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Notch Interaction with Glycogen Synthase Kinase-3β in Neurodegenerative Disease
Traub, Teodor ; Mašek, Jan (advisor) ; Rozbeský, Daniel (referee)
Glycogen synthase kinase-3βeta (GSK-3β) is a serine/threonine protein kinase involved in a number of signaling processes. Pharmacological inhibition of GSK-3β has been shown to have neuroprotective effects, and its dysregulation is present in a variety of neurodegenerative, neuromuscular, developmental, and psychiatric disorders. GSK-3β is an essential component of the canonical Wnt pathway, which is involved in nervous system development. Notch signaling, like Wnt, plays a key role in development, but its relationship to GSK-3β remains unclear. The existing literature indicates that GSK-3 phosphorylates Notch intracellular domain but contradicts whether GSK3β affects Notch positively or negatively. Thus, such molecular "cross-talk" is highly complex and interactions may exist at multiple levels beyond simple phosphorylation with other components being involved. Myotonic dystrophy is a genetic neuromuscular disease that causes dysregulated protein expression of many proteins, including GSK-3β, and features developmental, muscle, and neurological symptoms. Studying molecular interactions in the context of myotonic dystrophy may help uncover effects that GSK-3β and Notch have on development and disease of nervous and muscular systems. This thesis presents a review of previous studies concerning the...
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Recombinant expression, purification and characterisation of the surface immunoreceptor CD47 (integrin associated protein)
Kuchyňová, Sarah ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
The human immunoreceptor CD47, also designated <Integrin Associated Protein= is a novel therapeutic target for cancer treatment. When bound to one of its ligands, a protein found on the surface of immune cells, SIRP, the phagocytosis of the CD47 expressing cell is inhibited. This interaction is used by malignant cells to escape detection by the immune system, thus making CD47 a promising candidate molecule for cancer immunotherapy. The literary overview focuses on the characterisation of CD47 in terms of its structure, localization, and function. Additionally, this sections thoroughly delves into the aforementioned interaction with SIRP as well as existing mechanisms for the targeting of the CD47- SIRP axis. The following experimental section centers around the preparation of the plazmid pTT28_hCD47. The process by which the gene for the extracellular portion of human CD47 was inserted into the linearized pTT28 vector is noted and its transfection into a mammalian expression system to produce the protein. Moreover, the section divulges into the purification of the recombinant protein by affinity purification using Ni-NTA agarose as well as gel permeation chromatography, resulting in the isolation of CD47 proteins differing in glycosylation. Their functionality was validated via SPR, in a study...
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Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2
Kalábová, Dana ; Obšilová, Veronika (advisor) ; Pavlíček, Jiří (referee) ; Žáková, Lenka (referee)
Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2 Abstract Caspase-2 is a protease standing apically in the cascade of reactions leading to apoptosis. Properly functional apoptosis eliminates damaged cells, autoreactive lymphocytes or redundant groups of cells in ontogeny. The process of caspase-2 activation must be precisely regulated. One of the described ways of caspase-2 regulation causing its inhibition is posttranslational modification phosphorylation with subsequent binding of the regulatory scaffold protein 14-3-3. The aim of this dissertation is to explain the molecular mechanism of this regulation. To understand the interaction between the proteins, it was necessary to first identify the phosphorylation sites in the caspase-2 molecule recognized by the 14-3-3 protein and then describe the detailed structure of the binding complex. The structure was characterized by a number of biochemical and biophysical methods, such as analytical ultracentrifugation, native electrophoresis in TBE buffer, polarization-fluorescence assay, hydrogen/deuterium exchange coupled to mass spectrometry, or crystallization; and the results led to stimulating conclusions. Activation of caspase-2 begins with its binding to adaptor proteins, cleavage and dimerization of the catalytic subunits. The...
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Investigation of HSP70 oligomerization by structural mass spectrometry
Melikov, Aleksandr ; Novák, Petr (advisor) ; Jeřábek, Petr (referee)
Heat shock cognate protein 70 (HSC70) is a 71 kDa chaperone protein belonging to the ubiquitous family of heat shock proteins 70 (Hsp70). The representatives of this protein family are considered as molecular machines with ATP-hydrolase activity facilitating correct folding of spatial protein structure, both in normal and stressful conditions (hypoxia, heat shock, pH fluctuations etc.) In addition, HSC70 was identified as an uncoating enzyme for triskelion meshwork on the surface of clathrin-coated vesicles. Among other roles, HSC70 prevents protein aggregation and assists the polypeptide maturation, it facilitates the protein transport into organelles, such as endoplasmic reticulum and mitochondria. It is involved in targeting proteins for lysosomal degradation and in many other dramatically important cellular processes related to protein homeostasis. Therefore, the regulation of HSC70 and other HSP70 proteins is believed to be dramatically important, especially in a context of cellular stress. Based on the experimental observation, the mechanism of inactivation through oligomerization was hypothesized. The dimer and trimer species of Hsp70 proteins were identified both in case of prokaryotic and eukaryotic homologs. It was also speculated that Hsp40 cofactors promote oligomerization to even...
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Study of protein-protein interactions of human E3 ubiquitin ligase Nedd4-2.
Joshi, Rohit Ashok ; Obšilová, Veronika (advisor) ; Krůšek, Jan (referee) ; Holoubek, Aleš (referee)
Posttranslační modifikace prostřednictvím ubikvitinace hrají klíčovou roli v regulaci membránových proteinů. Nedd4-2, lidská HECT E3 ubikvitin ligáza, je poslední komponentou ubikvitinační kaskády, která přenáší molekuly ubikvitinu a spouští endocytózu svých následných cílových molekul. Dysregulace Nedd4-2 může způsobovat různé poruchy, včetně epilepsie, respirační úzkosti a Liddleova syndromu. Přestože se na regulaci Nedd4-2 podílejí různé adaptorové proteiny, v tomto výzkumu jsme se zaměřili na konzervované proteiny 14-3- 3, známé negativní regulátory Nedd4-2. V této studii jsme provedli biofyzikální charakterizaci konstruktů Nedd4-2190-581 a Nedd4-2186-975 v komplexu s 14-3-3, abychom získali další náhled do dynamiky této interakce. Naše výsledky časově rozlišené fluorescenční spektroskopie odhalily, že vazba 14-3-3 ovlivňuje emisní vlastnosti a pohyblivost specifických WW domén (WW3 a WW4) Nedd4-2, zatímco ostatní (WW1) šetří. Zajímavé je, že katalytická doména HECT prochází při tvorbě komplexu konformačními změnami a zvýšenou exponovaností rozpouštědlu. Předpokládáme, že sterická inhibice domén WW3 a WW4 v kombinaci s konformačními změnami v katalytické doméně může být základem regulačního mechanismu zprostředkovaného vazbou proteinem 14-3-3. Chemické zesítění spolu s hmotnostní spektrometrií...
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Analysis of IFI16 protein binding to DNA
Kratochvilová, Libuše ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
This diploma thesis deals with the binding of interferon gamma-inducible protein 16 (IFI16) to DNA with the potential of G-quadruplex formation. The IFI16 protein contains two tandemly located DNA-binding HIN domains showing differential binding to DNA structures. IFI16 protein has been shown to preferentially bind G-quadruplex structures over other nucleic acid secondary structures. G-quadruplexes are secondary local structures of DNA (or RNA) that are easily formed under physiological conditions in a number of important regulatory regions of the genome, or are part of the genomes of a number of viruses and pathogens. The ability to recognize, specifically bind and stabilize G-quadruplex structures explains the involvement of the IFI16 protein in the cellular processes of replication, transcription and translation and the establishment of innate immune responses. In the first part of the thesis, the sequences of synthetic oligonucleotides with the potential for G-quadruplex formation were characterized by selected biophysical methods and the full-length IFI16 protein was isolated, which was subsequently used for in vitro binding and competitive binding experiments with characterized oligonucleotides. In the last part of the work, isogenic yeast strains differing in the sequences of the responsive element were transformed with plasmid vectors for the expression of p53 and IFI16 proteins with constitutive and GAL inducible promoters, and the one-hybrid yeast system model was optimized for the study of IFI16 protein interactions in vivo. The results show that most of the analyzed sequences are able to form G-quadruplex structures in vitro, even in the presence of only one run of three or more G-bases. While the presence of several G-runs separated by a single nucleotide spacer led to the formation of intermolecular G-quadruplex structures, mutation in the original G-quadruplex sequence induced the formation of intramolecular structures with different conformations. In vitro binding and competitive binding experiments demonstrated specific binding of the IFI16 protein to G-quadruplex structures without differences in protein binding preference to a particular G-quadruplex conformation. Stabilization of G-quadruplex structures in vivo behind the transcription factor responsive element (p53) in the gene promoter induced repression of the transcription of the given gene. In the absence of any binding site of the IFI16 protein, a protein-protein interaction between the IFI16 and p53 proteins occurred, which led to an increase in the transactivation potential of the p53 protein, while the binding of the p53 protein and initiation of reporter gene transcription was influenced not only by the presence of the G-quadruplex motif and its stabilization, but and the DNA sequence adjacent to the p53 responsive element.
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Structural characterization of influenza A polymerase PA subunit domains in complex with novel inhibitors
Radilová, Kateřina ; Kožíšek, Milan (advisor) ; Rumlová, Michaela (referee) ; Obšil, Tomáš (referee)
Influenza RNA-dependent RNA polymerase is a heterotrimeric complex and has an essential role in the life cycle of the virus. It is responsible for viral replication and transcription. One of its subunits, the polymerase acidic protein, interacts with the PB1 subunit via a crucial protein- protein interaction at its C-terminal domain. This 310 helix-mediated intersubunit interaction is required for the whole heterotrimer assembly. The N-terminal domain carries the endonuclease active site with two manganese ions. Both domains are considered promising drug targets. Current strategies to fight the influenza virus are limited to seasonal vaccines, and there are only a few anti-influenza drugs targeting mostly other viral proteins. Many used antivirals are susceptible to rapid resistance mutations development or cause severe side effects. This thesis provides structural insights into the two domains of the PA subunit. The first part is devoted to the characterization and optimization of a PB1-derived minimal peptide interacting with the C-terminal domain. Results from this part may be considered as a starting point for the rational design of first-in-class anti-influenza inhibitors of the PA-PB1 protein-protein interaction. In the other half, we have explored the inhibitory potency of flavonoids and...
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Characterization of the binding interface between transcription factors FOXO4 and p53
Brzezina, Adam ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
This work deals with the study of human transcription factors FOXO4 and p53. FOXO4 is a member of the "O" subfamily of FOX transcription factors. Genes encoding FOXO proteins are evolutionarily conserved across species. FOXO transcription factors regulate the expression of genes involved in the control of metabolism, cell cycle and cell proliferation, cell survival and stress resistance. They are considered tumour suppressors because of their ability to arrest the cell cycle and induce apoptosis. However, their function in tumorigenesis appears to be more complicated, as recent studies indicate a poorer prognosis for the development of tumours that express higher levels of FOXO4. The p53 protein is a thoroughly studied naturally occurring tumour suppressor. The cellular response after its activation is somewhat similar to that of FOXO4, it can also block cell cycle progression or induce apoptosis depending on the cell type and severity/type of cellular stress. Both FOXO4 and p53 appear to be key molecules affecting aging. Under stress conditions, p53 and FOXO4 interact with each other and together increase the expression of p21 protein, thereby inducing the transition of cells to a senescent state. The accumulation of senescent cells is recognised as one of the main causes of ageing and the...
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Modulation of DNA Binding Affinity of Transcription Factors FOXO and p53 Through Protein-protein Interactions
Hofmanová, Adéla ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract The forkhead box "O" (FOXO) proteins are a subclass of the Forkhead family of transcription factors that play a critical role in a variety of cellular processes such as response to cellular stress, gluconeogenesis, cell cycle control, apoptosis, senescence, and repair of DNA damage. They are generally considered to be tumor suppressors. However, it has been shown that they can promote tumorigenesis and induce resistance to the chemotherapeutic agents. Despite many years of research into the biological role of FOXO proteins, a number of questions remain to be answered. For example, whether the slight structural differences observed in the otherwise highly homologous DNA-binding domains of individual FOXO transcription factors affect their DNA binding affinity. Furthermore, it is unclear how protein-protein interactions affect DNA binding affinity of FOXO proteins. Recent study has described the interaction of FOXO transcription factors with the p53 protein. Protein p53 is called the guardian of the genome due to its ability to mediate the response to acute DNA damage. The interaction of FOXO and p53 proteins appears to have a major effect on the DNA binding affinity of both these proteins. Based on this, DNA-binding domains of the human transcription factors FOXO1, FOXO3 and FOXO4 (FOXO1(144-270),...
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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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