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The Role of Bmh Proteins in the Regulation of Yeast Enzyme Neutral Trehalase Nth1.
Macáková, Eva ; Obšilová, Veronika (advisor) ; Krůšek, Jan (referee) ; Žáčková, Markéta (referee)
118 10. Summary Neutral trehalase 1 is a yeast enzyme from the family of hydrolases, which catalyzes hydrolysis of trehalose to two glucose molecules. Trehalose is a non-reducing disacharide, which serves as a carbon source in a yeast cells as well as a stress metabolite. When a cell is under stress conditions it accumulate trehalose and through the recovery process the trehalose is hydrolyses by trehalases. The main subject of our study was Nth1 from S. cerevisiae. It was published earlier (Panni, S., et al., 2008), that Nth1 must be phosphorylated by PKA and in the presence of 14-3-3 protein to be active. The activity of Nth1 also slightly increases in the presence of Ca2+ ions (Franco, A., et al., 2003). 14-3-3 proteins are family of acid regulatory proteins, which participates in variety of processes in the cells, like regulation of the cell-cycle, cell metabolism, transcription, apoptosis etc. They have more than 400 known binding partners, which include transcription factors, signalling molecules, enzymes and others. They control the regulation of their binding partners through phosphorylated motives in sequence by changing conformation of the binding partner, revealing or masking specific sequence or by mediation of protein-protein interactions. There are many isoforms of 14-3- 3 proteins through all...
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Study of structural differences among 14-3-3 protein isoforms.
Macáková, Eva ; Obšil, Tomáš (advisor) ; Gryčová, Lenka (referee)
The 14-3-3 proteins are a family of important regulatory proteins, found in all eukaryotes, which are involved in many cellular processes. In this diploma thesis, we studied structure/function relationships of 14-3-3 proteins, in this case it was the influence of the structure of H8-H9 loop on the binding affinity in barley isoform hv 14-3-3A and human isoform 14-3-3ζ. According to former results, hv 14-3-3A binds to a ligand with lowest affinity, which could be caused by present of a glycin in H8-H9 loop, while in other isoforms there is a serin on the same position. We measured the binding affinity in protein hv 14-3-3A WT and its mutant, which contained the serin instead of the glycin in H8-H9 loop. For comparison, we also measured the binding affinity of human isoform 14-3-3ζ containing the serin in H8-H9 loop and its mutant, where the the serin was replaced by the glycin. Proteins were expressed in E. coli cells strain BL21(DE3) and then purified. The dissociation constant for the binding of peptide pRaf-259 labeled with fluorophores FITC and ATTO was measured using both the fluorescence correlated spectroscopy and the steady-state fluorescence intensity. Our results showed that in both isoforms the mutation of H8-H9 loop causes decrease in the binding affinity.
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The Role of Bmh Proteins in the Regulation of Yeast Enzyme Neutral Trehalase Nth1.
Macáková, Eva ; Obšilová, Veronika (advisor) ; Krůšek, Jan (referee) ; Žáčková, Markéta (referee)
118 10. Summary Neutral trehalase 1 is a yeast enzyme from the family of hydrolases, which catalyzes hydrolysis of trehalose to two glucose molecules. Trehalose is a non-reducing disacharide, which serves as a carbon source in a yeast cells as well as a stress metabolite. When a cell is under stress conditions it accumulate trehalose and through the recovery process the trehalose is hydrolyses by trehalases. The main subject of our study was Nth1 from S. cerevisiae. It was published earlier (Panni, S., et al., 2008), that Nth1 must be phosphorylated by PKA and in the presence of 14-3-3 protein to be active. The activity of Nth1 also slightly increases in the presence of Ca2+ ions (Franco, A., et al., 2003). 14-3-3 proteins are family of acid regulatory proteins, which participates in variety of processes in the cells, like regulation of the cell-cycle, cell metabolism, transcription, apoptosis etc. They have more than 400 known binding partners, which include transcription factors, signalling molecules, enzymes and others. They control the regulation of their binding partners through phosphorylated motives in sequence by changing conformation of the binding partner, revealing or masking specific sequence or by mediation of protein-protein interactions. There are many isoforms of 14-3- 3 proteins through all...
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Study of structural differences among 14-3-3 protein isoforms.
Macáková, Eva ; Gryčová, Lenka (referee) ; Obšil, Tomáš (advisor)
The 14-3-3 proteins are a family of important regulatory proteins, found in all eukaryotes, which are involved in many cellular processes. In this diploma thesis, we studied structure/function relationships of 14-3-3 proteins, in this case it was the influence of the structure of H8-H9 loop on the binding affinity in barley isoform hv 14-3-3A and human isoform 14-3-3ζ. According to former results, hv 14-3-3A binds to a ligand with lowest affinity, which could be caused by present of a glycin in H8-H9 loop, while in other isoforms there is a serin on the same position. We measured the binding affinity in protein hv 14-3-3A WT and its mutant, which contained the serin instead of the glycin in H8-H9 loop. For comparison, we also measured the binding affinity of human isoform 14-3-3ζ containing the serin in H8-H9 loop and its mutant, where the the serin was replaced by the glycin. Proteins were expressed in E. coli cells strain BL21(DE3) and then purified. The dissociation constant for the binding of peptide pRaf-259 labeled with fluorophores FITC and ATTO was measured using both the fluorescence correlated spectroscopy and the steady-state fluorescence intensity. Our results showed that in both isoforms the mutation of H8-H9 loop causes decrease in the binding affinity.
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