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Expression of ubiquitin ligases in gastrointestinal tract
Pícková, Markéta ; Sedláček, Radislav (advisor) ; Čermák, Lukáš (referee)
Ubiquitin (Ub) ligases are important regulatory and signalling molecules, which are involved in majority of cellular processes such as differentiation, DNA repair, and regulation of energetic metabolism or immune response. E3 Ubiquitin ligases are also responsible for pathophysiological changes in the organism and their activity is associated with many human diseases including cancers. This makes E3 Ubiquitin ligases to be new diagnostic markers and interesting pharmaceutical targets. Based on previous studies, these enzymes evince very specific expression in the level of tissues or cell populations. Determination of this specific expression is important for a better understanding of their biological function. In this diploma thesis we systematically screened presence of 370 genes of E3-Ub ligases in gastrointestinal tract under physiological conditions and during acute inflammatory damage of distal colon. Obtained data allowed us to select genes, which can play important role in homeostasis as well as pathophysiology and regeneration of gastrointestinal tract. The screening was based on the expression profiling using qPCR, followed by in situ hybridization to determine the exact localization of the gene expression within tissues. From qPCR analysis was predicted hundred thirty seven candidates for...
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Searching for a physiological partner of Ddi2 (DNA damage-inducible protein homolog 2) protein
Kurfürst, Jaroslav ; Grantz Šašková, Klára (advisor) ; Vaněk, Ondřej (referee)
One of the most important cellular processes, essential not only for protein degradation, is the so called ubiquitin-proteasome system. A key player in this system is ubiquitin, a small protein with unique ability to form various chains. Cellular proteins marked for degradation via ubiquitin, are recruited to the proteasome either by a direct interaction with one of the intrinsic proteasomal receptors, or by adaptor proteins. These proteins typically possess ubiquitin-like domain and ubiquitin associated domain that predispose them for delivering ubiquinated proteins to the proteasome. Adaptor protein called Ddi2 differs from other members of this family by possessing additional domain called the retroviral protease like domain. This domain is structurally similar to HIV protease and its proteolytic function has been discovered only recently. Due to the presence of this proteolytic domain one could expect that Ddi2 might be a deubiquitinase. Here we therefore tested the possible cleavage of diubiquitin chains by recombinantly prepared Ddi2 protein. We can conclude that Ddi2 did not show any deubiquitinating activity in given conditions.
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Ddi1-like proteins: a novel family of retroviral-like aspartyl proteases
Šmilauerová, Kristýna ; Grantz Šašková, Klára (advisor) ; Šmahel, Michal (referee)
Ubiquitin-proteasome system is one of the key pathways which maintain cell homeostasis. Its purpose is to degrade damaged, misfolded or unnecessary proteins. It is also involved in multiple other processes such as DNA damage repair, cell cycle control or signaling. The entire system consists of multiple components, which are mutually strictly regulated. Important part of this system is group of so called proteasome adaptor proteins. Their role is to recognize and bind targeted substrates and transport them to the proteasome for degradation. Ddi1-like (abbrev. from DNA damage-inducible protein 1) protein family, a group of proteins with retroviral aspartyl protease-like domain, belongs to proteasome adaptor proteins. Global biological role of this protein family is only partially understood the most studied member is Ddi1 protein from Saccharomyces cerevisiae, and it is thus a subject of active research. This thesis summarizes published information about this protein family, describes its general characteristics and known functions, situates them in the context of cell processes and thereby might suggest the course of further study.
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Studies towards biological function of ubiquitin E3 ligase Rnf121 in vivo and in vitro
Škarabellová, Kateřina ; Sedláček, Radislav (advisor) ; Čermák, Lukáš (referee)
Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...
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Effect of sperm ubiquitination on the early embryonic development in pig
Kroumanová, Kristýna ; Krylov, Vladimír (advisor) ; Nevoral, Jan (referee)
The ubiquitin-proteasomal system which is the major pathway for intracellular protein degradation is also involved in sperm quality control in the mammalian epididymis. Defective sperm become surface- ubiquitinated during epididymal passage. The level of sperm surface ubiquitination negatively correlates with their quality. Hypothetically it is possible that after fertilization, highly ubiquitinated sperm, naturally present in mammalian ejaculates, would be actively recognized by oocyte (probably via 26S proteasomal complex). Subsequent partial or total sperm degradation should negatively affect the development of the potentially defective embryo. In this study, we examined the effect of sperm ubiquitination on the early embryonic development in pig (Sus scrofa f. domestica) using the method of intracytoplasmic sperm injection (ICSI). In vitro embryonic development to the blastocyst stage after ICSI was comparable with other laboratories. In this study, no significant difference was observed in the formation of pronuclei between oocytes fertilized by lower and highly ubiquitinated sperm cells. On the other hand, significantly better embryonic development to the blastocyst stage was observed in oocytes fertilized by sperm with lower surface ubiquitination (17 %) compared with oocytes fertilized by highly...
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Effect of ubiquitin-proteasome system on spermiogenesis in mammals
Kroumanová, Kristýna ; Petelák, Aleš (advisor) ; Krejčová, Tereza (referee)
Spermiogenesis is a complicated process in which the haploid spermatids differentiate into morphologically mature sperm. Morphological changes include condensation of chromatin and histone-to-protamine replacement, fusion of Golgi-derived vesicles to form acrosomal cap, flagellum formation, reduction of cytoplasmic volume and organelles rearrangement. Ubiquitin-proteasome system plays a key role in these processes. Ubiquitination is a posttranslational modification, leading to the labelling of intracellular proteins targeted for degradation by 26S proteasome. Importance of sperm ubiquitination, is supported by the fact, that deficiency of ubiquitin-proteasome system can lead to infertility at various degrees. During the subsequent sperm maturation in the epididymis extracellular ubiquitination of abnormal sperm and sperm quality control take place. Ubiquitin-proteasome system plays a key role during fertilization, when the sperm 26S proteasome is co-responsible in zona pellucida penetration. The purpose of this assay is to describe the effect of ubiquitin- proteasome system during different stages of spermiogenesis in mammals.
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Proteasomes and DNA virus infection
Vinšová, Barbora ; Drda Morávková, Alena (advisor) ; Motlová, Lucia (referee)
The development of virus infection depends on virus - host interactions. Millions of years of ongoing virus - host coevolution led to formation of many antiviral defense mechanisms as same as virus evasion strategies. Viruses have learned to intervene in the various cellular processes, modify it and take advantage of particular cellular components. One of those cellular components widely utilised by viruses is the ubiquitin-proteasome system. Proteasomes are multisubunit protein structures that under normal conditions provide degradation of damaged, missfolded or redundant cellular proteins. With their function proteasomes contribute to regulation of various cellular processes and maintain balance of proteins ratio. Viruses utilise those structures for protein degradation in order to evade host immunity system and deregulate cell cycle, to entry and unpacking of virions or in order to favor virus replication. This thesis is conceived to briefly summarize interactions of cellular ubiquitin-proteasome system and DNA viruses.
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Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...
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