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Cytochrome c and its role in apoptosis
Rajsiglová, Lenka ; Kalous, Martin (advisor) ; Švadlenka, Jan (referee)
Cell energetic metabolism and cell survival are strictly controlled by pathways in which cytochrome molecules play a central role, in particular cytochrome c. It is localized in the mitochondrial intermembrane space with other molecules cooperating in keeping energetic metabolism. Permeabilization of outer mitochondrial membrane by proteins from Bcl-2 family or changes in Ca2+ levels causes cytochrome c release into cytosol. In cytosol cytochrome c interacts with other pro-apoptotic proteins (Apaf-1, procaspase-9) cooperating to form apoptosome and phosphatidylserine. As a result of these interactions, the cell is going to apoptosis. This bachelor thesis summarizes the current state of knowledge of these processes. In the first part it focuses on the biosynthesis of cytochrome c, further on the mechanisms of its releasing from mitochondria and its interactions with other proteins within apoptosis including options of regulation of these processes.
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The role of sumoylation in cellular senescence
Kopová, Ivana ; Hodný, Zdeněk (advisor) ; Švadlenka, Jan (referee)
Organisms with renewable tissues require mechanisms to prevent the development of cancer. One such mechanism is cellular senescence, which irreversibly arrests the growth of cells at risk for neoplastic transformation. In this study, we show that 100 μM 5-bromo-2-deoxyuridine, 0.5 μM camptothecin, 0.5 μM aphidicolin and 2.5 mM thymidine cause chemically-induced premature senescence in different human cancer cell lines and they induce an increasing conjugation of SUMO-2/3 isoforms. Chemically- induced premature senescence also induces formation of SUMO-1 and SUMO-2/3 foci, which are colocalized with PML nuclear bodies. In addition, we describe that aphidicolin induces premature senescence in stable HeLa cell line expressing His6-tagged SUMO-1. HeLa-His6-SUMO-1 cell line has decreased number of PML bodies, which do not colocalize with SUMO-2/3 foci. Moreover, the number of PML bodies in HeLa cell line with ectopic expression of His6-SUMO-1 is not increasing during aphidicolin-induced senescence. This demonstrates that increasing number of PML nuclear bodies is not essential for aphidicolin-induced senescence. Absence of SUMO-2/3 foci and increased number of PML nuclear bodies support the theory that SUMO-1 acts as a SUMO-2/3 polymeric chain terminator. On the other hand, in stable HeLa cell lines expressing...
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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan ; Anděra, Ladislav (advisor) ; Forstová, Jitka (referee) ; Stopka, Tomáš (referee)
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan ; Anděra, Ladislav (advisor) ; Forstová, Jitka (referee) ; Stopka, Tomáš (referee)
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Cytochrome c and its role in apoptosis
Rajsiglová, Lenka ; Kalous, Martin (advisor) ; Švadlenka, Jan (referee)
Cell energetic metabolism and cell survival are strictly controlled by pathways in which cytochrome molecules play a central role, in particular cytochrome c. It is localized in the mitochondrial intermembrane space with other molecules cooperating in keeping energetic metabolism. Permeabilization of outer mitochondrial membrane by proteins from Bcl-2 family or changes in Ca2+ levels causes cytochrome c release into cytosol. In cytosol cytochrome c interacts with other pro-apoptotic proteins (Apaf-1, procaspase-9) cooperating to form apoptosome and phosphatidylserine. As a result of these interactions, the cell is going to apoptosis. This bachelor thesis summarizes the current state of knowledge of these processes. In the first part it focuses on the biosynthesis of cytochrome c, further on the mechanisms of its releasing from mitochondria and its interactions with other proteins within apoptosis including options of regulation of these processes.
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The role of sumoylation in cellular senescence
Kopová, Ivana ; Švadlenka, Jan (referee) ; Hodný, Zdeněk (advisor)
Organisms with renewable tissues require mechanisms to prevent the development of cancer. One such mechanism is cellular senescence, which irreversibly arrests the growth of cells at risk for neoplastic transformation. In this study, we show that 100 μM 5-bromo-2-deoxyuridine, 0.5 μM camptothecin, 0.5 μM aphidicolin and 2.5 mM thymidine cause chemically-induced premature senescence in different human cancer cell lines and they induce an increasing conjugation of SUMO-2/3 isoforms. Chemically- induced premature senescence also induces formation of SUMO-1 and SUMO-2/3 foci, which are colocalized with PML nuclear bodies. In addition, we describe that aphidicolin induces premature senescence in stable HeLa cell line expressing His6-tagged SUMO-1. HeLa-His6-SUMO-1 cell line has decreased number of PML bodies, which do not colocalize with SUMO-2/3 foci. Moreover, the number of PML bodies in HeLa cell line with ectopic expression of His6-SUMO-1 is not increasing during aphidicolin-induced senescence. This demonstrates that increasing number of PML nuclear bodies is not essential for aphidicolin-induced senescence. Absence of SUMO-2/3 foci and increased number of PML nuclear bodies support the theory that SUMO-1 acts as a SUMO-2/3 polymeric chain terminator. On the other hand, in stable HeLa cell lines expressing...
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