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Studies on structure and biological functions of NKR-P1 receptors
Rozbeský, Daniel ; Novák, Petr (advisor) ; Konvalinka, Jan (referee) ; Drbal, Karel (referee)
Natural killer (NK) cells play a significant role in the detection and destruction of virally infected and tumor cells. The NKR-P1 receptors regulate NK cell function by an alternative missing-self recognition system. Although the NKR-P1 receptors were among the first surface NK receptors identified on rodent NK cells more than 20 years ago, there is still very little known about their biological function and their physiological ligands. Furthermore, no three-dimensional structure of any of the NKR-P1 family receptors has been published so far. To understand the functional architecture of mouse NKR-P1 receptors, we developed a simple and efficient protocol providing large amounts of pure soluble NKR-P1 proteins. The crystal structure of mouse NKR-P1A, determined at 1.7 A resolution, is the first structure of a representative of the NKR-P1 family. Crystal structure is formed by a compact C-type lectin-like domain and an extended loop that participates in domain swapping. A potential role of the swapped loop has been suggested in natural ligand binding by in silico studies. However, chemical cross-linking and H/D exchange in combination with high resolution mass spectrometry revealed this loop in close proximity to the compact core in solution. The discrepancy between the crystal and solution structure...
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Derivation and characterization of tumor cell lines labeled with fluorescent proteins
Majerová, Miriam ; Šmahel, Michal (advisor) ; Drbal, Karel (referee)
The effective treatment of cancer is hindered by the mechanisms of tumor cells allowing them to escape from immunosurveillance. One such mechanism is the downregulation of MHC I expression by tumor cells. As a result, CD8+ T lymphocytes are not able to eliminate tumor cells. These cells are often characterized by different expression of MHC I, which leads to the heterogeneity of the tumor environment. This thesis describes a production of a model of MHC I heterogeneity in tumors. Expression plasmids carrying genes for FP were created. Tumor cell lines TC-1, TC-1/A9 and TC-1/dB2m with different expression of MHC I molecules were successfully labeled with these plasmids. When monitoring stability of FP expression by these cell lines, a decrease was observed both in vitro and in vivo. The assumption that the cytokine environment of the tumor induces FP expression could not be confirmed because of unstable FP expression. In a tumor from a mixture of TC-1+TC-1/dB2m cell lines, it was possible to distinguish between these two lines based on the expression of β2m. In a mixture of TC- 1/A9+TC-1/dB2m lines, that could not be done due to the heterogeneity of TC-1/A9 MHC I expression. The use of combined immunotherapy showed the greatest impact on immune cell infiltration in tumors from a mixture of TC-1+...
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Comparative genomic prediction of novel transmembrane adaptor proteins and their expression analysis.
Pitule, Pavel ; Drbal, Karel (advisor) ; Novotný, Marian (referee)
Transmembrane adaptor proteins play an important role in signal transduction cascades emanating from immune cell receptors leading to cellular effector mechanisms. Despite the lack of enzymatic function, their sequence contains interaction motifs which organize other proteins in time and space and initiates building of the signaling complexes. Functionally the most important and for the longest time known TRAPs are the ones associated with immune receptors. Their deficiency has severe impacts on the signal transduction and knock-out mice show dramatic phenotypes. The deficiency of Lat - the first discovered TRAP, which is not constitutively associated with immune receptors - has comparable effects on signaling in lymphocytes. The difference between the first group of TRAPs and Lat is not only the type of association with receptors, but also the lack of tyrosine - based activation motifs (ITAMs) in Lat, but Lat contains several other binding motifs also based on the phosphorylation of tyrosine. The group of TRAPs similar to Lat up today contains eight members, but all of them, except of Lat, has only very mild or undetectable phenotype in the knock-out mice. In this work I searched the protein databases for proteins with the characteristics of TRAP using the bioinformatic tools. Based on our...
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Vliv chemoterapie a genotoxického stresu na imunologické vlastnosti nádorových buněk.
Horňáková, Michaela ; Reiniš, Milan (advisor) ; Drbal, Karel (referee)
Cancer treatment includes the use of chemotherapeutic agents, which have various effects on tumour cells, such as direct toxicity to cancer cells, immunogenic cell death induction and changes in cancer cells phenotype. Throughout the last decade many researchers have been focusing on the induction of genotoxic stress and cellular senescence, which chemotherapy can trigger. Even though induction of senescence in cancer cells represents an important mechanism for tumour suppression, there has been increasing evidence that shifting cancer cells into a senescent state by chemotherapy is not always beneficial. Senescent cells are associated with a specific secretory phenotype, which allows such cells to alter their microenvironment, modulate anti-tumour immunity, induce tumour suppression and even promote cancer development. Therefore, senescent cells elimination by innate or specific immunity, which can be boosted by immunotherapy, can be an important barrier preventing tumour growth. Powered by TCPDF (www.tcpdf.org)
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Therapeutic use of alternative protein binders targeting tumor biomarkers in clinical testing of oncology patients
Tauš, Petr ; Drbal, Karel (advisor) ; Lepšík, Martin (referee)
Almost until the end of the last century, antibodies (aka immunoglobulins) were considered the only class of specific binding proteins. The discovery of hybridoma technology in 1975 had enabled the production of monoclonal antibodies and after twenty years some of them have entered clinical practice. Meanwhile, the first non-immunoglobulin protein scaffold, in which new specific binding sites could be introduced was discovered. To date, many different alternative scaffolds have been described, but only a few of them are being further developed for diagnostics, therapeutics or tools in basic research. Since these structures are overcoming the drawbacks of immunoglobulin structure, which are big size, expensive production and difficult rational design, they have potential to replace and exceed them. In this bachelor's thesis all the alternative scaffolds in development are summarized. Moreover, their advancements in clinical trials are described and compared with approved therapeutics based on immunoglobulin structure.
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FcεRI and Kit signal to actin cytoskeleton via different pathways
Šimíček, Michal ; Dráber, Petr (advisor) ; Drbal, Karel (referee)
Mast cells are key effector cells of the immune system whose exact physiological functions have been the subject of much debate. They have been mentioned mostly as the drivers of allergic and inflammatory reactions via allergens triggering their high affinity receptors for IgE (FcεRI). However, recent findings implicate their important role also in other innate immune functions like host protection from bacterial and viral infections. Agent recognized by surface receptor initialize various intracellular signaling pathways leading to cell response. Thus, better understanding of signal transduction in mast cells is important in development of new therapeutic approaches. Early phases of mast cell activation mediated by FcεRI and/or Kit, a receptor for stem cell factor (SCF), involve phosphorylation of the transmembrane adaptor protein Non-T cell activation linker (NTAL) and mast cell spreading. Morphological studies of mast cells derived from bone marrow of mice deficient in NTAL (NTAL knock out, NTAL -/-) revealed markedly reduced spreading on fibronectin after stimulation with antigen (Ag) alone or in combination with SCF, when compared to wild type (WT) cells. Subsequent quantification of cell area and analysis of other mophological parameters confirmed these observations. Mast cell activation was...
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Studies on interactions between natural killer cell lectin receptors and their protein ligands.
Hernychová, Lucie ; Novák, Petr (advisor) ; Drbal, Karel (referee)
NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.
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Analysis of candidate transmembrane adaptor proteins
Šulcová, Jitka ; Drbal, Karel (advisor) ; Černý, Jan (referee)
Transmembrane adaptor proteins (TRAPs) function as a scaffolding anchor for numerous signaling molecules and facilitate formation of large signaling complexes near the plasma membrane. They regulate spatio-temporal organization of signaling events in order to transduce fast and effective signal from surface receptors into the cell nucleus. The importance of described TRAPs in the development and proper functioning of immune cells in relation to their localization within lipid rafts is still under debate. The simple reason is the lack of a profound phenotype in knock-out mice. The described phenotypes observed in animals deficient in ITAM-containing adaptors of immunoreceptors as well as lipid raft adaptor Lat all lead to the block in the T-cell development and impaired TCR signaling. The subject of my diploma thesis is to perform bioinformatic search and filter candidate TRAP genes according to predicted and published data. Selected TRAP candidates were subjected to functional characterization. I checked for their localization to the plasma membrane and correlate their ectopic overexpression with the expression of CD69 surface Tcell activation marker. From 14 candidate TRAPs, only a single Pdzk1ip1 protein is probably localized to the plasma membrane but its expression does not influence CD69...
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Surface phenotype of human carcinoma cancer stem cells (CSC)
Bočková, Marie ; Drbal, Karel (advisor) ; Čermák, Vladimír (referee)
Tumor is composed of a heterogenous mass of cells. Similar to normal healthy organs and tissues, these can be divided into individual cellular subpopulations according to morphology, function and expression patterns. A subpopulation of cells that are able to give rise to all of these cellular lineages is referred to as cancer stem cells (CSC). CSCs have the capabilities of normal stem cells such as the self-renewal and the ability to give rise to a heterogenous population of differentiated cells. Usually, this is the most resistant subpopulation within a tumor, highly non-responsive to therapy. Doing so, they are the cause of residual disease. Characterisation of CSC markers of individual tumor types is beneficial since it enables higher therapy efficacy via targeting this cell population. The -omics approaches to characterisation of the surface proteome bring a broader view into the field when searching for a unique gene signature of specific cancer stem cell types. It has been found that these cells can be identified based on the high expression levels of CD44, CD90 and CD49f. Among other markers, CD47 is an important marker for its immunosuppressive function.
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