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Role of the WIP1 phosphatase in the nucleolus
Palková, Natálie ; Macůrek, Libor (advisor) ; Sztacho, Martin (referee)
Protein phosphatase 2C delta (known as WIP1) is an important negative regulator of the DNA damage response (DDR) signalling. As a chromatin-bound protein, it dephosphorylates and thus inactivates ATM kinase and the transcription factor p53. Increased expression of WIP1 suppresses the function of the tumour suppressor p53 and contributes to the development of several cancer types, including breast and brain tumours. In recent years, it has been shown that WIP1 can also regulate cellular processes that are not directly linked to DDR, such as ensuring telomere stability. However, alternative functions of the WIP1 protein have not yet been fully understood. In this work, we described a novel role of the WIP1 phosphatase in the nucleolus, the organelle responsible for ribosome biogenesis. We found that WIP1 associates with many nuclear proteins, and using deletion mutants, we identified a nucleolar localisation sequence (NoLS) at the C-terminus of the protein. Using super-resolution microscopy, we detected the localisation of WIP1 phosphatase in the fibrillar centres of the nucleolus. We employed an inducible Cas9 system for generating double-strand breaks in ribosomal DNA and found that WIP1 has an impact on recruitment of DNA repair factors to the nucleolar caps. Analysis based on quantitative...
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The role of truncated PPM1D/Wip1 phosphatase in cancer
Martiníková, Andra-Stefania ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Mistrík, Martin (referee)
When encountering damage, the cells activate the DNA Damage Response (DDR) pathway and stop the cell cycle until the DNA is repaired. PPM1D/WIP1 phosphatase resumes the cell cycle after the damage has been repaired, by directly dephosphorylating DNA damage markers. The DDR pathway prevents genome instability or cancer development. Mutations in the Ppm1d gene encoding PPM1D result in an overstable and truncated protein observed both in cancer patients and in cancer cell lines. In this thesis, we used an "in-house" transgenic mouse model in which mutations in the exon 6 of the Ppm1d gene resulted in a truncated PPM1D protein. First, we observed high PPM1D levels and impaired DDR to gamma ionizing radiation (IR) in the mouse thymi having truncated PPM1D (Ppm1dT/+ ). We then bred the Ppm1dT/+ mice with the Trp53+/- heterozygote knock-out mice which are prone to thymic lymphoma. The Ppm1dT/+ Trp53+/- double-mutants had a higher frequency of developing IR-induced T-cell lymphomas, compared to the single Trp53+/- mutants. Moreover, truncated PPM1D leads to a defective cell cycle checkpoint activation in human non-transformed RPE cells (RPE1), which then proliferate despite the presence of DNA damage. RPE1 cells also display increased proliferation after replication stress. RPE1 or U2OS cells with...
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Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
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In search of DUSP specificity
Sladeček, Stanislava ; Novotný, Marian (advisor) ; Martínková, Natália (referee)
Dual specificity phosphatases (DUSP) are enzymes that dephosphorylate both phosphoserine/threonine and phosphotyrosine residues on one substrate. Most of them specifically dephosphorylate family mitogen-activated protein kinases (MAPK). Number of DUSPs increases with complexity of organisms and in human genome there are 25 DUSPs described. Some DUSPs can dephosphorylate only one protein while other interact with wider spectrum of substrates. Except for substrate specificity DUSPs differ in expression, subcellular localization etc. Although first DUSPs were described about 20 years ago, a clear factor responsible for their substrate specificity is not known. This works uses in silico methods to discover and describe similarities and differences between DUSPs which may be important in determining DUSP specificity. Key words: phosphatase, kinase, DUSP, MAPK, substrate specificity, conservation of residues, phylogenetic tree, in silico methods
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Characterization of phosphatases in tobacco plants (Nicotiana tabacum L.)
Růžičková, Kateřina ; Ryšlavá, Helena (advisor) ; Tichá, Marie (referee)
Phosphateses (EC 3.1.3.x) are a group of enzymes that hydrolyze phosphoesters. That way they affect the energetic metabolism of a cell and its regulation. Phosphatases that dephosphorylate proteins are an integral part of signaling pathways, stress responses and they modulate enzymatic activity. This thesis deals with the study of phosphatases obtained from tobacco leaves (Nicotiana tabacum, L.). Solution of enzymes was prepared by extraction in both acidic and alkaline buffers. Through the use of the chromogenic substrate pNP-phosphate it was determined that there is a higher phosphatase activity in the glycosylated fraction than in the fraction that did not bind to Con A Sepharose. The research of the ions effect on the phosphatase activity has determined that Mg2+ and Ca2+ show positive effect on the phosphatase activity while the effect of Co2+ and Mn2+ is inhibitory. The Zn2+ ions have shown no effect whatsoever. The glycosylated phosphatases also dephosphorylated low-weight-molecular substrates phosphoserine, ATP and glucose-6-phosphate. The research of protein phosphatase activity discovered the affinity to the substrate phosvitin, although neither to casein nor its tryptic cleaves. Detailed experiments have shown that the pH optima for all the substrates lie from pH 5 to 6. Glycosylated phosphatases...
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Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
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In search of DUSP specificity
Sladeček, Stanislava ; Novotný, Marian (advisor) ; Martínková, Natália (referee)
Dual specificity phosphatases (DUSP) are enzymes that dephosphorylate both phosphoserine/threonine and phosphotyrosine residues on one substrate. Most of them specifically dephosphorylate family mitogen-activated protein kinases (MAPK). Number of DUSPs increases with complexity of organisms and in human genome there are 25 DUSPs described. Some DUSPs can dephosphorylate only one protein while other interact with wider spectrum of substrates. Except for substrate specificity DUSPs differ in expression, subcellular localization etc. Although first DUSPs were described about 20 years ago, a clear factor responsible for their substrate specificity is not known. This works uses in silico methods to discover and describe similarities and differences between DUSPs which may be important in determining DUSP specificity. Key words: phosphatase, kinase, DUSP, MAPK, substrate specificity, conservation of residues, phylogenetic tree, in silico methods
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Characterization of phosphatases in tobacco plants (Nicotiana tabacum L.)
Růžičková, Kateřina ; Ryšlavá, Helena (advisor) ; Tichá, Marie (referee)
Phosphateses (EC 3.1.3.x) are a group of enzymes that hydrolyze phosphoesters. That way they affect the energetic metabolism of a cell and its regulation. Phosphatases that dephosphorylate proteins are an integral part of signaling pathways, stress responses and they modulate enzymatic activity. This thesis deals with the study of phosphatases obtained from tobacco leaves (Nicotiana tabacum, L.). Solution of enzymes was prepared by extraction in both acidic and alkaline buffers. Through the use of the chromogenic substrate pNP-phosphate it was determined that there is a higher phosphatase activity in the glycosylated fraction than in the fraction that did not bind to Con A Sepharose. The research of the ions effect on the phosphatase activity has determined that Mg2+ and Ca2+ show positive effect on the phosphatase activity while the effect of Co2+ and Mn2+ is inhibitory. The Zn2+ ions have shown no effect whatsoever. The glycosylated phosphatases also dephosphorylated low-weight-molecular substrates phosphoserine, ATP and glucose-6-phosphate. The research of protein phosphatase activity discovered the affinity to the substrate phosvitin, although neither to casein nor its tryptic cleaves. Detailed experiments have shown that the pH optima for all the substrates lie from pH 5 to 6. Glycosylated phosphatases...
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