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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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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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In Vitro Selection of Aptamers for Methionine Sulfoxide
Jureček, Matěj ; Míšek, Jiří (advisor) ; Bařinka, Cyril (referee)
Oxidation of methionine to methionine sulfoxide in proteins is considered one of important post-translational modifications of proteins. This modification can activate and also inhibit functions of many proteins and it is a part of regulation mechanisms of various (patho)physiological processes. For further research of the effects of methionine oxidation in proteins it would be very helpful to find its bioindicator. So far however, there has not been found any such antibody, nor any of its alternatives. This thesis was concerned with the search of ssDNA aptamer specific for methionine sulfoxide by the method of in vitro selection (SELEX). Several conditions for in vitro selection of methionine sulfoxide were tested in this diploma thesis. None of them led to the enrichment of the starting oligonucleotide pool and no selective aptamer for methionine sulfoxide has been found. Such results don't necessarily point to the impossibility of finding such aptamer, but the conventional methods used in this thesis weren't suitable for this task. In a control in vitro selection there has been found an enriched ssDNA pool for sulforhodamine B as a ligand. Sequencing of clones of this enriched pool has shown oligonucleotides with G-rich sequences, which is typical for already published aptamers for sulforhodamine B.
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Molecular mechanisms and components controlling the Wnt signaling pathway output
Krausová, Michaela ; Kořínek, Vladimír (advisor) ; Drbal, Karel (referee) ; Bryja, Vítězslav (referee)
Beyond its essential roles in embryonic development, the Wnt-mediated signal transduction cascade is critically implicated in homeostasis of adult tissues. In the gastrointestinal epithelium, the threshold of active Wnt signaling is kept in a physiological range by a spectrum of regulatory networks and loops, thereby balancing the opposing processes of cell fate determination, proliferation and stem cell self-renewal. Furthermore, compelling evidence undoubtedly link an aberrant Wnt activity to the onset of bowel cancer. Understanding the principle causes and effects secondary to excessive Wnt signaling can provide valuable insights into the pathology of the malignant transformation of the colorectum. The proposed thesis attempts to focus on novel modes of the Wnt pathway modulation; both general and context-specific nuances of the Wnt level adjustment are thereby delineated. The results are presented in three distinct research publications and one review article. The first study examines the contribution of the distinct post-translational modifications, which the Wnt proteins undergo, to their proper processing, secretion and signaling activity. First, we investigated the sequential order and mutual interdependence of cysteine and serine-linked fatty acylation and N-linked glycosylation of murine...
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MAP code and regulation of microtubule-based processes
Karhanová, Adéla ; Lánský, Zdeněk (advisor) ; Tomášová, Štěpánka (referee)
Microtubule associated proteins (MAPs) are considered as key regulators of molecular trafficking in cells. Even though their malfunctioning results in severe pathologies, such as neurodegenerative disorders, the regulatory roles of these proteins remain under debate. Since MAPs bind to the cytoskeleton, this structure has to be vital for the function of MAPs. Microtubules, a highly dynamic type of cytoskeletal structure, have been given extra attention due to their association with cell division and vital functions in neurons. Microtubules can undergo post-translational modifications that affect molecular motors as well as binding of other proteins, such as MAPs. Whether post-translational modifications of microtubules regulate the distribution of MAPs is so far not sufficiently documented. However, MAPs have been shown to cooperatively form cohesive envelopes on the microtubules and thereby regulate the access of motors and severing enzymes. As there are many types of MAPs and they are mutually exclusive, a hypothesis of a regulatory 'MAP code' emerged recently in the literature. Using available literature, this review will try to introduce the new model of MAP code and provide some background information on previous research on this topic.
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Proteome analysis of anti-cancer drug effects and characterisation of drug resistance
Hrabáková, Rita
Despite significant progress in the development of anti-cancer drugs, there is still a need for novel therapeutic strategies that would improve the outcome of cancer patients. Using proteomic technologies and cell lines with different phenotype of p53 tumour suppressor, we monitored cancer cell response to anti-cancer treatment with focus on the development of drug resistance. The different levels of metabolic proteins were identified in our study which may help to explain different anti-cancer activity of drugs with only a subtle difference in structure. More importantly, proteins associated with the development of drug resistance were identified and such expression changes have become a focus of interest. Our findings demonstrate a higher protein level of serine hydroxymethyltransferase, serpin B5 and calretinin in cancer cells resistant to Aurora kinase inhibitors. Such proteins promote the tumour growth with no apparent impact of p53 phenotype whilst voltage-dependent anion-selective channel protein 2 contributes to the development of resistance only in cells with functional p53 which is accompanied by the decreased level of elongation factor 2. On the other hand, cancer cells with loss of p53 appear to amplify alternative mechanisms such as protection against oxidative stress. The results...
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Mechanisms of regulation of inhibitory factor IF1
Sklenář, Filip ; Dlasková, Andrea (advisor) ; Zelenka, Jaroslav (referee)
Inhibitory factor 1 (IF1) is one of the major regulators of mitochondrial ATP synthase activity, a key enzyme of energy metabolism. Its inhibitory effects are known in conditions such as hypoxia or starvation, but the hypothesis that IF1 inhibits ATP synthase activity even under physiological conditions is still not entirely accepted. Disorders of ATP synthase regulation can be fatal to the cell and have been described, for example, in carcinogenesis and ischemia. It has also been found that silencing of the IF1 gene in pancreatic β-cells increases insulin secretion, and thus, IF1 may be important in the pathogenesis of type 2 diabetes. The goal of this work is to summarize the current knowledge about the IF1 protein and to obtain new results that will help elucidate the mechanism by which this protein regulates mitochondrial ATP synthase. Specifically, this work deals with the ratio of IF1 protein to ATP synthase in pancreatic β-cells, depending on different culture conditions. It further investigates the occurrence of post-translational modifications of the IF1 protein in pancreatic β-cells (INS- 1E model cells), which may play a role in the regulation of IF1 activity. It also deals with the cellular ATP/ADP ratio, which is one of the key factors for insulin secretion by pancreatic β-cells. An...
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Role of post-translational modifications, O-GlcNAcylation and Phosphorylation, in neurodegenerative disorders and brain hypometabolism
Špundová, Tereza ; Růžička, Jiří (advisor) ; Čočková, Zuzana (referee)
Post-translational modifications are major mechanisms that highly increase the variability in protein function. O-GlcNAcylation and phosphorylation are among the most extensively studied post-translational modifications in research to date. In physiological conditions, O- GlcNAcylation acts as a metabolic sensor that links glucose metabolism to normal neuronal functioning. Reversible phosphorylation is one of the mechanisms that can downregulate metabolism by regulating the rates of flux through metabolic pathways. The impairments in the regulation of these modifications are linked to with neurodegenerative disorders and hypometabolism. This thesis focuses on the crosstalk and correlation between these two modifications, their reciprocal relationship and their mutual impact on their regulations in models of neurodegenerative diseases and disease non-related models. Keywords: hypometabolism, O-GlcNAcylation, phosphorylation, post- translational modifications, neurodegenerative disorders, hibernation, caloric restriction, memory, learning
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Molecular mechanisms of Wnt signalling in mammalian cells
Lukáš, Jan ; Kořínek, Vladimír (advisor) ; Kohoutová, Milada (referee) ; Macůrková, Marie (referee)
Wnt signalling represents an important mechanism participating in control of cellular and developmental processes, including establishment of cell polarity, cell fate specification, stem cell self-renewal, tissue patterning and organogenesis, homeostasis maintenance and regeneration. Misregulation of the Wnt signalling during embryogenesis leads to developmental defects while aberrant activation later in development is associated with degenerative diseases and a number of cancers. The presented PhD thesis is based on four original publications that deal with the post-translational modifications of Wnt ligands and molecular mechanisms contributing to the regulation of a transcriptional profile of the so-called canonical Wnt pathway. Wnt signalling pathway is used repetitively both in time and different cellular contexts throughout development of multicellular organisms. Inevitably, in each single situation -catenin/TCF complexes, the downstream effectors, induce only subsets of all potential target genes. How this differential tissue- and stage-specific control over various subsets of target genes is achieved with such a limited number of nuclear effectors is not fully understood. Along with the expression of specific LEF/TCF family members or their variants containing different functional domains...
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