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Molecular mechanisms and functions of 14-3-3 proteins
Šilhán, Jan ; Obšil, Tomáš (advisor) ; Krůšek, Jan (referee) ; Schneider, Bohdan (referee)
Závěr Hlavním cílem této doktorské práce bylo objasnění molekulárních mechanismů funkce 14-3-3 proteinů a vlivu na proteiny FOXO4 a tyrosinhydroxylasu. V první časti této práce (publikace I) byla potvrzena předložená hypotéza polohy Cterminálního konce molekuly 14-3-3. Bylo ukázáno, že v nepřítomnosti ligandu se Cterminální konec nachází ve vazebném místě a brání tak vstupu ligandů. Po vazbě fosforylovaných ligandů, dochází k velmi silné vazbě a vytěsnění C-terminálního konce 14-3- 3 proteinu z vazebného místa. Tyto výsledky jsou v souladu s původními pracemi, které navrhly důležitost tohoto segmentu jako inhibitoru nepatřičných ligandů. Druhá část této doktorské práce poskytuje rozsáhlý popis vlivu 14-3-3 proteinů na transkripční faktory FOXO4. S použitím stacionární a časově-rozlišené fluorescence byla studována interakce 14-3-3 proteinu s fosforylovaným transkripčním faktorem FOXO4. Navázání 14-3-3 proteinu způsobuje rozpad komplexu FOXO4:DNA. Tato část práce charakterizuje interakci 14-3-3 proteinu s DNA-vazebnou doménou FOXO4. Výsledky neprokázaly výrazné konformační změny v rámci DNA-vazebné domény. Spíše dochází ke sterickému bránění vazby DNA (publikace II). Ve třetí části se práce zabývá studiem interakcí 14-3-3 s fosforylovaným ligandem odvozeným od C-konce enzymu serotonin N-acetyltransferasa...
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Study of the action of ivermectin on purinergic P2X4 receptor
Jelínková, Irena ; Teisinger, Jan (advisor) ; Šťastný, František (referee) ; Krůšek, Jan (referee)
The P2X4 receptor is ATP-gated cation channel. It is the only mamrnalian purinergic receptor which is modulated by extracellularly applied ivennectin (IVM). rVM is an allosteric modulator that has several effects on receptor [unction: it increases sensitivity to agonists, potentiates maximum current amplitude and prolongs the deactivation kinetics of the channel after agonist washout. The aim of this study was to localize IVM binding site and using its positive allosteric effect to get new inforrnatioll about the structure and function of P2X. receptor. Initially we focused on identification of regions and residues responsible for IVM effect on channel function. We used several chimeras of P2X2 and P2X. receptors and P2X. receptors with single point mutatioll. Experiments with chimeric receptors revealed that extracellular sequence V49-V61 but not tbe sequ nce V64-Y315 is important for the effects af IVM on channel deactivation. Receptor-specific alanine mutations placed in transmembrane domains 029-V61 and N338-L358 showed the importance of residues W50, V61 and V357 for TVM effect Oll channel deactivation. We tested further the irnportance of aH residues in transmembrane domains. Cysteine scanning mutagenesis supported the relevance of previously identified W50 residue and showed the importance ofresidues...
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Identification of the binding sites on transient receptor potential cation channel TRPC6 for Calmodulin and S100A1
Bílý, Jan ; Teisinger, Jan (advisor) ; Krůšek, Jan (referee) ; Pavlíček, Jiří (referee)
Identification of the binding sites on transient receptor potential cation channel TRPC6 for Calmodulin and S100A1 The TRP (transient receptor potential) group of ion channels represents a large subset of membrane receptors. A part of this supergroup are canonical TRPC channels with a sequence homology analogical to TRP receptor first discovered at fruit fly (Drosophila melanogaster). These membrane channels are involved in a variety of physiological functions in different cell types and tissues. TRPC6 is a non-selective cation channel that modulates the calcium level in eukaryotic cells (including sensory receptor cells) in response to external signals. TRPC6 channel contains binding domain CIBR (Calmodulin inositol binding region), which is also able to adapt to calcium binding protein S100A1. Characterisation of the integrative binding site for calmodulin (CaM) and S100A1 at the C-tail of TRPC6 is presented in this work. Using site-directed mutagenesis, soluble protein fragments TRPC6 CT (801-787) were prepared with intentional changes in amino acid sequence. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by measurement of fluorescence anisotropy influence and their participation in the calcium-dependent binding of CaM and/or S100A1 to...
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The comparison of the performace of carbocyanine dyes disC3(3) a diSC3(5) in fluorescent probing of yeast cell membrane potential.
Matunová, Petra ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
Membrane potential represents a voltage across a membrane and it is an important parameter that helps to describe processes in cells. Carbocyanine fluorescent probes diS-C3(3) and diS-C3(5), for which a common short chemical name 3,3'- dipropylthiadicarbocyanine iodide is used, are suitable for monitoring membrane potential changes of cells in which microelectrodes can not be used because of a small size of the cells. These changes can be measured on the scale of mV. A spectral analysis of cell suspensions containing a fluorescent probe makes it possible to determine the ratio of extracellular and intracellular concentrations of the probe. Using it we can calculate the value of membrane potential changes which can be induced by an outer stimulus. This Bc. thesis presents a comparison of the rate of accumulation of the above mentioned fluorescent probes in yeast cells, as well as experiments aimed for studying an inuence of different substances and their various concentrations on free and bound component of the dye.
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Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors
Kaniaková, Martina ; Krůšek, Jan (advisor) ; Chaloupka, Roman (referee) ; Zemková, Hana (referee)
Title: Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors Author: Mgr. Martina Kaniaková Department: Institute of Physiology AS CR, v.v.i. Supervisor: RNDr. Jan Krůšek, CSc., Institute of Physiology AS CR, v.v.i. Abstract: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. We use the whole-cell patch-clamp technique to study functional and pharmacological properties of muscle and neuronal nicotinic receptors. Rat neuronal receptors were heterologously expressed in COS cells and human embryonic muscle receptors were studied in TE671 cells. Lobeline, a plant alkaloid with a long history of therapeutic use, interacts with the classical agonist-binding site of nAChRs. The final result of this interaction depends on the receptor subtype, lobeline and other agonists concentrations and the time schedule of application. Generally, lobeline is a very weak partial agonist eliciting deep desensitization at several subtypes of nAChRs. In combination with other agonists, lobeline acts as a competitive antagonist or coagonist. Using point mutation procedure we studied the functional role of negatively charged amino acids in the F-loop of β2 and β4 subunits of neuronal receptors. Neutralising mutations in β4 subunit led to up to eighteen-fold increase in the...
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Modulation of nociceptive synaptic transmission
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee) ; Hejnová, Lucie (referee)
Modulation of synaptic transmission in the spinal cord dorsal horn plays an important role in development and maintenance of pathological pain states. The indisputable part of this modulation is conducted via activity of the transient receptor potential cation channel subfamily V member 1 (TRPV1) and the cannabinoid receptor 1 (CB1), expressed on presynaptic endings of primary afferents in the superficial spinal cord dorsal horn. Under physiological conditions, activation of TRPV1 receptors is pronociceptive while CB1 receptor activation leads to attenuation of nociceptive signalling. However, both receptors share also one endogenous agonist anandamide (AEA) that may be produced from N-arachidonoyl phosphatidylethanolamine (20:4-NAPE). Main objective of this thesis focuses on the effect of 20:4-NAPE on nociceptive synaptic transmission in spinal cord slices under naïve and inflammatory conditions and consequent on the possible interaction of TRPV1 and CB1 receptors. First, 20:4-NAPE application induced significant release of anandamide from spinal cord slices under in vitro conditions. Next, patch- clamp recordings of excitatory postsynaptic currents (mEPSC and sEPSC) from superficial dorsal horn (DH) neurons in acute spinal cord slices were used. 20:4-NAPE application under the physiological...
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Topology and function of the transmembrane domain of colicin U produced by Shigella boydii
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Krůšek, Jan (referee)
Colicin U is a protein produced by strains of bacterium Shigella boydii. It exhibits antibacterial activity against some bacterial strains Shigella and Escherichia. Based on sequence homology with colicins A, B and N, the colicin U is classified as a pore-forming colicin. Interaction of colicin U with attacked bacteria is ensured by three-step mechanism: 1) First colicin U interacts with surface receptors OmpA, OmpF and core of LPS. 2) Thereafter the colicin is translocated to periplasm through interaction with Tol proteins. 3) Finally colicin U interacts with the inner membrane of the attacked bacteria causing its depolarization. In this thesis I demonstrated pore-forming features of colicin U and further observed characteristics and properties of these pores. Using methods of measuring on black lipid membranes I determined a single channel conductance (19 pS), ion selectivity, the influence of various conditions on the behaviour of the pores. These findings, in many cases, correspond to the findings on other related colicins. Furthermore, I successfully determined the pore diameter of colicin U ( ≈ 0,8 nm). The next section of the thesis focuses on creation of single cysteine mutations of colicin U. Subsequently I produced five mutant variants of colicin U and verified their functionality so that...
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Study of the relations between the structure and function of C - terminal vanilloid receptor TRPV1
Gryčová, Lenka ; Obšil, Tomáš (advisor) ; Heřman, Petr (referee) ; Urbánková, Eva (referee) ; Krůšek, Jan (referee)
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labeling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations. Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the Cterminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT's binding to CaM, with arginine R785...
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The role of TRPV1 receptors in chemokine CCL2 induced modulation of nociceptive synaptic transmission at spinal cord level
Adámek, Pavel ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn is a significant mechanism in the development and maintenance of different pathological pain states. Accumulating evidence indicates that the TRPV1 (transient receptor potential vanilloid 1) receptor and chemokine CCL2 (C-C motif ligand 2) may play a critical role in this process. The aim of this diploma thesis was to investigate the CCL2 induced modulation of nociceptive synaptic transmission in the dorsal horn of spinal cord and the role of the TRPV1 receptors. To investigate this aim patch-clamp recordings of spontaneous and miniature excitatory postsynaptic currents (sEPSC, mEPSC) from superficial dorsal horn neurons in acute rat lumbar spinal cord slices were used. After acute application of CCL2 on the slice preparation from naïve animals, a frequency increase of both sEPSC and mEPSC was present. This CCL2 induced increase in both sEPSC and mEPSC frequency was prevented by the TRPV1 receptor antagonist SB366791 application. No changes were observed in the amplitudes of sEPSC or mEPSC after application of the CCL2, SB366791, or co-application of CCL2 and SB366791. This suggests that the observed changes were mediated predominantly by presynaptic mechanisms. The preliminary results indicate that after chronic constriction...
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