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Glutamate Receptors and Endoplasmic Reticulum Quality Control
Tachezy, Ruth ; Horák, Martin (advisor) ; Adámek, Pavel (referee)
Quality control (QC) is a collection of processes taking part in the biogenesis and architecture of proteins. The objective of this thesis is to describe these processes in detail. QC takes place on many different levels in various compartments of the cell. The focus is on the endoplasmic reticulum (ER) QC interconnected with cytosolic QC. There are multiple steps involved in ERQC: several types of protein translocation to the ER lumen, glycosylation, disulfide bond formation via protein disulfide isomerase, chaperones that assist to achieve a correct conformation, and ER- associated degradation pathway for retranslocation of misfolded proteins back to the cytoplasm, where they are degraded. Cytosolic QC is interconnected with the ERQC through various ways of translocation of proteins to the ER membrane or lumen. Proteins that are retranslocated from ER to the cytosol are ubiquitinated and subsequently degraded in the proteasome. Ubiquitination is a process of targeting a protein for degradation. Cytosolic chaperones and other cellular structures, such as aggresomes, juxtanuclear compartments, and insoluble protein deposits, take part in the ubiquitination. Calcium dysregulation that is linked to QC and correct protein folding in ER is also described. Some of the possible consequences of protein...
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Functional and structural study of thermally activated TRP ion channels: The role evolutionarily conserved motifs in the TRPA1 modulation
Kádková, Anna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee) ; Obšilová, Veronika (referee)
Ankyrin receptor TRPA1 is an ion channel widely expressed on primary afferent sensory neurons, where it acts as a polymodal sensor of nociceptive stimuli. Apart from pungent chemicals (e. g. isothiocyanates, cinnamaldehyde and its derivatives, acrolein, menthol), it could be activated by cold temperatures, depolarizing voltages or intracellular calcium ions. TRPA1 channel is a homotetramer in which each subunit consists of cytoplasmic N and C termini and a transmembrane region. The transmembrane part is organized into six alpha- helices connected by intra- and extracellular loops. The N terminus comprises a tandem set of 16 to 17 ankyrin repeats (AR), while the C terminus has a substantially shorter, dominantly helical structure. In 2015, a partial cryo-EM structure of TRPA1 was resolved; however, the functional roles of the individual regions of the receptor have not yet been fully understood. This doctoral thesis is concerned to elucidate the role of highly conserved sequence and structural motifs within the cytoplasmic termini and the S4-S5 region of TRPA1 in voltage- and chemical sensitivity of the receptor. The probable binding site for calcium ions that are the most important physiological modulators of TRPA1 was described by using homology modeling, molecular-dynamics simulations,...
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Role of glycosylation of ionotropic glutamate receptors in mammalian neurons
Danačíková, Šárka ; Horák, Martin (advisor) ; Kriška, Ján (referee)
Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system. There are two distinct types of glutamate receptors, ionotropic and metabotropic, present in the mammalian excitatory synapses. My thesis is focused on the ionotropic glutamate receptors, which play critical roles in learning and memory formation. The main subtypes of ionotropic glutamate receptors are α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate receptors. All types of the ionotropic glutamate receptors, which are assembled as tetramers, contain many glycosylation sites, which can be modified by glycans or monosaccharides. The glycans and monosaccharides attached to the ionotropic glutamate receptors have been shown to regulate key processes such as folding of the subunits, transport to the cell surface as well as their functional properties. Recent literature also suggests that many neurological and psychiatric disorders such as schizophrenia exhibit abnormal glycosylation of ionotropic glutamate receptors. Thus, understanding of the molecular mechanisms, which regulate the glycosylation of the ionotropic glutamate receptors, may be important for developing new therapies for the patients with altered functioning of the glutamatergic synapses in the...
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Structural basis for interspecies differences in the TRPA1 receptor activation
Synytsya, Viktor ; Šulc, Miroslav (advisor) ; Hudeček, Jiří (referee)
Ankyrin transient receptor potential channel TRPA1 is an excitatory ion channel that transduces nociceptive information on primary aferent sensory nerves of mammals and other organisms. Structure function studies on TRPA1 are valuable for understanding the mechanisms of channel activation and for specific drug discovery efforts, however, significant interspecies differences hamper direct transfer of findings in animals to human. On the other hand, the interspecies differences may prompt identification of many important functional domains. The aim of this bachelor thesis is to give an overview of recent evidence regarding the functional and structural properties of human TRPA1 ion channels from the point of view of the comparison of the most important interspecies differences among TRPA1 orthologs. The experimental part is focused on the comparison of activation properties of human TRPA1 channel with a chimera in which the fifth transmembrane region was replaced by that from Drosophila melanogaster. The presented results obtained by electrophysiological technique patch-clamp demonstrate that outward membrane currents induced by depolarizing voltage are significantly reduced in chimera, which indicates an important role of the fifth transmembrane domain in TRPA1 channel gating (In Czech). Key words:...
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Role of variable chains at the interface between subunits in forming ATP-binding pocket and function of P2X4 receptor
Tvrdoňová, Vendula ; Zemková, Hana (advisor) ; Novotný, Jiří (referee) ; Vlachová, Viktorie (referee)
7 ABSTRACT Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. The role of these domains in forming of ATP-binding pocket and receptor function was investigated by using alanine scanning mutagenesis of the R203- L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor and by examination of the responsiveness to ATP and orthosteric analog agonists 2- (methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4- benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'- triphosphate. ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. These experiments, together with homology modeling, indicate that residues of the first group located in the upper part of...
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Functional role of cytoplasmic domains in the gating of TRPA1 channel
Vašková, Jana ; Vlachová, Viktorie (advisor) ; Zemková, Hana (referee)
The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in a subset of primary afferent neurones where it is activated by a variety of pungent and chemically reactive compounds such as allyl isothiocyanate or cinnamaldehyde. This voltage- dependent channel is activated through covalent modification of cytoplasmic cysteines and, from the cytoplasmic side, is also critically regulated by calcium ions. Both, amino (N-) and carboxyl (C-) termini have been shown to be involved in these processes. Using electrophysiological and molecular-biology techniques, we explored the role of specific cytoplasmic domains in the activation of TRPA1. By measuring chemically-, voltage-, and calcium-activated membrane TRPA1-mediated currents, we identified highly conserved serine and threonine residues along the N-terminal ankyrin repeat domain, mutation of which strongly affected responses of the channel. In addition, using C-terminally truncated construct previously reported to be involved in calcium regulation, we present a new finding that the distal C-terminal tail contributes to voltage-dependent activation of TRPA1.
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Molecular mechanisms of activation and modulation of TRPV3 receptor
Chvojka, Štěpán ; Vlachová, Viktorie (advisor) ; Novotný, Jiří (referee)
Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...
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The mechanism of regulation of NMDA receptors transport to the cell surface
Lichnerová, Katarína ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are a class of ionotropic glutamate receptors, involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They form heterotetrameric complexes composed of GluN1, GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Previous reports showed that different subunits of NMDA receptors, especially the GluN2 subunits, confer different functional and pharmacological properties on the receptor complexes. However, the subunit-dependent differences in the regulation of intracellular processing and transport of NMDA receptor subtypes has not been clearly elucidated. The aim of this work was to clarify the mechanisms of regulation of the NMDA receptor transport. In our experiments we performed immunocytochemistry of receptors on heterologous COS-7 cells and cultured cerebellar granule cells (CGC), both expressing recombinant NMDA receptors. The results of my work show that the transport of NMDA receptors is regulated by presence of GluN2A and GluN2B subunits. Our results further showed that transport of the GluN1/GluN2C receptors is regulated by three specific areas of the GluN2C subunit: i) the A2 segment within the amino- terminal domain, ii.) the M3 domain, and iii.) the proximal part of the C-terminus containing the...
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Functional and structural study of thermally activated TRP ion channels: The role evolutionarily conserved motifs in the TRPA1 modulation
Kádková, Anna
Ankyrin receptor TRPA1 is an ion channel widely expressed on primary afferent sensory neurons, where it acts as a polymodal sensor of nociceptive stimuli. Apart from pungent chemicals (e. g. isothiocyanates, cinnamaldehyde and its derivatives, acrolein, menthol), it could be activated by cold temperatures, depolarizing voltages or intracellular calcium ions. TRPA1 channel is a homotetramer in which each subunit consists of cytoplasmic N and C termini and a transmembrane region. The transmembrane part is organized into six alpha- helices connected by intra- and extracellular loops. The N terminus comprises a tandem set of 16 to 17 ankyrin repeats (AR), while the C terminus has a substantially shorter, dominantly helical structure. In 2015, a partial cryo-EM structure of TRPA1 was resolved; however, the functional roles of the individual regions of the receptor have not yet been fully understood. This doctoral thesis is concerned to elucidate the role of highly conserved sequence and structural motifs within the cytoplasmic termini and the S4-S5 region of TRPA1 in voltage- and chemical sensitivity of the receptor. The probable binding site for calcium ions that are the most important physiological modulators of TRPA1 was described by using homology modeling, molecular-dynamics simulations,...
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Photocontrolled Biomolecules
Planer, Jakub ; Bartošík, Miroslav (referee) ; Vácha,, Robert (referee) ; Kulhánek, Petr (advisor)
This work is focused on molecular dynamics simulations of artificial photosensitive ion channel and AFM probe. To assemble this ion channel, DFT methods were employed for reparametrization of the GAFF force field describing the bridged azobenzene, which was used as a light controlled molecular switch. We proved by molecular dynamics simulations that newly developed parameters correctly describe behavior of assembled model of ion channel in a lipid bilayer. We also constructed a model of AFM probe and observed formation of water meniscus between the AFM probe and surface, both made of -quartz, by employing molecular dynamics simulations. A contribution of this work is the set of new parameters extending GAFF force field for description of the bridged azobenzene. We also verified functionality of ion channel model and model of AFM probe, which can be used for the further water meniscus studies.
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