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Functional Characterization of SCFFBXO38 Ubiquitin Ligase-dependent Protein Degradation
Dibus, Nikol ; Čermák, Lukáš (advisor) ; Konvalinka, Jan (referee) ; D´Angiolella, Vincenzo (referee)
Ubiquitin ligases are responsible for the specific recognition of proteins targeted for proteasome-dependent degradation. This project focused on the molecular and functional characterization of the SCFFBXO38 ubiquitin ligase. As with many others, its biological function has not yet been elucidated in detail, although it is the only ubiquitin ligase whose mutations lead to the onset of a distal form of muscle atrophy. In the first part of our project, we identified new substrates for this ubiquitin ligase, the nuclear proteins ZXDA and ZXDB, with insufficiently characterized functions. Using genetic and biochemical methods, we have shown that ZXDA/B proteins act as positive regulators of centromeric chromatin integrity and that experimental inactivation of the SCFFBXO38 ubiquitin ligase resulted in a ZXDA/B-dependent stabilization of CENP-A and CENP-B proteins in the centromeric regions. In the second part of the project, we focused on analyzing the mouse model deficient in the Fbxo38 gene. We demonstrated that loss of Fbxo38 leads to growth retardation affecting various organs, including the male reproductive system. A detailed histological examination revealed pathological alterations in the seminiferous tubules, accompanied by a lower number of spermatozoa and decreased fertility. We have shown...
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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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