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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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Preparation of contructs for transgenic expression of DPP-IV and FAP
Košek, Dalibor ; Bezouška, Karel (advisor) ; Křepela, Evžen (referee)
Preparation of contructs for transgenic expression of DPP-IV and FAP Bc. Dalibor Košek Abstract: DASH (Dipeptidyl peptidase-IV Activity and/or Structure Homologues) protein group involves multi-funcional molecules typically bearing enzymatic activity similar to the Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with lymphocyte differentiation antigen CD26). In general, they cleave multiple regulatory as well as structural peptides and proteins, possessing proline residue on the penultimate position from the N-terminus. We focused on two members of this group: canonical DPP-IV and Fibroblast activation protein alpha (FAP-α). Both are typically type II plasma membrane proteins with specific tissue distribution. Soluble extrecellular forms have also been identified. Available knowledge suggest important roles of these proteins in oncogenesis, executed by their enzymatic activity but also by non-proteolytic interactions. To study their role in gliomagenesis we designed several experimental models exploiting astrocytoma cell lines with defined DPP-IV or FAP-α phenotype. Enzymatically inactive forms and analogues with different subcellular distribution will also be included. These models will allow to assess the impact of DPP-IV and FAP-α on the glial tumor development and the importance of their...
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Optimization of recombinant protein expression in HEK293 cell line
Bláha, Jan ; Bezouška, Karel (advisor) ; Šácha, Pavel (referee)
Mammalian cells have become the dominant system for recombinant expression of pharmaceutical proteins. This system is becoming suitable also for structural biology, with the advances in methodology of transient transfection of mammalian cells. This work dealt with optimization of recombinant expression in HEK293T and HEK293-6E cell lines in various media using easily quantifiable markers - secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). Emphasis was placed on optimizing key factors behind the creation of transfection complex - the ratio of DNA to polyethyleneimine and the amount of DNA used. The positive influence of histone deacetylases inhibitor valproic acid and also of casein hydrolysate Tryptone N1 was also studied. (The thesis is written in Czech.)
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Analysis of influence of glycosylation on assembly and molecular architecture of enzymes.
Horáková, Jana ; Bezouška, Karel (advisor) ; Ryšlavá, Helena (referee)
Our laboratory studies the influence of glycosylation on protein architecture and the biological activity of fungal hexosaminidases. I studied the enzyme from Penicillium oxalicum which has an advantage of quite a homogenous glycosylation. The propeptide of this enzyme is naturally glycosylated only at two places - asparagine 11 and serine 66. Catalytic subunit and propeptide of the hexosaminidase enzyme from Aspergillus oryzae were separated from each other for reconstitution experiments. In the propeptide part of the enzyme separated from the Penicillium oxalicum hexosaminidase, asparagine 11 and serine 66 were swapped for cysteine by targeted mutagenesis. The newly synthesized propeptide modified with cellobiose, which is a compound most similar to naturally occurrency saccharide chitobiose, promised a significant chance of restoration of enzyme activity. The glycosylated propeptide was separated from the nonglycosylated. In reconstitution experiments we observed the influence of glycosylation on the enzyme structure and activity using different glycosylated propeptides. The highest efficiency occurred during the reconstitution by the original propeptide. Next we used combinations of artificially glycosylated propeptides of which the highest reconstitution efficiency had cellobiose(Asn11...
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Immunological testing of combined LELTE / saccharide dendrimers
Kádek, Alan ; Bezouška, Karel (advisor) ; Man, Petr (referee)
Immunological testing of combined LELTE / saccharide dendrimers. (in Czech) Alan Kádek (Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic) CD69 is a widespread receptor of the immune system cells. Although a physiological ligand for this receptor is still unknown, in Laboratory of protein architecture its affinity towards a range of compounds including calcium, carbohydrates and charged compounds such as carboxylated calixarenes, has been proved. Moreover, a pentapeptide sequence LELTE derived from a mycobacterial chaperonine protein hsp65 has been recently identified as a ligand for CD69 representing a "danger" signal for the immune system. However, this peptide is not immunoactive per se, but only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using bi- or polyfunctional reagents. In this thesis I evaluate for four newly synthesized compounds their in vitro immunological effects on cellular signalization, proliferation, natural killing of tumors and on induction of apoptotic cell death in immunocytes. The tested compounds present LELTE peptide through attachment to a cyclopeptidic RAFT scaffold (K-K-K-P-G)2 through the ε-amino groups of lysine residues, alone or in combination with a...
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The possibilities to influence the response of innate immune cells to gliadin
Drašarová, Hana ; Tučková, Ludmila (advisor) ; Bezouška, Karel (referee)
Gluten sensitive entheropathy - celiac disease is a lifelong, genetically predisposed, immunologically mediated susceptibility to dietary wheat gluten, most frequently demonstrated by small-bowel damage and malabsorption syndrome. Strict adherence to gluten-free diet is the sole rational therapy of the disease. One of the possible therapeutic strategy for the treating of celiac disease is to utilize the synthetic polymer P(HEMA-co-SS). This polymer is capable specifically bound gliadin in gastrointestinal tract and by this way to neutralize the damaging effect of this alimentary protein on mucosa of small intestine in celiac patients. The in vitro study on human PBMC and specimens of small intestinal biopsies of celiac patients in our laboratory demonstrated that putative therapeutic ability of P(HEMA-co-SS) is substantially influenced by degree of proteolytic processing of gliadin and P(HEMA- co-SS) and also by different timing of per os administration of both components in organism. Another putative adjuvant therapy of celiac disease is employing of the beneficial probiotic bacterial strains. Our experiments were based on the findings of Prof. Y Sánz and her group demonstrating the significant differences in the composition of bacterial microflora in patients with active form of celiac disease,...
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