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RNAi of the a subunit of human translation initiation factor 3 (eIF3).
Peclinovská, Lucie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Translation initiation is the first step of protein synthesis that captures the flow of gene expression pathway in all living organisms. The advantage of regulation of gene expression at the level of translation initiation is that it allows for more rapid changes in the proteome and serves as the rate limiting step under certain conditions such as stress. This process is masterminded by many initiation factors. One of them, a multisubunit eukaryotic initiation factor 3 (eIF3), is a very efficient player in this field taking a part in the most of the initiation steps. The largest subunit of the eIF3 complex is called eIF3a p170 and TIF32 in mammals and yeast, respectively, and at least in yeast, it was shown to represent an essential constituent of the translational machinery. This work is based on all that has been learned about the eIF3a roles in translation initiation in the model organism of yeast Saccharomyces cerevisiae in effort to examine the degree of the functional conservation with its human ortholog. This is achieved by the RNAi-mediated knock-down of eIF3a in HeLa and HEK cell lines followed by variety of well established assays to monitor translational status of eIF3a depleted cells. In the first part, I describe optimization of the RNA interference protocol with respect to the choice...
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Analysis of substrate specificity and mechanism of GlpG, an intramembrane protease of the rhomboid family.
Peclinovská, Lucie ; Stříšovský, Kvido (advisor) ; Konvalinka, Jan (referee)
Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...
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RNAi of the a subunit of human translation initiation factor 3 (eIF3).
Peclinovská, Lucie ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Translation initiation is the first step of protein synthesis that captures the flow of gene expression pathway in all living organisms. The advantage of regulation of gene expression at the level of translation initiation is that it allows for more rapid changes in the proteome and serves as the rate limiting step under certain conditions such as stress. This process is masterminded by many initiation factors. One of them, a multisubunit eukaryotic initiation factor 3 (eIF3), is a very efficient player in this field taking a part in the most of the initiation steps. The largest subunit of the eIF3 complex is called eIF3a p170 and TIF32 in mammals and yeast, respectively, and at least in yeast, it was shown to represent an essential constituent of the translational machinery. This work is based on all that has been learned about the eIF3a roles in translation initiation in the model organism of yeast Saccharomyces cerevisiae in effort to examine the degree of the functional conservation with its human ortholog. This is achieved by the RNAi-mediated knock-down of eIF3a in HeLa and HEK cell lines followed by variety of well established assays to monitor translational status of eIF3a depleted cells. In the first part, I describe optimization of the RNA interference protocol with respect to the choice...
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