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Novel substrates of cullin-RING ubiquitin ligases: identification and functional characterisation
Liďák, Tomáš ; Čermák, Lukáš (advisor) ; Grantz Šašková, Klára (referee) ; Mašek, Jan (referee)
Selective protein degradation by the ubiquitin-proteasome system is essential for cellular homeostasis and the regulation of diverse biological processes. The selectivity of this system is imparted by hundreds of ubiquitin ligases that specifically recognise substrates and catalyse their ubiquitination, thereby targeting them for degradation. Among ubiquitin ligases, multisubunit cullin-RING ubiquitin ligases constitute the largest group. However, despite significant advances in understanding their assembly, regulation, and molecular architecture, the substrates and functions of most of them remain unknown. This thesis focuses on two ubiquitin ligases from the cullin-RING ubiquitin ligase 4 (CRL4) subfamily: CRL4DCAF4 and CRL4DCAF12 . To identify their candidate substrates and to address their biological roles, several different approaches have been employed. First, proteomic screening revealed a wide range of candidate substrates. Next, detailed characterisation of the identified interactions and exploration of the condition under which candidate substrates undergo degradation was performed. Finally, knockout human cell lines and mice with a targeted disruption of genes encoding DCAF4 and DCAF12 were generated to explore the physiological roles of CRL4DCAF4 and CRL4DCAF12 . In summary, the herein...
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Dynamic of PER2 clock protein degradation detected by real time bioluminescence assay in the tissue explant of the circadian clock of mPER2Luc mouse
Stanislavová, Faustýna ; Sumová, Alena (advisor) ; Doležel, David (referee)
Suprachiasmatic nuclei are the main oscillator of circadian rhythms. Using clock genes and their protein products (forming transcription-translation feedback loops), suprachiasmatic nuclei play an important role in the control of many physiological functions. Bioluminescence (the amount of hourly protein PER2) was measured by a method using transgenic organisms. In this work, cycloheximide was used to inhibit proteosynthesis and to subsequently monitor the degradation of PER2 in real time. The protein was then measured in explants of suprachiasmatic nuclei of adult mice, in suprachiasmatic nuclei of fetuses and in placentas. Furthermore, the effects of glycogen synthasykinase 3b inhibitors on the dynamics of PER2 protein degradation were compared. A selective inhibitor CHIR-99021 and a non-specific inhibitor lithium chloride were used. The experiment shows that the CHIR inhibitor slows down protein degradation in all tissues used. In contrast, the effect of a non-specific lithium chloride inhibitor has not been clearly demonstrated. In fetal nuclei, its effect on the dynamics of degradation was slowing, while in adult nuclei, degradation was significantly accelerated. No significant results were observed in placental explants. Research focusing on the influence of these clock genes, respectively...
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Protein quality control in the secretory pathway of eukaryotic cells
Bařinková, Markéta ; Stříšovský, Kvido (advisor) ; Černý, Jan (referee)
More than 30 % of the cellular proteome enters the secretory pathway during biogenesis in eukaryotic cells. The secretory pathway then ensures that these proteins are correctly folded, undergo necessary post- translational modifications, and reach their target site in membrane organelles or outside of the cell. Since a significant number of the nascent proteins in the pathway are or become dysfunctional, the cell must possess quality control mechanisms by which to weed them out. As proteins travel through the secretory pathway they may be degraded by various pathways in the endoplasmic reticulum, Golgi apparatus, endosomes, or at the plasma membrane. These degradatory pathways utilize a number of molecules including chaperones, ubiquitin ligases, and many others. They are coordinated by a unifying principle - the unfolded protein response, which acts as a support mechanism in case the degradation pathways are overwhelmed. The study of protein quality control mechanisms is necessary as they help us understand the production of a significant portion of the cellular proteome. Furthermore, defects in these degradation pathways are linked to several human diseases such as cystic fibrosis or some neurodegenerative diseases. These protein degradation pathways have been studied for decades, but thanks to...
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Narušení metabolismu proteinů a jeho efekt na růst rostlin
Luklová, Markéta
Recent years have shown that plant hormones operate through circuits in which targeted, conditional turnover of regulator proteins via proteasome are of critical importance. Thus, a disruption of the ubiquitin-proteasome system (UPS) would have a significant impact on plant growth and development. This thesis entitled Protein metabolism disruption and its effect on plant growth reviews the mechanisms of proteasome-mediated targeted degradation and analyzes its role in plant hormone cytokinin signaling. The experimental part is divided into two sections (i) growth response analyzes and (ii) an LC-MS profiling of diurnal variations in transgenic Arabidopsis seedlings RV 86-5 carrying modified inducible ubiquitin that prevents protein degradation via the UPS. The results present evidence that an intricate balance between the UPS and cytokinin modulates the growth response output of cytokinin signaling. On the proteome level, an LC-MS profiling identified >4,000 proteins, 61 of which were previously identified in response to proteasome inhibition.
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Interakce rostlin s pathogeny: význam proteasomu a cytoskeletu
Zelená, Pavla
Modification of substrate proteins by ubiquitin is one of the major regulatory events in eukaryotic cells, and plants use this modification to react to exogenous and endogenous cues. Mounting evidence indicates that the ubiquitin-proteasome system is also a key component in response to biotic stressors. This thesis entitled "Plant-pathogen interactions: The role of proteasome and cytoskeleton" reviews the role of proteasome in plant signaling and briefly summarizes interactions between cytoskeleton and pathogen and outlines the role of cytokinin in plant-pathogen interactions. In the experimental part, proteasome-mediated signaling is probed using growth response assay, identification of proteasome substrates and confocal microscopy of cytoskeleton structures. The results present evidence that the employed methodology will be useful for plant-pathogen interaction analyses.
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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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