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Localization and function of phosphoinositides in the cell nucleus
Kalasová, Ilona ; Hozák, Pavel (advisor) ; Stopka, Tomáš (referee) ; Šolc, Petr (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids. Their inositol head can be phosphorylated at three positions generating seven differently phosphorylated species. Cytoplasmic phosphoinositides regulate membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generate second messengers. In the nucleus, PIs are implicated in pre-mRNA processing, DNA transcription and chromatin remodelling. However, their nuclear functions are still poorly understood. Here we focus on nuclear phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We describe their localization and interaction with proteins involved in regulation of DNA transcription. PI(4)P localizes to nuclear membrane, nuclear speckles and nucleoplasm. The majority of nuclear PI(4)P is associated with chromatin and colocalizes with H3K4me2. PI(4,5)P2 localizes to nucleoli and nuclear speckles. Besides, 30 % of nuclear PI(4,5)P2 forms small nucleoplasmic PI(4,5)P2 islets. They have carbon rich core, which is probably formed by lipids, and are surrounded by proteins and nucleic acids. The active form of RNA polymerase II associates with PI(4,5)P2 islets and DNA is actively transcribed in the vicinity of PI(4,5)P2 islets. Moreover,...
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On the role of the first transmembrane domain in desensitization kinetics of the P2X4 receptor.
Kalasová, Ilona ; Zemková, Hana (advisor) ; Krůšek, Jan (referee)
Extracellular adenosin-5'-triphosphate (ATP) is an important signalling molecule. Cells of eukaryotic tissues release ATP and express responding purinergic receptors. Ionotropic P2X receptors are trimeric ion channels permeable for K+, Na+ and Ca2+ ions. Each subunit consists of two transmembrane domains (TM1 and TM2), an extracellular loop and intracellular N- and C- termini. The transmembrane region is formed by six helical domains. According to the known crystal structure of zfP2X4 receptor, TM1 helixes are oriented peripherally and stabilize TM2 helixes which form the ion gate. However, eletrophysiological studies revealed that TM1 might also participate in channel gating and forming of the ion pore in the open state. The aim of this work was to investigate the role of TM1 in the process of desensitization of rat P2X4 receptor using cystein-scanning mutagenesis. Mutation of two residues (in Asn32 and Tyr42) prolonged desensitization of P2X4 receptor. Moreover, experiments with a partial agonist α,β-methylenadenosin-5'-triphosphate (αβ-meATP) proved that conformation change of TM domains in the process of desensitization is independent on conformation change caused by an agonist binding. Conserved residue Tyr42 is located in the proximity of TM2 of neighbouring subunit. It probably interacts with Met336...
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