamate Carboxypetidase II
Tužil, Jan ; Konvalinka, Jan (advisor) ; Novák, Petr (referee)
English Abstract The incoming paradigm of the network (or systems) biology calls for a new high throughput tool for a wide scale study of protein-protein interactions. Mass spectrometry-based proteomics have experienced a great progress in recent years and have become an indispensable technology of elementary as well as clinical research. Glutamate carboxypeptidase II (GCPII; EC 3.5.17.21) is a transmembrane protein with two known enzymatic activities. Its expression is highly upregulated in some solid tumors and also in tumor-associated neovasculature in general. Nevertheless, none of the two enzymatic activities were shown to be physiologically relevant to these cells. Some facts point at a possible receptor function of GCPII, however, no specific binding partner has been found yet. In the search for potential binding partners and/or ligands of GCPII, a series of methods have been employed, including pull-down experiment, immunoprecipitation and mass spectrometry. Sample preparation and mass spectrometry data processing methodology was specifically developed in order to identify potential binding partners. As one of the outcome of that methodology, the interaction of β-subunit of F1 ATP synthase was selected for further detailed analysis as a putative ligand of GCPII.
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Digestive proteases of termites
Čermáková, Markéta ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)
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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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Vascular Endothelial Growth Factor Expression and its Application in Vascular Tissue Engineering
Mikulová, Barbora ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
This paper deals with the expression of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and its use in tissue engineering of vascular wall. During the work interaction of endothelial cells with the modified fibrin-based biomaterial into which vascular endothelial growth factor (VEGF-A121) has been incorporated was monitored. This modification supported the adhesion and growth of endothelial cells. Vascular endothelial growth factor VEGF-A121 is signal glycoprotein that activates transmembrane receptors on endothelial cells. VEGF-A121 is a key regulator in vasculogenesis, angiogenesis, proliferation, migration and survival of endothelial cells. In this work, this protein was heterologously expressed at a thioredoxin fusion partner in an expression system of E. coli Origami B (DE3). Recombinant VEGF-A121 was additionally coexpressed with bacterial chaperones GroEL/GroES for potential increase of its solubility and biological activity. In the next part of this work thin fibrin network was prepared by catalytic action of thrombin on the polystyrene-bound monolayer of fibrinogen. This network has been further enriched by vascular endothelial growth factor (VEGF-A121), which was covalently incorporated in it by enzyme activity of transglutaminase (factor XIIIa). The last...
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Nové inhibitory HIV proteasy: návrh, synthesa a testování aktivity
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
More than 20 years after its discovery HIV protease still remains one of the primary targets in HIV treatment. Currently there are 9 approved protease inhibitors on the market. However, due to immense replication rate and the high error prone nature of reverse transcriptase, resistance to each of them has already been described. Therefore, the search for new protease inhibitors with different binding mode is still active. A novel type of protease inhibitors (1, 4-benzodiazepine analogs) was recently discovered in our laboratory. Even though this new class of inhibitors is highly potent (Ki' in range of 10-9 ), it also has several undesirable qualities, such as low solubility and a high number of stereogenic centers. Primary objective of this study was to try to prepare more soluble compounds with lower number of possible stereoisomers, enzymologically characterize its binding to the wild-type and mutated HIV protease and to determine its structure in the complex with the enzyme. A small library of 1, 4-benzodiazepine inhibitors of HIV protease was synthesized and fully characterized using NMR spectroscopy and mass spectroscopy. The number of stereogenic centers was successfully reduced from 4 to 2 without loosing activity of the inhibitor. The improvement in solubility was always associated with a...
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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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Production of recombinant cathepsin C from human blood fluke
Illichová, Hana ; Konvalinka, Jan (advisor) ; Martínková, Markéta (referee)
Blood flukes of the genus Schistosoma cause schistosomiasis, a serious parasitic disease occurring in tropical and subtropical areas. Cathepsin C (EC 3.4.14.1) is a digestive enzyme of the blood flukes which participates in the degradation of hemoglobin through its dipeptidyl aminopeptidase activity. This enzyme is critical for metabolism of the parasite and represents a potential target for the development of antischistosomal drugs. Cathepsin C has not yet been studied in detail. This bachelor thesis is focused on the development of expression systems for production of recombinant cathepsin C of Schistosoma mansoni (SmCC). The yeast Pichia pastoris system was used for the expression of an inactive SmCC precursor (zymogen) whose proteolytic stability was analysed. Furthermore, the expression system for SmCC in the protozoan Leishmania tarentolae was employed, and four different SmCC constructs were prepared to optimize production.
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