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The effect of variations in GRIN genes on the biogenesis and functional properties of the NMDA receptor
Kuchtiak, Viktor ; Balík, Aleš (advisor) ; Rozbeský, Daniel (referee) ; Ladislav, Marek (referee)
The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the central nervous system. The major class are the calcium-permeable NMDA receptors that are fundamental for the various forms of synaptic plasticity, a key mechanism in the process of learning and memory formation. NMDA receptors are heterotetrameric and are represented by three types of subunits: GluN1, GluN2A-D, and GluN3A-B. Each subunit consists of four domains, with the intracellular C-terminal domain accounting for up to half of the entire NMDA receptor subunit (GluN2A/2B). A body of evidence indicates that the hypofunction of the NMDA receptor plays an important role in the pathogenesis of several neuropsychiatric disorders, including schizophrenia. Schizophrenia is characterised by a high degree of heritability, but its genetic background is not yet fully understood. Previous studies have identified in the human genome several individual loci that contribute to disease susceptibility, including the GRIN genes encoding NMDA receptors. Using a sequencing approach, we identified and annotated genetic variations across all GRIN genes in a cohort of schizophrenia patients and control subjects. The submitted doctoral thesis focuses on the functional analysis of the genetic...
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The role of structural motifs in the localisation of T-cell plasma membrane proteins
Glatzová, Daniela ; Cebecauer, Marek (advisor) ; Brábek, Jan (referee) ; Rozbeský, Daniel (referee)
Plasma membrane of T cells is abundant in diverse receptors and other molecules orchestrating immune responses. Numerous studies demonstrate that the localisation of proteins in the cell is non-random and that mislocalisation either in the context of plasma membrane at nanoscale or with respect to the cell interior can lead to the protein malfunction and subsequent aberrant T- cell response. In my first Ph.D. project we focused mainly on the role of the transmembrane domain length and amino acid composition, proximal sequences and the presence or absence of palmitoylation on the localisation of transmembrane adaptor proteins LAT, PAG and NTAL in T cells. We showed that plasma membrane localisation of PAG and NTAL is controlled by the amino acid composition of their TMD and is palmitoylation independent. We propose that NTAL localisation to the plasma membrane is, despite its suboptimal length, facilitated by the electrochemical asymmetry of its TMD. Among transmembrane adaptor proteins, LAT was the most interesting one. Dependency of LAT plasma membrane localisation on palmitoylation in combination with unusual amino acid composition of its TMD led us to investigate it in a separate project. My first author Ph.D. project was thus to elucidate the role of highly conserved helix-breaking amino acids,...
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Quantitative fluorescence microscopy techniques to study three-dimensional organisation of T-cell signalling molecules.
Chum, Tomáš ; Cebecauer, Marek (advisor) ; Lánský, Zdeněk (referee) ; Brameshuber, Mario (referee)
10 SUMMARY Proteins represent one of the basic building blocks of all organisms. To understand their function at the molecular level is one the critical goals of current biological, biochemical and biophysical research. It is important to characterise all aspects that affect the localisation of proteins into different compartments with specific functions, the dynamic structure of proteins and their role in multiprotein assemblies, because altering these properties can lead to various diseases. Most of the proteomic studies are nowadays performed using biochemical approaches that allow us to study multicellular organism or tissue at once. The disadvantage of these methods is complex preparation of sample and the need for a large number of cells, which leads to the loss of information at the molecular level and in individual cells. On the contrary, microscopy can provide rather detailed information about proteins of interest and at the level of a single cell. A variety of fluorescence microscopy methods in combination with recombinant DNA techniques were applied to elucidate subcellular localisation of transmembrane adaptor proteins (TRAPs) in human lymphocytes and their nanoscopic organisation at the plasma membrane. Linker of activation of T lymphocytes (LAT), phosphoprotein associated with...
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Localisation of CD4 coreceptor and its variants in human T cells
Glatzová, Daniela ; Cebecauer, Marek (advisor) ; Drbal, Karel (referee)
CD4 co-receptor of main T cell receptor (TCR) is essential for proper development of T lymphocytes and their function in adaptive immune responses. It is believed that CD4 stabilizes the interaction of TCR with antigenic ligand, peptide-MHC, and thereby improves T cell-dependent responses during immune reaction. CD4 is transmembrane glycoprotein with a number of structural motifs in its intracellular domain which do not dramatically affect its sorting to the plasma membrane but can influence its local organization at nanoscale. CD4 was shown to transiently accumulate in the immunological synapse formed between T cell and antigen-presenting cell. Such accumulation is rapidly followed by its internalization and/or delocalization outside the synapse. This is in contrast with TCR which accumulates strongly in the immunological synapse and is later found enriched in the central area of this structure. It is therefore unclear how TCR and its CD4 co-receptor function together when binding to their common ligand during the initiation of signaling in T cells. We aim to study localization of CD4 at nanoscale using advanced fluorescence microscopy techniques achieving significant improvements in resolution. In this work, CD4 and its mutant variants, potentially causing its different localization at the...
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