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The effect of 14-3-3 protein on intradomain interaction of ubiquitin ligase Nedd4-2
Pohl, Pavel ; Obšilová, Veronika (advisor) ; Žáková, Lenka (referee) ; Pavlíček, Jiří (referee)
EN The human ubiquitin ligase Nedd4-2 (NEDD4L) ubiquitinates a wide range of membrane proteins and receptors, playing a key role in maintaining homeostasis. This enzyme is regulated by phosphorylation and subsequent interaction with 14-3-3 proteins, which primarily affects its ability to interact with various substrates. However, very little is known about the molecular basis of this protein-protein interaction. In this work, we focused on biophysical characterization of the role of individual phosphorylation sites and also on mapping the structural changes in the Nedd4- 2 protein induced by 14-3-3 protein binding. Our experiments using analytical ultracentrifugation methods revealed that two phosphorylation sites Ser342 and Ser448 are primarily required for stable binding of Nedd4-2 to 14-3-3 proteins. The crystal structure of the 14-3-3ηΔC:Nedd4-2335-455 T367A complex than revealed the simultaneous binding of both phosphorylated residues to the binding groove of 14-3-3 protein. Subsequent modeling based on small-angle X-ray scattering and chemical cross-linking data combined with mass spectrometry indicated extensive structural changes in the individual domains of the Nedd4-2 protein. Binding of 14-3-3η protein blocks the WW3 domain of Nedd4-2 in the central channel of 14-3-3 protein, while...
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Role of RAD18 in ubiquitin signaling at DNA double-strand breaks
Palek, Matouš ; Macůrek, Libor (advisor) ; Čermák, Lukáš (referee)
RAD18 is an E3 ubiquitin ligase that prevents the replication forks from collapsing caused by damaged DNA. As an important factor controlling replication, its dysregulation was shown to be associated with some human tumours. However, the clinical relevance of this finding is unknown. The aim of the thesis was evaluation of selected RAD18 variants that had been identified in breast and ovarian cancer patients. This work revealed functional defects of RAD18 variants not only in replication fork protection but also in repair of DNA double-strand breaks. This unconventional role of RAD18 is known to be dependent on upstream ubiquitination events, however, its contribution to the repair per se is not understood. This work aimed to elucidate the function of RAD18 in DNA double-strand break repair by homologous recombination focusing especially on its relationship with 53BP1. Data presented here show that RAD18 effectively disrupts 53BP1 accumulation in the repair foci by competition for the same binding partner and thus promotes resection of DNA ends. This antagonistic function of RAD18 is restricted both spatially (to the vicinity of the repair centre) and temporarily (to S phase). Moreover, it seems to be regulated by existence of RAD18 in two distinct complexes. Potential models for this regulation...
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Cancerogenic and metastatic potential of cancer cells with non-functional CRL4 ubiquitination complex
Slámová, Monika ; Procházka, Jan (advisor) ; Grantz Šašková, Klára (referee)
Ubiquitination complex CRL4 (Cullin Ring Ligase) attracts a lot of attention due to its involvement in physiological and pathological processes, especially in the development of cancer. Cullin4 a/b proteins are reported to serve as oncoproteins in various malignancies. Due to their role in the regulation of cancer drugs targeting CRL4 have been identified, including thalidomide and its derivatives inhibiting one of the substrate receptors of the complex, the Cereblon protein. The adapter protein within the CRL4 complex - DDB1, which is involved i.a. in DNA repair, also has a role in cancer. However, the mechanism of this function has not yet been fully elucidated. The subject of this master thesis was to study the effects of elimination and suppression of CRL4 complex functions in the prostate cancer cell line LNCaP. Significantly variable changes in cell proliferation and migration have been observed if the complex functions were affected by thalidomide. The creation of the LNCaP cell line with conditionally suppressed DDB1 function was used to study tumor dynamics in a mouse model. Results show that suppression of DDB1 function has an inhibitory effect on tumor cell proliferation but increases their ability to invade adjacent tissues. Complete deletion of the DDB1 gene in the LNCaP cell line...
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Dysregulation of E3 ubiquitin ligases in inflammatory bowel diseases
Armerová, Eliška ; Petrezsélyová, Silvia (advisor) ; Červená, Klára (referee)
E3 ubiquitin ligases are a large family of enzymes involved in many cellular processes such as DNA damage repair, transport of membrane proteins, chromatin modification, cell cycle and apoptosis. E3 ubiquitin ligases have been shown to play a significant role in the maintenance of intestinal homeostasis, and their abnormal function associated with their deregulation contributes to inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. In recent years, GWAS has identified approximately 200 risk loci that are susceptible to these diseases. Including those encoding E3 ubiquitin ligases.The aim of this work is to compare the already identified E3 ubiquitin ligases associated with these diseases and to give an overview of them with a focus on the regulation of intestinal homeostasis. Key words: E3, ubiquitination, inflammatory bowel diseases, intestinal homeostasis, CD, UC
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Dysregulation of E3 ubiquitin ligases in inflammatory bowel diseases
Armerová, Eliška ; Petrezsélyová, Silvia (advisor) ; Červená, Klára (referee)
E3 ubiquitin ligases are a large family of enzymes involved in many cellular processes such as repair of damaged DNA, transport of membrane proteins, chromatin modification, cell cycle and apoptosis. E3 ubiquitin ligase has been shown to play a significant role in the maintenance of intestinal homeostasis, and their abnormal function associated with their deregulation contributes to inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. In recent years, approximately 200 risk loci have been identified that are susceptible to these diseases, including those encoding E3 ubiquitin ligase. 10 of them have been identified. The aim of this work is to compare the already identified E3 ubiquitin ligases associated with these diseases and to give an overview of them with a focus on the regulation of intestinal homeostasis. Key words: E3, ubiquitination, inflammatory bowel diseases, intestinal homeostasis, CD, UC
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Efekt inhibice proteasomu na proteom Arabidopsis thaliana
Dufek, Martin
Proteasome is highly conserved in its structure among all eukaryotes. The main function of this multi-protein complex is to facilitate protein turnover and degrade misfolded, altered, aged or unneeded proteins. Proteasome-dependant degradation predominantly operates in cooperation with a complex system that conjugates proteins destined for degradation to ubiquitin. Emerging evidence suggests that the proteasome-dependant degradation is crucially involved in plant hormone signalling and as such could play a central role in plant growth and development. This bachelor thesis entitled "Effects of proteasome inhibition on Arabidopsis thaliana proteome" reviews proteasome-ubiquitin pathway with the focus on plant hormone signalling. In the experimental part, the effects of proteasome inhibition were investigated via LC-MS profiling. Altogether, abundances of more than 1400 proteins were followed. PCA analysis and a detailed pair-wise comparison of MG-132 and mock treated seedlings provided an insight into the molecular mechanism behind processes in the response to proteasome inhibition.
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