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Analysis of Zika and Dengue virus proteases
Novotný, Pavel ; Konvalinka, Jan (advisor) ; Vondrášek, Jiří (referee)
in English Zika and Dengue flaviviruses are transmitted by mosquitoes in human populations living in tropical areas. They cause fevers which in the case of Dengue can lead to life threatening haemorrhagic form. There is a possible relationship between pregnant women being infected by Zika virus and higher risk of microcephaly in new-borns. The infection is currently treated mainly symptomatically. However, there is an effort to develop compounds which block viral life cycle and viral spread through organism. Viral enzymes, such as flaviviral proteases, are regarded as suitable targets for this effort. These serine proteases with chymotrypsin fold are heterodimers which consist of flaviviral non- structural proteins NS2B and NS3. NS3 domain also contains a helicase, which can be removed by gene recombination for study purposes. NS2B is a transmembrane protein, but only a hydrophilic 40 amino acid peptide is important for the interaction with NS3 domain. This peptide has a chaperon function and participates in substrate binding to the active site. In this study, six variants of recombinant proteins containing activating peptide of NS2B and protease domain of NS3 were expressed and purified. Four variants were characterized in enzymologic studies including testing of possible inhibitors. A dipeptide...
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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Konvalinka, Jan (advisor) ; Brynda, Jiří (referee)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Characterisation of recombinant mouse glutamate carboxypeptidase III
Janoušková, Karolína ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Glutamate carboxypeptidase II (GCPII, PSMA, NAALADase) is transmembrane metalopeptidase and due to cleavage of substrates β-citryl-L-glutamate (BCG), N-acetyl-L-aspartyl-L-glutamate (NAAG) and polyglutamylated folates (Pte-Glun) is being studied as potential therapeutic target. Enzymes, which could compensate for enzyme activity and functions of GCPII, are thus relevant targets of enzymology as well. One of GCPII's homologs with similar enzyme activity is mouse glutamate carboxypeptidase III (GCPIII, NAALADase II). Enzymatic cleavage has not been determined using recombinant mouse GCPIII yet. It is important to kinetically characterize mouse GCPIII so that we can compare enzyme activity with human ortolog. Then we can find out whether mouse model is comparable with human. Recombinant mouse GCPIII was kinetically characterized. Kinetic parameters (KM, kcat) for recombinant mouse GCPIII were measured for substrates NAAG and BCG using radioactive assay. Experiments with the substrate Pte-Glu2 were analyzed using HPLC method. Although human GCPIII is more effective than mouse ortolog at clearage of NAAG, both enzymes are comparable during hydrolysis of BCG. Those results can contribute to better understanding of the role of GCPIII in the most commonly used animal model.
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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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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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Expression, characterisation and biological role of Ddi II, putative protein partner of proteasomal complex
Sivá, Monika ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Cell homeostasis is maintained via strictly regulated processes. One of the important regulation systems is ubiquitin-proteasome proteolytic pathway. Proteins to be degraded are posttranslationally modified with polyubiquitin chains and targeted to the proteasome for degradation. Ubiquitin-proteasome system consists of several processes: ubiquitination of target substrates via set of enzymes, substrate transfer and degradation in the 26S proteasome. There are two ways of ubiquitinated substrate recognition via proteasome. It is either directly by proteasomal receptors or by protein shuttles. Shuttling factors bind polyubiquitinated target substrate and transfer it to the entrance of proteasomal cavity thanks to their typical domain architecture. The N-terminal ubiquitin-like domain binds to regulatory particle of the proteasome and the C-terminal ubiquitin-associated domain binds polyubiqitinated chains on substrates. This thesis focuses on the human DNA damage-inducible protein homolog 2 (Ddi2), a potential member of protein shuttles of humans, and on the interaction of its ubiquitin-like domain with its putative interaction partner, a proteasomal subunit PSMD2. PSMD2 has been cloned, expressed and purified in sufficient yields for further experiments. "Cold" as well as isotopically labeled UBL domain of...
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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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