|
| |
|
| |
|
| |
|
|
Contribution of ten ectodomain cysteine residues to function of ATP-gated P2X4 receptor
Tvrdoňová, Vendula ; Zemková, Hana (advisor) ; Teisinger, Jan (referee)
Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...
|
|
| |
|
|
Peripheral metabolism of glucocoricoids in immune cells
Ergang, Peter ; Pácha, Jiří (advisor) ; Kalous, Martin (referee) ; Teisinger, Jan (referee)
4 Abstract Glucocorticoids are hormones that regulate a variety of homeostatic processes including metabolism, cell proliferation, differentiation and immune functions, including inflammation. Acute inflammatory response is associated with an increase in glucocorticoid levels via the stimulation of pro-inflammatory cytokines and activation of the hypothalamo- pituitary-adrenal axis. Within target cells or tissues the glucocorticoid action depends not only on the plasma level of the hormone, its receptors and receptor-effector coupling, but also on the local metabolism of glucocorticoids. Two distinct types of this enzyme have been cloned and characterized. Type 1 (11HSD1) is a NADP+ (H)-dependent enzyme whose reductase activity predominates in intact cells. This enzyme activates cortisol and corticosterone from their 11-keto derivatives and thus increases the local concentration of active glucocorticoid. In contrast, type 2 (11HSD2) requires NAD+ as a co-substrate and possesses only dehydrogenase activity, thereby inactivating endogenous glucocorticoid hormones. We have demonstrated that inflammation (arthritis or experimental colitis) is accompanied by elevated 11-reductase activity and the expression of 11HSD1 mRNA, moreover in the case of colitis also with a decrease in the expression of 11HSD2....
|
|
| |
|
|
Study of the interaction between the C-terminus of DNA-binding domain of FOXO4 and DNA
Zusková, Iva ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
Forkhead transcription factors are structurally similar molecules containing approximately 110-amino-acid-long DNA-binding domain known as a forkhead domain. Protein FOXO4 is a member of subgroup "O" of forkhead transcription factors. Members of this subgroup play a key role in many biologically important processes. For example, FOXO factors participate in metabolism control, cell-cycle control, apoptosis and oxidative stress resistance. The forkhead domain (DNA-binding domain) consists of three α-helices (H1, H2 and H3), three β-strands (S1, S2 and S3) and two flexible loops (called wings W1 and W2). The role of the wing W2 in FOXO binding to the target DNA is still elusive. Wing W2 probably interacts with the DNA in the region upstream of the core motif. It has been speculated that the FOXO DNA-binding affinity depends on A-T content (number of A-T pairs) in the region upstream of the core motif. In order to investigate this hypothesis, DNA- binding domain of the FOXO4 protein was expressed and purified and it was determined its binding affinity for three molecules of double stranded DNA containing different number of A-T pairs in the region upstream of the core motif using steady-state fluorescence anisotropy- based method. Our results show no significant differences between obtained FOXO4...
|
|
|
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...
|
|
|
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...
|
guest ::
