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Molecular basis of interactions between Dishevelled 3 (Dvl3) and Protein Regulator Of Cytokinesis 1 (PRC1)
Kropáčková, Veronika ; Bařinka, Cyril (advisor) ; Macůrková, Marie (referee)
Scaffolding protein Disheveled (Dvl) is a key component of Wnt signaling cascades. Dvl participates in a number of biological processes, such as cell proliferation, differentiation and migration, determination of cell polarity, and also stem cell self-renewal. It is therefore indispensable for the correct embryo development and tissue homeostasis in adulthood. The protein regulator of cytokinesis (PRC1) is a microtubule-associated protein. PRC1 is involved in spindle midzone formation during cell division. Spindle midzone precedes the contractile ring assembly and is essential for normal cell cleavage. In our laboratory, PRC1 was identified as a putative interaction partner of DVL3. This master thesis is focused on delineation of the interaction interface between DVL3 and PRC1 using TIRF microscopy (Total Internal Reflection Fluorescence microscopy). To this end, full-length DVL and PRC1 proteins together with their truncated variants were designed, expressed and purified. It was discovered that PRC1 interacts with all three DVL isoforms and the N-terminal part of PRC1 is required for the interaction between PRC1 and DVL3. Furthermore, the DEP domain of DVL3 is likely involved in PRC1interactions. Key words: Dishevelled 3, DVL3, Protein regulator of cytokinesis 1, PRC1, interaction interface, TIRF...
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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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Molecular basis of interactions between Dishevelled 3 (Dvl3) and Protein Regulator Of Cytokinesis 1 (PRC1)
Kropáčková, Veronika ; Bařinka, Cyril (advisor) ; Macůrková, Marie (referee)
Scaffolding protein Disheveled (Dvl) is a key component of Wnt signaling cascades. Dvl participates in a number of biological processes, such as cell proliferation, differentiation and migration, determination of cell polarity, and also stem cell self-renewal. It is therefore indispensable for the correct embryo development and tissue homeostasis in adulthood. The protein regulator of cytokinesis (PRC1) is a microtubule-associated protein. PRC1 is involved in spindle midzone formation during cell division. Spindle midzone precedes the contractile ring assembly and is essential for normal cell cleavage. In our laboratory, PRC1 was identified as a putative interaction partner of DVL3. This master thesis is focused on delineation of the interaction interface between DVL3 and PRC1 using TIRF microscopy (Total Internal Reflection Fluorescence microscopy). To this end, full-length DVL and PRC1 proteins together with their truncated variants were designed, expressed and purified. It was discovered that PRC1 interacts with all three DVL isoforms and the N-terminal part of PRC1 is required for the interaction between PRC1 and DVL3. Furthermore, the DEP domain of DVL3 is likely involved in PRC1interactions. Key words: Dishevelled 3, DVL3, Protein regulator of cytokinesis 1, PRC1, interaction interface, TIRF...
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Study of protein-protein interaction in bacterial pathogenesis: PIXL (photo-induced cross-linking) methodology
Žídek, Radek ; Šulc, Miroslav (advisor) ; Ječmen, Tomáš (referee)
Gramnegativní bakterie druhu Bordetella pertussis jsou původci smrtelného lidského onemocnění označovaného jako pertuse, častěji jako černý kašel. Tyto bakterie produkují adenylátcyklázový toxin (ACT), který se váže na povrch makrofágů a umožňuje vpravit do cytosolu hostitelské buňky přes cytoplazmatickou membránu adenylátcyklázovou doménu (dAC). Abychom mohli studovat kovalentní interakce mezi proteiny pomocí fotochemického zesítění, byl náš studovaný protein exprimován s foto-methioninem (kyselinou L-2-amino-5,5- azi-hexanovou) v kultivačním médiu v bakteriálním kmenu Escherichia coli B834 (DE3). Foto- methionin je netoxickým analogem L-methioninu, takže je normálně inkorporován pomocí aminoacyl-tRNA syntház do struktury adenylátcyklázové domény. Maximální míry inkorporace foto-methioninu do struktury dAC bylo dosaženo po optimalizaci celého expresního protokolu. Celková míra inkorporace foto-methioninu do struktury proteinu po optimalizaci zjištěná hmotnostně-spektrometrickou analýzou byla až 80 %. Získaný protein s inkorporovaným foto- methioninem byl izolován. Byly provedeny síťovací experimenty s kalmodulinem a vláknitým hemaglutininem. Při těchto experimentech bylo provedeno jak fotochemické, tak i chemické zesítění. Vzniklé kovalentně zesítěné produkty byly rozděleny pomocí SDS-PAGE a...
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Expression and purification of protein photo-initiated nanoprobe: tool to study clinically relevant protein-protein interactions
Knížek, Antonín ; Šulc, Miroslav (advisor) ; Koblihová, Jitka (referee)
Cytochrome b5 is a key protein in the function and regulation of the mixed function monooxygenase (MFO) system in mammalian endoplasmic reticulum and is, therefore, a clinically relevant target for biochemical studies. To study its interactions within the MFO system using photo-initiated crosslinking, we have developed cytochrome b5 mutants with methionine in several key amino acid positions within the primary amino acid sequence, such as serine 23 and leucine 41. Also, naturally presented Met in positions 96, 126 and 131 were mutated to Leu with no effect to cytochrome b5 activity. Our protein was expressed in E. coli B834 auxotrophic type with L-2-amino-5,5-azi-hexanoic acid (photo-Met) present in the cultivation medium. This methionine analogue with photolabile diazirine ring is readily incorporated in Met positions into the primary sequence of proteins by aminoacyl-tRNA synthetases. The whole expression protocol was optimized to achieve maximal percentage of photo-Met incorporation into the expressed protein sequence. Up to 93.4% incorporation of photo-Met was achieved. The expressed protein was isolated and photo-Met incorporation was established with MALDI-TOF mass spectrometry. After reconstitution with its natural interaction partners - full-length cytochrome P450 2B4 (rabbit isoform) or...
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