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The role of CRL4 ubiquitin ligase complex in homeostasis and regeneration of gastro intestinal tract
Daněk, Veronika ; Procházka, Jan (advisor) ; Janečková, Lucie (referee) ; Čermák, Lukáš (referee)
Ubiquitination represents a central regulatory mechanism for protein function utilization. Dysfunction of ubiquitin ligases is associated with the development of various pathological conditions. However, the full complexity of the ubiquitinating network in tissue homeostasis and carcinogenesis remains to be elucidated, limiting its potential as a therapeutic target. In my PhD project, I aimed to reveal the role of E3 ubiquitin ligases, particularly CUL4A, in the alteration of regulatory pathways that result in gastrointestinal (GIT) homeostasis disorders and tumor expansion. By using a novel semantic biclustering technique, we performed expression profiling of the E3 ubiquitin ligases and uncovered the potentially redundant features of GIT-specific ubiquitin ligases based on their tissue-specific expression and ontological relationships. Testing the identified compensatory network on the mouse model showed that genes from the same ontology group simultaneously altered their expression pattern after induced epithelial damage, exposing their complementary role during tissue regeneration. Next, I focused on CUL4A, a scaffold of the Cullin4-RING E3 ubiquitin ligase complex (CRL4). We described that CUL4A is expressed by populations of secretory cells in the small intestine and colon. CUL4A deficiency...
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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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Ubiquitin-proteasome system in studies of its inhibition and its utilization in the cell-based assay measuring viral protease activity
Fürst, Eliška ; Majerová, Taťána (advisor) ; Čermák, Lukáš (referee)
and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...
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Roles of acrosomal proteins in fertilization
Macíčková, Eliška ; Stopka, Pavel (advisor) ; Čermák, Lukáš (referee)
The acrosome is a crucial organelle for sperm cells. Defects in the biogenesis of the acrosome often lead to the impaired development of sperm cells and consequently, to the inability to fertilize the oocyte. Although it is evident that the acrosome is an essential structure, its main function has not been solved yet. The initial hypothesis was that the acrosome helps sperm to pass through the extracellular layers of the egg. This statement is based on evidence that acrosome is full of proteolytic enzymes, and inhibition of these enzymes reduces the ability to fertilize the oocyte. However, this hypothesis began to be questioned when it was found that (i) acrosome-reacted sperm cells are still able to pass through extracellular layers and fertilize the oocyte (ii) most of the sperm cells undergo premature acrosomal reaction before they reach the oocyte. Therefore, we aimed to characterize proteomes of the acrosome and acrosome- reacted sperm cells, identify important proteins using advanced bioinformatics approaches and discuss the function of the acrosome as a vesicle that serves to store and process metabolites after the previous maturation process. In this work, we revealed new findings that contribute to the hypothesis that acrosome is a lysosome-related organelle. Key words: sperm, acrosome,...
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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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Defects in DNA repair and RNA metabolism associated with human neurological disorders
Cihlářová, Zuzana ; Hanzlíková, Hana (advisor) ; Čermák, Lukáš (referee) ; Roithová, Adriana (referee)
The human genome is constantly under the attack by various damaging agents, leading to the breakage of one or both strands of DNA that might interfere with RNA processing. Importantly, our cells have evolved diverse mechanisms to rapidly repair various DNA lesions, highlighting the importance of genetic integrity. Defects in DNA repair and/or RNA metabolism can lead to a variety of human hereditary diseases, with pathologies including growth and developmental defects, immunodeficiency, predisposition to cancer, and neurodegeneration. Mutations in the BRAT1 (BRCA1-associated ATM activator-1) protein have been associated with neurological disorders characterized by heterogenous phenotypes with varying levels of clinical severity ranging from microcephaly, hypertonia, epilepsy, seizures, and early death in the first two years of life to mild cerebellar atrophy and ataxia. Previously, BRAT1 protein has been implicated in the cellular response to DNA double-strand breaks and ATM signalling. However, the exact mechanism/s by which mutations in BRAT1 gene trigger neurological disorders are largely unknown. Recently, we have identified a homozygous missense c.185T>A (p.Val62Glu) variant in BRAT1 that markedly reduced the level of BRAT1 protein in patient-derived cell lines. Surprisingly, our data show that...
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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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The role of Fbxo38 ubiquitin ligase in mouse spermatogenesis
Zobalová, Eliška ; Čermák, Lukáš (advisor) ; Stopka, Pavel (referee)
Cullin-dependent ubiquitin ligases are responsible for the regulation of most cellular processes. Despite their mutated forms being the cause of many human diseases, their physiological roles are not sufficiently described. In the presented results, we focused on the physiological role of ubiquitin ligase SCFFBXO38 (SKP1-CULLIN1-FBXO38), whose mutated forms are responsible for the progression of distal neuropathy. Preparation of mouse model deficient in FBXO38 revealed that homozygous pups were born in a lower than expected ratio. Animals were growth-retarded, both at the level of the whole organism and individual organs, especially the liver and testes. Males with a deletion in the Fbxo38 gene had significantly lower reproductive capacity, which was associated with lower production of mature sperm and pathological changes in the structure of seminiferous tubules. We found that the FBXO38 protein is functionally expressed in Sertoli cells responsible for regulating spermatogenesis and seminiferous tubules integrity. Detailed analysis of spermatogenic populations revealed a defect at the level of spermatocyte differentiation. The dynamics of this differentiation depend on the hematotesticular barrier functional integrity formed by the intercellular junctions of Sertoli cells. We confirmed that the...
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BIOPHYSICAL AND FUNCTIONAL CHARACTERIZATION OF DDI1-LIKE ASPARTIC PROTEASES INVOLVED IN REPLICATION STRESS RESPONSE
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Čermák, Lukáš (referee)
Accurate, timely replication of a DNA molecule is a pivotal moment in the life cycle of every living organism. Any temporal or spatial defect putting the fine-tuned replication machinery off balance causes the so-called replication stress. As the replication machinery consists mainly of enzymes and other proteins, it is not surprising that many of the obstacles most severely blocking the replication machinery progress are of protein origin. Therefore, specialized proteases responsible for relieving replication stress matured during evolution. However, neither the full repertoire of proteolytic enzymes and their particular substrates taking place in countering the DNA replication stress nor detailed molecular mechanisms involved remain unknown. This thesis describes how conserved putative aspartic proteases of the Ddi1-like family engage in countering DNA replication stress via a proteolysis dependent mechanism. We structurally and biophysically characterized yeast and human members of the Ddi1-like family, explored their interactions with ubiquitin and polyubiquitin chains, and identified hypersensitivity to DNA replication inhibitor hydroxyurea in a yeast strain double deleted for DDI1 gene together with a DNA dependent metalloprotease WSS1. Detailed analysis of the DDI1 role in hydroxyurea...
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Ubiquitin-proteasome system in studies of its inhibition and its utilization in the cell-based assay measuring viral protease activity
Fürst, Eliška ; Majerová, Taťána (advisor) ; Čermák, Lukáš (referee)
and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...
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