National Repository of Grey Literature 41 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
Role of β-arrestin in μ-opioid and TRPV1 receptor signalling
Nagy Marková, Vendula ; Novotný, Jiří (advisor) ; Blahoš, Jaroslav (referee) ; Roubalová, Lenka (referee)
β-Arrestin belongs to the protein family which has a huge impact not only on GPCR signaling, but its role exceeds the function of the membrane channel, its own signaling cascade, or as a scaffold protein, etc. Here we aimed to study β-arrestin roles on the MOR behaviour in the plasma membrane or -opioid receptor (MOR) signaling and effect on adenylyl cyclase (AC) function using the siRNA to decrease the expression of β-arrestin isoforms. Furthermore, we focused on investigating the role of β-arrestin on the crosstalk between MOR and TRPV1 channels, which are important parts of pain transduction. For this purpose, we used HEK293 cells that stably expressed MOR-YFP or transiently transfected with TRPV1-CFP. We observed that both β-arrestin isoforms have an effect on the lateral mobility of MOR in the plasma membrane and the silencing of one or another β-arrestin isoforms abolishes the effect of MOR agonists to affect its diffusion in the plasma membrane. Interestingly, silencing of β-arrestin1 diminish the internalization of MOR induced by the endogenous agonist endomorphin-2. On the other hand, silencing of β-arrestin2 did not abolish the endomorphin-2 induced MOR internalization. Moreover, both isoforms exhibit a distinct impact on the inhibition of AC induced by the agonists of MOR....
Characterization of the effect of pregnanolone sulfate and its derivatives on NMDA receptors.
Švehla, Pavel ; Vyklický, Ladislav (advisor) ; Blahoš, Jaroslav (referee)
N-methyl-D-aspartate (NMDA) receptors are a subtype of receptors for major excitatory neurotransmitter glutamate in the central nervous system. Their activity is regulated by variety of allosteric modulators, including endogenous neurosteroids and their synthetic analogues. NMDAreceptor dysfunction is implicated in various forms of neurodegeneration and inhibitory neurosteroids have unique therapeutic potential to act as neuroprotective agens. The aim of this work is to investigate relationship between structure and function of neurosteroids with modifications in the D-ring region, using whole-cell patch clamp recording at recombinant GluN1/GluN2B receptors. In this work, we characterised inhibition effect of 19 neurosteroid analogues on NMDA receptor activity and found several of them to be potent NMDA receptor inhibitors. According to our results, there is a linear relationship of IC50 and lipophilicity of a neurosteroid compound, suggesting the plasma membrane plays an important role in neurosteroid access to NMDA receptor. Indeed, using capacitance recording configuration in combination with amphipathic molecule gamma-cyclodextrin, we were able to separate the kinetic of neurosteroid membrane binding from receptor binding. Moreover, these experiments showed that neurosteroid accumulation in the...
Metabotropic glutamate receptors: mechanism of activation
Hlaváčková, Veronika ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Martásek, Pavel (referee) ; Konvalinka, Jan (referee)
Any living organism receives constantly many signals that have to be evaluated and weighted to respond in an appropriate way. To perform all functions needed for precise control of homeostasis and for communication with the surrounding environment, signals coming from the outside are recognized and transferred into modulation of intracellular signaling cascades. These mediate response to the extracellular stimulus as well as intercellular communication. Cell communication is mediated by several types of receptors, located either intracellularly (including nuclear receptors) that modulate gene transcription and receptors localized on plasma membrane. Cell membrane receptors are transmembrane proteins that are divided into three superfamilies according to their structure and principles of signal transduction. These are ion channel-linked receptors, enzyme-linked receptors and G-protein-coupled receptors (GPCRs). GPCRs comprise the biggest family of membrane receptors and are one of the largest gene families in general. They are encoded by about 1% of genes in mammals. Many of them bind sensory ligands (rhodopsin, taste and olfactory receptors), but others also recognize ions, amino acids, nucleotides, peptides and large glycoproteins (1). They play a crucial role in such distant physiological functions as...
Structure and Function of Glutamate Carboxypeptidase II
Šácha, Pavel ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Blahoš, Jaroslav (referee)
4 Závěr GCPII je důležitý protein, který hraje roli v mnoha fyziologických i patologických procesech. Proto bylo třeba získat větší množství enzymaticky aktivního proteinu pro jeho další biochemický výzkum. Heterologní expresí v hmyzích buňkách S2 bylo exprimováno a následně purifikováno dostatečné množství velmi čistého a aktivního enzymu. To umožnilo jeho biochemickou charakterizaci, krystalizaci a později vedlo i k vyřešení krystalové struktury. GCPII je aktivní v širokém rozmezí pH 6 - 8, s maximem kolem pH 7,5. Zjistili jsme, že kromě přirozeného substrátu Ac-Asp-Glu GCPII štěpí také acetylované dipeptidy Ac-Asp-Met, Ac-Glu-Met, Ac-Glu-Glu, Ac-Ala-Glu a Ac-Ala-Met. U těchto substrátů byly změřeny kinetické parametry štěpení. Nalezení dalších substrátů může vést k objevení dosud nepopsaných fyziologických rolí GCPII. Také byly porovnány hodnoty IC50 inhibitorů známých z literatury u námi připravené GCPII a GCPII izolované z potkaních mozků. IC50 bylo u všech méně specifických inhibitorů nižší u rekombinantní GCPII než u GCPII izolované z mozků. Rozdíly ve výsledcích vysvětlujeme vazbou méně specifických inhibitorů na jiné proteiny preparátů z mozku. Vůbec nepochybujeme o tom, že data získaná za použití čistého rekombinantního proteinu jsou přesnější než ta, ktará byla získána ze špatně definovaných...
Study of Structure-Function Relationship of Temperature-Gated TRP Channels
Benedikt, Jan ; Vlachová, Viktorie (advisor) ; Kršiak, Miloslav (referee) ; Blahoš, Jaroslav (referee)
Sensory physiology research was heavily influenced by molecular identification of transient receptor potential (TRP) ion channel family. Discovery of these unique family of membrane receptors allowed detailed study of their structure-function relationship. TRP channel expression in sensory neurons, but also apparently in keratinocytes provides living organisms with the ability to fast and accurately detect noxious thermal and chemical stimuli and to transmit this noxious signaling to higher nervous system structures. Despite recent efforts to elucidate molecular mechanisms of temperature or chemical activation of these non-selective cation channels, there is still no unifying hypothesis that is able to explain complex behaviour of these receptors. This dissertation aims to investigate three aspects of the TRP channel function: 1. Molecular characterization of acute desensitization of vanilloid receptor TRPV1 and investigation of the role of phosphorylation sites for calmodulin kinase II. 2. To characterize mechanisms of etanol-induced inhibition of menthol receptor TRPM8 and to find out possible physiological consequences of this inhibition. 3. To explore the role of inner pore region in activation gating of ankyrin receptor TRPA1 and identify amino acids involved in this process. Our findings contribute to...
Characterization of molecular components in cannabinoid signaling pathways.
Hájková, Alena ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Maletínská, Lenka (referee)
The cannabinoid receptor 1 (CB1R), a member of the G-protein coupled receptors superfamily, is a key player in endocannabinoid signalling. The CB1R is found presynaptically in neurones where it modulates synaptic plasticity. Precise description of the molecular mechanisms of synaptic neurotransmission is crucial for understanding of brain diseases and development of new therapeutic aproaches. Possible pharmacological targets of CB1R signalling include the treatment of various ailments such as energy imbalance disorders (anorexia, obesity), drug addiction, pain, insomnia, and some psychiatric conditions. This study reveals the "Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1" (SGIP1) as a novel interacting partner of the CB1R. The SGIP1 is an intracellular neuronal protein localized predominantly in axon terminals and is involved in clathrin mediated endocytosis. The overexpression of SGIP1 imbalance energy homeostasis and leads to obesity. We show that SGIP1 affects CB1R signalling via ERK1/2 whereas G-protein signallization remains unaltered. The SGIP1 also hinders CB1R internalization from the cell surface and supports its interaction with β-arrestin2. Also, we demonstrated heterodimerization of the main splice variants of metabotropic glutamate...
Glutamatergic synaptic transmission, targeting and distribution of metabotropic glutamate receptor in neuronal cells
Techlovská, Šárka ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Fusek, Martin (referee)
Stránka 7│146 ABSTRACT An evolutionary developed eukaryotic proteins for transformation of extracellular signal into intracellular targets are receptors. There are known receptors integrated to intracellular organelle membranes e.g. inositol-3-phosphate receptor (Inositol-3-phoshpate receptor; IP3R) specific for endoplasmic reticulum or nuclear receptors e.g. receptors for gonad hormones transforming the intracellular signals, however the vast majority of receptors are present on plasmatic membrane and recognize an extracellular signals. High percentage of these receptors are included in family of proteins called G-protein coupled receptors, (G-protein coupled receptors, GPCRs) due to their signal mediators heterotrimeric G-proteins. Each receptor of this family has the preference to specific type of G-protein influencing the intracellular concentrations of Ca2+ , cyclic adenosinmonophosphate or inositol-3- phosphate. Some of them are able to signalise by dual manner or through G-protein independent pathways. Independently on cellular target membrane, GPCRs integration into membranes is enables by the content of GPRCs-specific 7-transmembrane domain (7-transmembrane domain, 7TM) which is consist of hydrophobic amino acids. The relation between the structure and functionality is very tightly related. Many...
Function of Zinc finger protein 644 (Zfp644) in mouse organism.
Szczerkowska, Katarzyna Izabela ; Sedláček, Radislav (advisor) ; Komrsková, Kateřina (referee) ; Blahoš, Jaroslav (referee)
ZNF644 (Zinc Finger Protein 644) is a C2H2 zinc finger gene encoding a putative transcription regulator, of which a point mutation (S672G) is associated with inherited high myopia in humans. It is also described to be a partner of the G9a/GLP (G9a- euchromatic histone- lysine N-methyltransferase 2, EHMT2; GLP - euchromatic histone-lysine N-methyltransferase 1, EHMT1) complex, known for its essential role in histone methylation, specifically H3K9me1and H3K9me2. It was reported that another transcription factor, WIZ (Widely-Interspaced Zinc Finger-Containing Protein), can bind to this complex and cooperate in gene silencing simultaneously. In order to study Zfp644 impact on myopia, we generated a mouse model, Zfp644S673G that mimics human mutation. In addition, a mouse with a persuasive truncated form of the protein, Zfp644Δ8 was created. Both mouse models went through an examination of retinal function and morphology. Moreover, with use of ultrasonography, different ocular parameters were examined. We conclude, that Zfp644 gene is causative for myopia in mice. Further examinations of Zfp644Δ8 animals show severe symptoms in metabolism and female fertility. To describe the impact of Zfp644 in mouse fertility we performed various experiments including analysis of expression of Zfp644 in reproductive...

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