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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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Heat-shock protein 90 (HSP90) in cell physiology.
Karmazin, Alina ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Heat-shock protein 90 (HSP90) is a molecular chaperone that represents one of the most important proteins for cellular homeostasis in all life domains. Chaperones are proteins that assist other proteins in proper folding and refolding. First discovered as a protein of a heat-shock response, HSP90 eventually emerged as a hub connecting multiple cellular functions, such as transcription, translation, DNA repair, immune response, cell signaling, etc. Unsurprisingly, HSP90 also plays a role in the pathogenesis of human diseases: various cancers, and neurodegenerative and respiratory diseases. For that reason, it became a target of medical research. HSP90 is a homodimer consisting of two protomers, each of which is composed of three domains: N-terminal domain, middle domain, and C-terminal domain. To fulfill its functions, HSP90 goes through an ATP-dependent conformational cycle, tightly regulated by a large group of assisting proteins-co-chaperones, and several post-translational modifications, such as phosphorylation and acetylation. Acetylation is known to affect HSP90 binding to nucleotides, clients, and co-chaperones, and thus it is suggested as a control mechanism of HSP90 function. Potentially, HSP90 acetylation can be utilized in the treatment of hormone-dependent cancers. Therefore, regulators of HSP90...
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The effect of 14-3-3 protein on intradomain interaction of ubiquitin ligase Nedd4-2
Pohl, Pavel ; Obšilová, Veronika (advisor) ; Žáková, Lenka (referee) ; Pavlíček, Jiří (referee)
EN The human ubiquitin ligase Nedd4-2 (NEDD4L) ubiquitinates a wide range of membrane proteins and receptors, playing a key role in maintaining homeostasis. This enzyme is regulated by phosphorylation and subsequent interaction with 14-3-3 proteins, which primarily affects its ability to interact with various substrates. However, very little is known about the molecular basis of this protein-protein interaction. In this work, we focused on biophysical characterization of the role of individual phosphorylation sites and also on mapping the structural changes in the Nedd4- 2 protein induced by 14-3-3 protein binding. Our experiments using analytical ultracentrifugation methods revealed that two phosphorylation sites Ser342 and Ser448 are primarily required for stable binding of Nedd4-2 to 14-3-3 proteins. The crystal structure of the 14-3-3ηΔC:Nedd4-2335-455 T367A complex than revealed the simultaneous binding of both phosphorylated residues to the binding groove of 14-3-3 protein. Subsequent modeling based on small-angle X-ray scattering and chemical cross-linking data combined with mass spectrometry indicated extensive structural changes in the individual domains of the Nedd4-2 protein. Binding of 14-3-3η protein blocks the WW3 domain of Nedd4-2 in the central channel of 14-3-3 protein, while...
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Roles of tubulin post-translational modifications in regulation of microtubule-based processes
Šliková, Pavlína ; Novák, Petr (advisor) ; Libusová, Lenka (referee)
Microtubular cytoskeleton plays crucial roles during diverse cellular processes, such as intracellular transport, cell motility and chromosome segregation during cytokinesis. Tubulin, the building block of microtubules, undergoes numerous post-translational modifications which affect microtubular dynamics and organization as well as their interaction with associated proteins. Understanding the role post-translational modifications play in the diversification of functions and properties of microtubules is key for our comprehension of the dynamics of the complex microtubule cytoskeleton. However, mechanisms behind the effect of post-translational modifications on microtubule cytoskeleton are not fully understood. In this work, we focus on the influence of post-translational modifications on microtubule polymerization and interaction with molecular motor kinesin-1. Using total internal fluorescence and interference reflection microscopy techniques, we here show that high levels of post-translational modifications on microtubules decrease the time of microtubule-kinesin interaction whereas binding affinity and median velocity are not significantly different on modified and unmodified microtubules. Further, we show that the absence of polyglutamylation on tubulin isotypes leads to a faster microtubule...
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Oxidative Post-translational Modifications and Their Importance
Šimková, Anna ; Míšek, Jiří (advisor) ; Petrák, Jiří (referee)
Aerobic life brings the inevitable exposure of living systems to reactive oxygen, nitrogen and chlorine species. A wide variety of oxidants can alter the structure of biomolecules such as proteins, lipids and DNA. Oxidative post-translational midifications play an important role in a number of cellular processes, for example, they are involved in redox signaling, gene transcription and metabolism. The increase of oxidants in cells leads to oxidative stress, which is associated with the development of neurodegenerative, cardiovascular and autoimmune diseases in humans. Considering reactive species as specific molecular agents can shed light on the complexity of the connection between redox processes and develop the emerging field of redox medicine.
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The role of posttranslational modifications in the molecular mechanism of the circadian clock
Janáčová, Klára ; Sumová, Alena (advisor) ; Sládek, Martin (referee)
The timing of the biological processes of organism is controlled by an endogenous circadian clock. The molecular clock is present in almost every cell and is synchronized with the external environment. The main mechanism of the clock is a transcription-translation feedback loop. The 24-hour circadian rhythm period is provided by reversible posttranslational modifications (PTMs) of the clock proteins and another regulators of the circadian clock. PTMs are further important for clock entrainment, their regulation by metabolic state in the cell, and reciprocal regulation of the circadian clock end cell cycle. Phosphorylation, histones PTMs, acetylation, SUMOylation, ubiquitination, O-linked N-acetylglucosamination and polyADP-ribosylation play a crucial role. The molecular mechanism of the biological clock is an evolutionarily conserved mechanism found in most organisms. This bachelor thesis summarizes the knowledge about the role of PTMs in the molecular mechanism of the mammalian and human circadian clocks. Key words: circadian clock, clock genes, clock proteins, posttranslational modifications
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Posttranlational protein modifications in response to DNA damage
Kroupa, Michal ; Hodný, Zdeněk (advisor) ; Novotný, Marian (referee)
- 5 - Abstract Thousands of DNA lessions occur in each cell every day of which the most toxic are double-strand breaks (DSBs). Signaling of their presence and subsequent repair are mediated by so-called DNA-damage response mechanism (DDR), which involves accumulation of many effector proteins into DSBs sites. These molecular accumulation at DSBs are termed DNA damage foci. Depending on presence of sister chromatid, DSBs are repaired by two major mechanisms: by homologous recombination and by non-homologous end joining. Both pathways lead to activation of checkpoint kinases (Chk1 or Chk2) which iniciate checkpoints in cell cycle and allow repair of damaged DNA. Signaling of DNA damage and activation of these pathways are regulated by posttranslational protein modifications. These enzymatic reactions involve mainly phosphorylation, ubiquitination and sumoylation. Recently it was shown that ubiquitination of damaged chromatin is a prerequisite for sumoylation of tumor supressors BRCA1 and 53BP1. Failure in DNA damage recognizing mechanisms caused by disorders such as modifications or mutations of 53BP1 and BRCA1 genes can lead to subsequent disruption of genomic integrity and then a high risk for selection of cell clones with tumorigenic potencial. Current research is focused on regulation of posttranslational...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Regulatory mechanisms of WNT signalling
Pospíchalová, Vendula ; Kořínek, Vladimír (advisor) ; Trka, Jan (referee) ; Bryja, Josef (referee)
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