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Regulation of LAT trafficking to the plasma membrane
Rakhimbekova, Anastasia ; Cebecauer, Marek (advisor) ; Černý, Jan (referee)
Linker for activation of T cells is a palmitoylated transmembrane adaptor protein, which is expressed in most of immune cells, but not in immature and mature B cells. It plays an important role in T-cell activation and maturation. LAT is synthesized in the endoplasmic reticulum. Its sorting to the plasma membrane is controlled with various determinants, such as properties of the transmembrane domain and structural motifs in the intracellular part of the protein. Some of those determinants are important for posttranslational modifications, export from the Golgi apparatus and, probably, membrane microdomain targeting, while others interact with COPII machinery and mediate protein export from the endoplasmic reticulum or targeting to endosomes. Keywords: LAT, functional motifs, protein sorting, plasma membrane, Golgi apparatus
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Modulatory effect of monovalent ions on δ-opioid receptors
Vošahlíková, Miroslava ; Svoboda, Petr (advisor) ; Jakubík, Jan (referee) ; Kršiak, Miloslav (referee)
The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.
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The role of membrane cholesterol in delta-opioid receptor signaling Correlation with plasma membrane structure
Brejchová, Jana ; Svoboda, Petr (advisor) ; Novotný, Jiří (referee) ; Teisinger, Jan (referee)
Study of HEK293 cells stably expressing fusion protein between delta opioid receptor (δ-OR) and pertussis toxin-insensitive mutant of Gi1α protein, δ-OR-Gi1α (Cys351 -Ile351 ), provided the following results. Decrease of plasma membrane cholesterol content (cholesterol depletion) induced by cyclic oligosaccharide β-cyclodextrin did not affect binding of specific δ-OR agonist, [3 H]DADLE. Neither the maximum number of binding sites nor the affinity of [3 H]DADLE binding was changed by cholesterol depletion. However, the ability of δ-OR to activate cognate trimeric G proteins was impaired. EC50 value of agonist-stimulated [35 S]GTPγS binding was an order of magnitude higher. This effect was observed in case of both control and pertussis toxin-treated cells. It means that cholesterol depletion markedly reduced the efficiency of functional coupling of δ-OR to endogenously expressed pertussis toxin-sensitive G proteins of Gi/Go family as well as covalently bound Gi1α (Cys351 -Ile351 ) protein. Unchanged plasma membrane cholesterol content is therefore important requirement for proper δ-OR function. Detection of the effect of cholesterol depletion on the functional activity of δ-OR was supported by the analysis of changes in biophysical state of plasma membrane using fluorescent membrane probes,...
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The effect of ethinylestradiol on Na+, K+ - ATPase
Kettnerová, Karolína ; Svoboda, Petr (advisor) ; Driák, Daniel (referee)
This diploma thesis is oriented to analysis of physiological effect of synthetic estrogen ethinylestradiol (EE), which represents the main component of steroid-based substance used in hormonal contraception. From wide range of physiologically important protein molecules, which might be effected by this steroid, thesis focuses to the study of the sodium plus potassium activated, magnesium dependent adenosinetriphosphatase (Na+, K+ - ATPase), which is selectively inhibited by cardiac glycosides such as ouabain (g strophantine). Na+, K+ - ATPase represents an important plasma membrane bound enzyme, which catalyzes the active transport of sodium and potassium across plasma membrane. In the first part of this work, Na+, K+ - ATPase was determined by binding of radioactively labeled selective inhibitor of this enzyme [3H]ouabain, used for this purpose. In the second part of this work, plasma membrane fluidity was analyzed by steady-state fluorescence anisotropy of DPH. The effect of EE on [3H]ouabain binding was studied first under in vitro conditions by using human embryonic kidney cells (HEK293) which were cultivated for 24 hours in the presence of EE in tissue culture medium. Second, the effect of EE was also studied under in vivo conditions, by subcutaneous application of EE to the female rats of Wistar...
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Cell signalling and molecular complexes of the TRH receptor
Drastichová, Zdeňka
The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained high amounts of Na+ ,K+ -ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM-enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH compared to control cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed effects of TRH on the...
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Cell signalling and molecular complexes of the TRH receptor
Drastichová, Zdeňka ; Novotný, Jiří (advisor) ; Hodný, Zdeněk (referee) ; Říčný, Jan (referee)
1 Summary The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained markedly increased the amount of Na,K-ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM- enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH than in control untreated cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed...
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Caveolae and caveosoms
Galica, Tomáš ; Forstová, Jitka (referee) ; Černý, Jan (advisor)
Caevolae are remarkably stable structures at the plasma membrane. They form specific domains distinct in lipid composition from the rest of plasma membrane. Many diverse functions are assigned to Caevolae. They play role in modulation of cellular surface, signalization and well regulated endocytosis. Caveosomes suppose to be large intracellular vesicular structures potentialy new membrane organels. They are derived from internalized caveolae. Tohether with caveolae they are proposed to form a separeted system of intracellular vesicles. However recent evidence suggests that caveolae can fuse with endosomes immediately after internalization. If this is true, then the system of vesicles derived from caveolae, including caveosomes, can be considered a regular component of endosomal system. Isolation of caveosomes from endosomes has been seen mainly in experiments where polyomavirus SV40 was used. Thus the question, if this isolation is not just a result of SV40 infection, arises. It has been shown recently that SV40 virus is capable of inducing caveosome-like structures even in the absence of caveolae. Consequently existence and properties of caveosomes are being questioned. The problem of high importance is the genesis of caveosomes and their existence in SV40 non-infected cells. In this thesis...
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