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Elucidating the interactions of interleukin-1alpha with components of the eukaryotic transcription machinery
Zámostná, Blanka ; Pospíšek, Martin (advisor) ; Černý, Jan (referee) ; Mělková, Zora (referee)
4 ABSTRACT Interleukin-1α (IL-1α) is a pleiotropic cytokine and a key mediator of host immune response. It is synthesised as a 31-kDa precursor, that is cleaved by the cysteine protease calpain into the 17-kDa mature IL-1α and the 16-kDa N- terminal peptide of IL-1α (IL-1αNTP). Although IL-1α can be secreted, act on target cells through the surface receptor IL-1RI and trigger the signal transduction pathway, increasing evidence points toward the involvement of IL-1α in certain nuclear processes. IL-1αNTP is highly conserved among higher eukaryotes and contains a nuclear localisation sequence; indeed, both the precursor and IL-1αNTP are found in the cell nucleus. Previously, a genetic interaction of IL-1α with nuclear histone acetyltransferase (HAT) complexes has been reported from mammalian cells and, interestingly, also from the heterologous yeast model. This thesis extends the research of the nuclear function of IL-1α and demonstrates that IL-1α physically associates with the HAT/Core module of yeast SAGA and ADA HAT complexes. Results of the HAT subunit gene knock-out experiments followed by a set of co-immunoprecipitations also suggest a novel model of the yeast SAGA complex assembly, in which ADA appears to represent only a partly functional HAT complex. In its natural milieu of mammalian cells, IL-1α...
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Influence of rRNA modifications on translation initiation in eukaryots
Kročová, Eliška ; Pospíšek, Martin (advisor) ; Kouba, Tomáš (referee)
Modifications of ribosomal RNA are present in every livivng organism. The function of rRNA modifications could be studied only when the process of modifications was described. Currently, scientists study not only individual modifications but also the importance of global level of modifications for maturation and function of ribosome. This thesis deals with the influence of 2'-O-methylation of citidine 1639 and adenosine 100 in 18S rRNA and uridine 2729 in 25S rRNA on initiation in yeast Saccharomyces cerevisiae with special attention of translation controlled by internal ribosome entry site (IRES). Strains with deletion in genes snR51, snR70 and duoble deletion in both genes were successfully created during my master study. Pilot experiments showed the importance of products of both genes in translation initiation.
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Preparation of yeast system for investigation of the human translation initiation
Holásková, Lucie ; Pospíšek, Martin (advisor) ; Cuchalová, Lucie (referee)
Protein synthesis is principally regulated at the initiation stage in which eIF4F complex plays an important role. The eIF4F complex contains three subunits - eIF4A, eIF4E and eIF4G. The eIF4E is cap binding protein, the eIF4A is RNA dependent helicase which unwinds secondary structures at mRNA and scaffolding eIF4G protein. The interaction with other translation initiation factors is important for protein synthesis. The goal of my thesis was to create a new Saccharomyces cerevisiae yeast strain with the human eIF4F factor. Firstly I replaced yeast eIF4E protein with human eIF4E protein. I used a cre/loxP recombination to prepare yeast strains with deleted genes eIF4GI (huΔ4G1) and eIF4GII (huΔ4G2). Characterization of the new yeast strains showed that the human eIF4E protein replaced yeast ortholog factor better than the eIF4E protein from yeast Candida albicans. First experiments showed putative role of the eIF4GII protein during the cell growth under the temperature and osmotic stress. Key words: translation initiation, eIF4E, eIF4G, Saccharomyces cerevisiae
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Interactome of IL-1α N-terminal domain
Dolečková, Denisa ; Pospíšek, Martin (advisor) ; Černý, Jan (referee)
Interactome of IL-1α N-terminal domain Cytokines are highly effective mediators produced by various cell types within and outside of the immune system with the aim to influence the orientation, intensity, and duration of the immune response and inflammatory process. Their biological effects mediated through binding the high-affinity membrane receptors and triggering the signal transduction pathway are usually well defined. However, as it is more and more frequently observed, in addition to the exocrine function, some cytokines may show intracrine effects. For this type of cytokines, the term "dual function cytokines" has been adopted. One of these cytokines is Interleukin-1α, in which the recent research has concentrated on determining its intracellular functions. The intracellular function of interleukin-1α has not been clearly defined so far. However, apart from the absence of the conventional hydrophobic sequence, its existence is supported by the fact that the N-terminal peptide included in its precursor is highly conserved and contains nuclear localization signal. The aim of this work is to define the conditions of localization of the interleukin-1α N- terminal domain in different cellular compartments and to study proteins potentially interacting with it using fluorescent microscopy. Key words:...
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Regulation of pre-mRNA splicing in S. cerevisiae: where RNA cooperates with proteins.
Gahura, Ondřej ; Půta, František (advisor) ; Pospíšek, Martin (referee) ; Staněk, David (referee)
Ondřej Gahura, PhD Thesis 2011 Regulation of pre-mRNA splicing in S. cerevisiae: where RNA cooperates with proteins Abstract Removal of introns from protein coding transcripts occurs in two splicing reactions catalyzed by a large nuclear complex, spliceosome. The spliceosome is an extremely intricate and dynamic machine, wherein contributions of small RNA molecules and multiple proteins are coordinated to meet the requirements of absolute precision and high flexibility. For an intimate understanding of pre-mRNA splicing, it is necessary to unravel roles of individual components and to dissect the partial mechanisms. In the first part of this work, we describe the role of the Prp45 splicing factor in Saccharomyces cerevisiae. Mapping of genetic interactions of a conditionally lethal allele prp45(1-169) suggests a relationship of Prp45 to the NTC complex and to the second transesterification. Two-hybrid assay and purification of spliceosomal complexes reveal a contribution of the Prp45 C-terminus in the Prp22 helicase recruitment and/or regulation. Numerous experiments with reporter substrates document the need of Prp45 for the efficient splicing of a specific subset of introns. Our observations suggest that the function of Prp45 in splicing is conserved in evolution. The second part is devoted to the role of...
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Role of the oncogenic microRNAs miR-17-92 and miR-155 in the regulation of hematopoietic differentiation and leukemogenesis
Pospíšil, Vít ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee) ; Machová Poláková, Kateřina (referee)
(English version): Hematopoietic differentiation is highly ordered multistep process, where generation of terminal blood cells is dependent upon coordinated regulation of gene expression by key regulators: transcription factors and mikroRNAs. PU.1 (Sfpi1) is a versatile hematopoetic transcription factor required for the proper generation of both myeloid and lymphoid lineages. MikroRNAs represent a novel class of ~22 nucleotide long non-coding posttranscriptional regulators that inhibit expression of genes by blocking protein translation or by mRNA degradation. In this PhD thesis I present research data documenting novel mechanisms of regulation and function of two oncogenic mikroRNAs, miR-17-92 cluster and miR-155 and myeloid transcriptional factors PU.1 upon macrophage differentiation of myeloid progenitors. The miR-17-92 cluster (Oncomir1) encodes seven related mikroRNAs that regulate cell proliferation, apoptosis and development and is overexpressed in number of malignancies including myeloid leukemia. Presented PhD thesis documents novel macrophage specific regulatory mechanisms involving the oncogenic cluster miR-17-92. Using transgenic PU.1-/- myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant, the transcription...
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To cap or not to cap? Eukaryotic translation initiation with a special interest in human opportunistic pathogen C. albicans
Feketová, Zuzana ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Vanáčová, Štěpánka (referee)
Candida albicans belongs to serious human opportunistic pathogens, causing severe health complications to immunocompromised patients. To my best knowledge, it is the only organism that survives with unmethylated cap structures found on the 5'ends of mRNA molecules. Using functional assay, I demonstrated that orf19.7626 codes for C. albicans translation initiation factor 4E (Ca4E). We couldn't prove our hypothesis, that Ca4E could be responsible for the unmethylated cap recognition in our model organism S. cerevisiae. Candida sp. possesses also another rather unusual feature - ambiguous CUG codon. In most of the cases, CUG is decoded as a serine, but sometimes also as a leucine. This gives rise to a so called "statistical proteome". One CUG codon is also part of the mRNA coding for Ca4E protein, therefore two versions of Ca4E-Ca4ELeu and Ca4ESer -might occur in C. albicans simultaneously. Both of them are able to rescue deletion of S. cerevisiae eIF4E gene, but they confer temperature sensitivity to the heterologous host. This phenotype is more pronounced with the Ca4ELeu version. We observed milder temperature sensitive phenotype after co-expression of Ca4E together with C. albicans eIF4G (Ca4G). Conformational coupling between eIF4E and eIF4G leads to enhanced affinity of eIF4E to the cap...
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Dissecting the nuclear function of the interleukin-1alpha
Novák, Josef ; Pospíšek, Martin (advisor) ; Vondrejs, Vladimír (referee)
Interleukin-1alpha (IL-1alpha) is a well-known proinflammatory mediator acting as a secreted molecule. However, in addition to its ability to activate its membrane-bound receptor, there is growing evidence on its noncanonical nuclear function, which classifies IL-1alpha as a "dual function cytokine". This nuclear action depends on the evolutionary conserved N-terminal domain of IL-1alpha. After proteolytic processing, the N-terminal domain of IL-1alpha translocates into nucleus. Histone acetyltransferase (HAT) complexes were previously identified as nuclear targets of IL-1alpha precursor. However, the specific protein which is responsible for the interaction between IL-1alpha and HAT complexes has not been identified yet. To dissect this interaction, the N-terminal domain of IL-1alpha was produced in yeast. Suitability of this experimental setup for testing the interaction between IL-1alpha and eukaryotic HAT complexes was evaluated in this study. IL-1alpha has been analyzed in this study using bioinformatics approaches as well. Putative amphipatic acidic helixes of IL-1alpha have been characterized. One of the potential binding partners of these domains is protein Ada2. Protein Ada2, mature IL-1alpha and IL-1alpha precursor in fusion with epitopes suitable for affinity purification were produced in...
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Screening for the HCV IRES interacting proteins
Roučová, Kristina ; Pospíšek, Martin (advisor) ; Kuthan, Martin (referee)
Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...
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Impact of the rRNA modifications on protein synthesis
Kročová, Eliška ; Pospíšek, Martin (advisor) ; Holá, Dana (referee)
A ribosome is a supramolecular structure, which mediates synthesis of all cellular proteins, and therefore is essential for cell life. The fact, that some nucleotides of ribosomal RNA are modified, is known for forty years. However only recently, successful deeper studies on how the individual modifications are synthesized and what is their effect on ribosome synthesis and function appear. Some particular nucleotide modifications are important for the ribosome formation (like m1 acp3 Ψ1191 SSU), some others influence proper function of the ribosome (e.g. Um2921, Gm2922, Ψ2923 LSU, m1 acp3 Ψ1191 SSU). Majority of modified nucleotides in eukaryotic rRNA is being recognized by small nucleolar RNA (snoRNA). Few nucleotides is, however, recognized and subsequently modified by specific proteins. These proteins also play crucial role in ribosome maturation. In thesis presented, current knowledge on the role of ribosomal RNA nucleotide modifications during their formation and maturation, and on their function is summarized and overviewed.
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