National Repository of Grey Literature 188 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Production and characterisation of human C1 inhibitor and Plasmodium falciparum PfMSP3.1 recombinant proteins for structural studies
Čápová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
PfMSP3.1 is one of the surface proteins of the intracellular parasite Plasmodium falciparum, which causes malaria. As one of the evasion strategies of the immunity system of the human host this protein interacts with one regulator of the complement system - C1 inhibitor. Determining the exact binding site and its structural assessment would help to better understand the interaction between the parasite and the host, which is necessary for the disease progression and thus for the development of a potential therapy. In the theoretical part of the thesis, the life cycle of Plasmodium falciparum, the role of the parasite stage called merozoite, the role of its surface proteins, including merozoite surface protein 3, in the attack of red blood cells by the parasite, are described in more detail. It also briefly describes the complement system, its activation pathways and the regulation of these pathways. The experimental part includes the cloning of plasmids to produce C1 inhibitor and various forms of merozoite surface protein PfMSP3.1, transfection of S2 insect cells with these plasmids, subsequent protein expression in S2 cells and their purification. In the second half of the experimental part, we tried to create complexes of C1 inhibitor with individual PfMSP3.1 forms and an attempt to crystallize...
Synthesis and characterization of binding ligands for the study of targeted lysosomal protein degradation
Sidej, Natan ; Konvalinka, Jan (advisor) ; Baszczyňski, Ondřej (referee)
Targeted protein degradation is a novel concept of chemical biology that has been formulated about 20 years ago. Its central postulate is based on the fact that instead of suppressing protein activity with low-molecular inhibitors, we can instead use molecular tools to hijack the host organism's own degradation pathways and force it to degrade chosen proteins by itself. This diploma thesis revolves around the preparation of biocompatible polymeric conjugates called "iBodies" that will be used to induce targeted lysosomal degradation of two model enzymes - Fibroblast activation protein α, and Glutamate carboxypeptidase II. First, a total of four low-molecular ligands were prepared and fully characterized by standard methods of organic synthesis. The first two are mannose-6-phosphonate derivatives that serve as the inducers of protein degradation via the cellular endosomal-lysosomal degradation pathway. The remaining two are known potent inhibitors of the chosen model enzymes that will serve as their targeting-ligands. The prepared compounds were then used to prepare a total of eight poly-N-(2-hydroxypropyl)methacrylamide conjugates called iBodies, after which the polymeric conjugates were fully characterized by standard means of macromolecular chemistry. Afterwards, the obtained conjugates will be...
Functional Characterization of SCFFBXO38 Ubiquitin Ligase-dependent Protein Degradation
Dibus, Nikol ; Čermák, Lukáš (advisor) ; Konvalinka, Jan (referee) ; D´Angiolella, Vincenzo (referee)
Ubiquitin ligases are responsible for the specific recognition of proteins targeted for proteasome-dependent degradation. This project focused on the molecular and functional characterization of the SCFFBXO38 ubiquitin ligase. As with many others, its biological function has not yet been elucidated in detail, although it is the only ubiquitin ligase whose mutations lead to the onset of a distal form of muscle atrophy. In the first part of our project, we identified new substrates for this ubiquitin ligase, the nuclear proteins ZXDA and ZXDB, with insufficiently characterized functions. Using genetic and biochemical methods, we have shown that ZXDA/B proteins act as positive regulators of centromeric chromatin integrity and that experimental inactivation of the SCFFBXO38 ubiquitin ligase resulted in a ZXDA/B-dependent stabilization of CENP-A and CENP-B proteins in the centromeric regions. In the second part of the project, we focused on analyzing the mouse model deficient in the Fbxo38 gene. We demonstrated that loss of Fbxo38 leads to growth retardation affecting various organs, including the male reproductive system. A detailed histological examination revealed pathological alterations in the seminiferous tubules, accompanied by a lower number of spermatozoa and decreased fertility. We have shown...
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...
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
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.
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 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...
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...
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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