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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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Role of PML in ribosomal stress
Kremserová, Petra ; Vašicová, Pavla (advisor) ; Sztacho, Martin (referee)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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Structural characterization of human protein kinase CaMKK2 and its interactions with binding partners
Koupilová, Nicola ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...
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Identification of small compounds disrupting protein-protein interaction in influenza A polymerase.
Hejdánek, Jakub ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...
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Study of interactions of forkhead box O (FOXO) transcription factors with DNA
Hofmanová, Adéla ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
This work studies the "O" subgroup of FOX transcription factors, which consists of four members (FOXO1, FOXO3, FOXO4 and FOXO6). They are important regulatory molecules that play a critical role in a number of physiological and pathological processes such as cell cycle control, the body's response to stress, differentiation and apoptosis. Due to their ability to induce cell death, they are generally considered to be tumor suppressors. However, recent studies have shown that they can also induce an opposite effect, i.e. to promote tumor progression or induce resistance to drugs used in the therapy of certain types of tumors. Despite intensive research, a number of questions regarding the function of FOXO proteins still remain unanswered. One question is whether the small structural differences observed in the highly conserved DNA-binding domains (DBD) of FOXO transcription factors affect their DNA- binding affinities. Furthermore, it is unclear whether the recently described protein-protein interaction of FOXO-DBD with the transcription factor p53 affects their DNA-binding affinity. Moreover, the role of the binding site for Mg2+ ion which was found in the crystal structure of FOXO4-DBD:DNA, is also still not understood. To clarify these questions, the DNA-binding domains of the human transcription...
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Role of PML in ribosomal stress
Kremserová, Petra ; Vašicová, Pavla (advisor) ; Sztacho, Martin (referee)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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