|
|
Study of molecular mechanisms of the signaling proteins regulation
Kylarová, Salome ; Obšilová, Veronika (advisor) ; Mikšík, Ivan (referee) ; Novák, Petr (referee)
EN The aim of this study was to investigate the regulatory mechanisms of two important signaling proteinkinases and promising therapeutic targets, ASK1 and CaMKK2. ASK1 kinase is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-JNK kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress. The function of ASK1 is associated with the activation of apoptosis and thus plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration or cardiovascular diseases. The natural inhibitor of ASK1 is a ubiquitous oxidoreductase, thioredoxin, which is probably bound to N-terminus of ASK1, thus preventing a homophilic interaction and subsequent ASK1 activation. It has been suggested, that upon oxidative stress and oxidation of thioredoxin active site, thioredoxin dissociates from ASK1, but the structural basis of this interaction remains unclear. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of CaM kinase pathway that activates CaMKI, CaMKIV and AMPK involved in gene expression regulation or apoptosis activation. Function of this protein is often associated with neuropathology, carcinogenesis and obesity. CaM kinases are activated via binding Ca2+ sensor protein...
|
|
|
Study of mechanism of action of anticancer drug tamoxifen and its toxic side effects
Kylarová, Salome ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
Anti-estrogen therapy is used for treatment of hormone (estrogen) receptor positive breast cancer. The rise of this type of cancer is associated with a prolonged exposure to these hormones throughout life. Tamoxifen is one of the most used hormonal drugs, which blocks the effects of these hormones in breast cancer tissue by competitive binding to hormonal receptors. The affinity of tamoxifen to these receptors is not sufficient, therefore it has to be activated to metabolites having greater affinity, namely 4-hydroxytamoxifen and endoxifen. The formation of these intermediates is catalysed by cytochromes P450. In the second phase of its biotransformation hydroxylated metabolites of tamoxifen are primarily sulphated by sulphotransferases and eliminated from the body. In addition to these active intermediates, which inhibit the growth of breast tumor tissue, there are metabolites causing negative effects in the others. The most important metabolite is α-hydroxytamoxifen, which forms covalent DNA adducts in liver tissue of rats and endometrium of females. Tamoxifen therapy is associated with numerous side effects, but the greatest attention is focused to formation of endometrial cancer and induction of tumor's resistance to this therapy. Effects of tamoxifen therapy are dependent on the activity of...
|
|
|
Study of molecular mechanisms of the signaling proteins regulation
Kylarová, Salome ; Obšilová, Veronika (advisor) ; Mikšík, Ivan (referee) ; Novák, Petr (referee)
EN The aim of this study was to investigate the regulatory mechanisms of two important signaling proteinkinases and promising therapeutic targets, ASK1 and CaMKK2. ASK1 kinase is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-JNK kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress. The function of ASK1 is associated with the activation of apoptosis and thus plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration or cardiovascular diseases. The natural inhibitor of ASK1 is a ubiquitous oxidoreductase, thioredoxin, which is probably bound to N-terminus of ASK1, thus preventing a homophilic interaction and subsequent ASK1 activation. It has been suggested, that upon oxidative stress and oxidation of thioredoxin active site, thioredoxin dissociates from ASK1, but the structural basis of this interaction remains unclear. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of CaM kinase pathway that activates CaMKI, CaMKIV and AMPK involved in gene expression regulation or apoptosis activation. Function of this protein is often associated with neuropathology, carcinogenesis and obesity. CaM kinases are activated via binding Ca2+ sensor protein...
|
|
|
Preparation and characterization of binding partners of phosducin.
Kylarová, Salome ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
AABBSSTTRRAACCTT Phosducin (Pdc) is a highly conserved acidic phosphoprotein, which plays an important role in the regulation of G-protein signalization in intact retina. It binds to Gβγ dimer of heterotrimeric G-protein transducin thereby decreases the pool of available transducin resulting in modulation of signal. Function of phosducin is negatively regulated by its phosphorylation followed by interaction with the 14-3-3 protein. Besides this established way of regulation, we were interested in other putative interaction partners of phosducin, like SUG1 and CRX. SUG1 is a subunit of 26S proteasome with a large scale of biological functions, especially a degradation of many transription factors. Its role in regulation of phosducin is still unclear, but is probably involved in targeting of phosducin to 26S proteasome for its degradation. Subsequently, we prepared four different expression constructs of full-length protein in order to find the best expression and purification strategy. These results suggest that all purified fusion proteins of SUG1 form stable and soluble high molecular weight oligomers. This behaviour was confirmed by dynamic light scattering and analytical ultracentrifugation measurements. In addition, this observation is consistent with previous studies of its bacterial counterpart, PAN...
|
|
|
Study of mechanism of action of anticancer drug tamoxifen and its toxic side effects
Kylarová, Salome ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
Anti-estrogen therapy is used for treatment of hormone (estrogen) receptor positive breast cancer. The rise of this type of cancer is associated with a prolonged exposure to these hormones throughout life. Tamoxifen is one of the most used hormonal drugs, which blocks the effects of these hormones in breast cancer tissue by competitive binding to hormonal receptors. The affinity of tamoxifen to these receptors is not sufficient, therefore it has to be activated to metabolites having greater affinity, namely 4-hydroxytamoxifen and endoxifen. The formation of these intermediates is catalysed by cytochromes P450. In the second phase of its biotransformation hydroxylated metabolites of tamoxifen are primarily sulphated by sulphotransferases and eliminated from the body. In addition to these active intermediates, which inhibit the growth of breast tumor tissue, there are metabolites causing negative effects in the others. The most important metabolite is α-hydroxytamoxifen, which forms covalent DNA adducts in liver tissue of rats and endometrium of females. Tamoxifen therapy is associated with numerous side effects, but the greatest attention is focused to formation of endometrial cancer and induction of tumor's resistance to this therapy. Effects of tamoxifen therapy are dependent on the activity of...
|
guest ::
