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The role of nuclear-encoded subunits of cytochrome c oxidase in mitochondrial metabolism
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Gahura, Ondřej (referee) ; Ugalde, Cristina (referee)
Mitochondria, 'the powerhouses of the cell', house the integral metabolism pathway of oxidative phosphorylation to produce the majority of cellular energy. Mammalian cytochrome c oxidase, also called complex IV (cIV), is indispensable for the overall oxidative phosphorylation function as the terminal oxidase, and for its regulation to sustain energetic needs. Since cIV is a multimeric enzyme composed of subunits encoded by nuclear and mitochondrial genomes, its biogenesis is a complicated process, which needs to be coordinated to complete a fully functional complex. Further, the setup of individual nuclear-encoded subunits isoforms of cIV may fine-tune cIV function based on the tissue or the environment context. Despite the physiological and pathological relevance of cIV composition, biogenesis, and the secondary deficiencies triggered by cIV defects, nuclear-encoded subunits' function remains poorly understood. At first, mammalian COX4 subunit isoforms with tissue- and oxygen-dependent expression were studied in the HEK293 cellular model with an exclusive expression of COX4I1 or COX4I2 isoform. Remarkably, the COX4I2 isozyme showed lower affinity to oxygen, which may imply regulation of cIV activity under hypoxia, and is of physiological relevance for the oxygen-sensing mechanism. Further,...
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The regulation of cofilin by the ERK signaling cascade
Rasl, Jan ; Vomastek, Tomáš (advisor) ; Gahura, Ondřej (referee)
Cofilin is small ubiquitous actin binding protein, which is required for polymerization and depolymerization of actin fibers. Cofilin is involved in numerous cellular processes where the remodeling of actin cytoskeleton is required, such as cell division and cell migration. In order to precisely and dynamically regulate the cofilin activity, cells utilize large network of interconnected signaling pathways. One of these signaling pathways is the MAP-kinase cascade ERK (extracellular signal-regulated kinase), although the molecular mechanisms by which ERK regulates cofilin activity are not fully understood. Much evidence suggests that ERK controls the cofilin activity mainly through the regulation of Rho family of small GTPases. The ERK signaling cascade can modulates the Rho GTPase pathway signaling components, such as GAPs (GTPase activating proteins), GEFs (guanine nucleotide exchange factors) or Rho GTPases effectors. The ERK signaling cascade utilizes two different mechanisms for the regulation of Rho GTPases signaling pathways. The first mechanism is on transcriptional and translational level, where ERK regulates the transcription and subsequently translation of key regulatory proteins. Second mechanism, which is far more dynamic, occurs at the level of posttranslational modification,...
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The Role of Lipid Rafts in Translocation of the Adenylate Cyclase Toxin of B. pertussis across Cytoplasmatic Membrane
Chvojková, Věra ; Šebo, Peter (advisor) ; Gahura, Ondřej (referee)
The bacterium Bordetella pertussis is the causative agent of pertussis or whooping cough. The main virulent factor of this gram-negative bacterium is adenylate cyclase toxin, a member of the RTX protein family. After secretion, the toxin binds to CD11b/CD18 integrin receptor of myeloid phagocytic cells and consequently translocates its adenylate cyclase enzyme (AC) into the cytosol of the host cells. In the cytosol, the AC is activated by calmodulin, followed by rapid conversion of ATP into the signaling molecule cAMP, resulting in paralysis of bactericidal functions of phagocytic cells. Recently it was shown that translocation of the catalytic AC domain into the cytosol proceeds in two steps. After binding of CyaA to the receptor, the influx of calcium ions into the cell occurs. High local concentration of calcium induces the translocation of the CyaA- CD11b/CD18 complex into the lipid raft, where the translocation of adenylate cyclase enzyme into cytosol occurs. This work is aiming on the description of the new established paradigm dealing with membrane translocation of RTX toxins.
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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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