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Interaction partners of protein eIF4E2 in human cells
Pospíšilová, Klára ; Pospíšek, Martin (advisor) ; Hálová, Martina (referee)
Protein eIF4E2 belongs to the family of eukaryotic translation initiation factors 4E, but it does not participate in translation initiation under normal circumstances. Its main role lies in translational repression of specific mRNAs. Nevertheless eIF4E2 takes part in translation initiation as a subunit of a specific translation initiation complex in hypoxic conditions. The exact mechanism in which eIF4E2 takes part in either of these processes is not known. One way to study the role of eIF4E2 in the cell is to find out what other proteins does eIF4E2 interact with. The goal of this work was to seek out potential eIF4E2-interacting partners in the HEK293 cell line using immunoprecipitation followed by mass spek- trometry. Apart from finding individual proteins the goal was to identify eIF4E2-containig protein com- plexes in HEK293 cells. A second line of work was preparation of a system for screening inhibitors of the interaction between eIF4E2 and eIF4G3. The main result is finding potential new eIF4E2-intera- cting partners in human cells.
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Noncanonical functions of IL-1α
Novák, Josef ; Pospíšek, Martin (advisor) ; Černý, Jan (referee) ; Brdička, Tomáš (referee)
1α (IL 1α) is a multifunctional cytokine 1α is 1α independent on the receptor sig 1α is responsible for 1α to the plasma membrane. 1α activates express κB, binds to 1α 1α 1α to the plasma membrane 1α to signal 1α is required for membrane 1α exter 1α anchoring 1α 1α 1α with tumor suppressor p53 following genotoxic stress is further described in human cell 1α coloca
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Noncanonical human eIF4Es in and out of the RNA granules
Frydrýšková, Klára ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Valášek, Leoš (referee)
Eukaryotic translation initiation factor eIF4E1 (eIF4E1) plays a pivotal role in the control of cap-dependent translation initiation, occurs in P- bodies and is important for the formation of stress granules (SG). Human cells encompass two other non-canonical translation initiation factors capable of cap binding although with a lower affinity for the cap: eIF4E2 and eIF4E3. Here, I investigated the ability of individual eIF4E family members and their variants to localize to SGs and P-bodies in stress-free, arsenite and heat shock conditions. Under all tested conditions, both eIF4E1 and eIF4E2 proteins and all their variants localized to P-bodies unlike eIF4E3 protein variants. Under both arsenite and heat stress conditions all tested variants of eIF4E1 and the variant eIF4E3-A localized to SGs albeit with different abilities. Protein eIF4E2 and all its investigated variants localized specifically to a major part of heat stress-induced stress granules. Further analysis showed that approximately 75% of heat stress-induced stress granules contain all three eIF4Es, while in 25% of them eIF4E2 is missing. Large ribosomal subunit protein L22 was found specifically enriched in arsenite induced SGs. Heat stress-induced re- localization of several proteins typical for P-bodies such as eIF4E2, DCP-1, AGO-2...
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Splicing Factors in the Regulation of Gene Expression - the Relationship Between Splicing and Transcription in Saccharomyces cerevisiae
Hálová, Martina ; Folk, Petr (advisor) ; Pospíšek, Martin (referee) ; Staněk, David (referee)
Transcription and pre-mRNA processing, e.g., splicing, occur at the same place and time in the context of chromatin. A growing amount of evidence supports the hypothesis that these processes are interconnected. Prp45/SKIP is one of the factors which are believed to mediate the interconnection. The human ortholog, SKIP, is known for affecting mRNA formation on the levels of transcription initiation and elongation. Moreover, it interacts with chromatin modifiers and it is a splicing factor, too. The function of the Saccharomyces cerevisiae ortholog, Prp45, has been so far connected only to pre-mRNA splicing. In this work, we characterized the role of Prp45 in splicing and elaborated the results connecting Prp45 to transcription and chromatin modifications. RNA-seq results showed that pre-mRNA is accumulated in prp45(1-169) cells. This accumulation is not caused by the reduced activity of pathways responsible for RNA degradation. The extent of the splicing inefficiency in prp45(1-169) cells did not depend on either the canonicity of the 5' splice site and branch site or the distance between the branch site and the 3' splice site. Using chromatin immunoprecipitation, we found that prp45(1-169) mutation causes delay in U2 snRNP recruitment to assembling spliceosome. This delay transfers to the later...
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Regulation of translation iniitiation in yeast saccharomyces cerevisiae
Mašek, Tomáš ; Pospíšek, Martin (advisor) ; Krásný, Libor (referee) ; Hašek, Jiří (referee)
IV. Shrnutí výsledků 63 64 Denaturační RNA elektroforéza v TAE agarózových gelech 60% koncentrace formamidu postačuje k dostatečné denaturaci RNA pro elektroforetickou separaci. Denaturační RNA elektroforéza v TAE pufru vykazuje stejné separační rozlišení RNA molekul jako nejčastěji používaná RNA elektroforéza v MOPSovém pufru a navíc je rychlejší. Denaturační RNA elektroforéza v TAE pufru je použitelná nejen k separaci čisté směsi RNA molekul, ale i směsných vzorků obsahujících také DNA a proteiny (např. buněčných lyzátů). Denaturační RNA elektroforézu v TAE pufru lze kombinovat s kapilárním přenosem a následnou hybridizací (pro blotování lze použít jak konveční 10xSSC pufr, tak levnější 8 mM NaOH). Tento elektroforetický protokol poskytuje levnější a rychlejší alternativu k RNA elektroforéze v MOPSovém pufru, snižuje expozici laboratorních pracovníků toxickým látkám a je vhodný i pro laboratoře, které s RNA běžně nepracují. Rck2 se zapojuje do reprogramování ribozómů během oxidativního stresu Oxidativní stres inhibuje translaci stejnou měrou jako stres osmotický. Lze pozorovat pokles množství aktivně translatujících ribozómů. Tento pokles je proporcionální ke zvětšení ploch 40S, 60S a 80S "vrcholů" v polyzomálním profilu. Aplikace t-BOOH vede k vyšší disociaci polyzomálních komplexů u rck2Δ...
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The role of HOG MAPK signaling pathway during osmotic stress in Saccharomyces cerevisiae
Vrbová, Michaela ; Pospíšek, Martin (advisor) ; Janderová, Blanka (referee)
THE ROLE OF HOG MAPK SIGNALING PATHWAY DURING OSMOTIC STRESS IN SACCHAROMYCES CEREVISIAE Budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high- osmolarity glycerol or HOG pathway) during conditions of increased external osmolarity. It evokes cellular responses necessary to permit continued growth. Activation of HOG pathway with Hog1 MAP kinase results in production of glycerol to prevent dehydration and up regulation of other Hog1 dependent genes because of cell adaptation. We were trying to find difference in translation between wild-type cells and two mutants of hog1 gene before and after 0,4 M NaCl osmotic stress (2, 6, 30 min). We used deletion mutant hog1Δ and hog1-as mutant with point mutation which allows inhibition of Hog1 MAPK during presence of specific AS inhibitor. We tested AS inhibitor by plate test and have found optimal concentration of 5 μM for blocking Hog1 MAPK in hog1-as mutant. Translation profiling proves that osmotic stress decreases translation in general. Hog1Δ mutant and hog1-as AS inhibited mutant behave similarly and their translation recovers slower than the wild-type's. That confirms that HOG1 gene is important for cell recovery from the osmotic stress. Microarray analysis shows that Hog1 dependent...
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Interactome of the translation initiation factor eIF4E2
Lettrich, Patrik ; Pospíšek, Martin (advisor) ; Zeman, Jakub (referee)
Regulation of gene expression at the level of translation is one of the most prominent processes when it comes to maintaining cellular vital functions in physiolgical norm. Initiation phase plays an especially important role in translational regulation. Majority of eukaryotic mRNAs possess methylguanosine cap on its 5' end. Binding of initiation factor eIF4E1 to cap is substantial part of canonical mechanism of translational initiation. eIF4E1 is component of bigger complex termed eIF4F, whose association with cap mRNA is essential for initiation of translation. Due to cap-binding ability of noncanonical initiation factors and their interaction partners, that are distinct from those of eIF4E1, there can occur specific regulation of translational initiation. One of these noncanonical factors is eIF4E2. In regulatory processes it mostly acts as a translational repressor. Binding of eIF4E2 to cap impedes association of eIF4F complex with mRNA, and therefore inhibits translation. However, eIF4E2 is also able to promote cap-dependent initiation of translation using an alternative mechanism in hypoxic conditions. This thesis represents the first complex analysis of protein-protein interactions and physiological functions of factor eIF4E2 and its homologs in several model organisms. Key words:...
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Inhibitors of the eukaryotic translation initiation factor eIF4E1
Pospíšilová, Klára ; Pospíšek, Martin (advisor) ; Roithová, Adriana (referee)
Protein synthesis is one of the most important processes that take place in a cell. Thus there are various mechanisms in a cell that regulate it. If that regulation fails it may lead to serious pathologies. An example of this is an abnormal increase in the production of eukaryotic translation initiation factor 4E1 which occurs in some types of cancer including head and neck squamous cell carcinoma, colorectal cancer, cervical cancer or lung cancer. Enhanced availability of factor 4E1 enables transformed cells to undertake a more intense translation. The expression of individual proteins is not increased to the same extent though. The enhancement in the level of factor 4E1 has a more significant effect on oncogenic proteins. Malignant transformation caused by an incorrect regulation of factor 4E1 can be precluded by an application of compounds that impair the activity of factor 4E1. A summarization of 4E1 inhibitors is the subject of this work.
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