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Specific heme interaction modulates the conformational dynamics and function of p53
Sergunin, Artur ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Tumor suppressor p53 is one of the most studied proteins in terms of cancer and the mechanism of its formation. The general function of p53 is based on the transcriptional regulation of various genes, which can differently influence numerous cellular processes. Recent studies revealed a relationship between p53 and iron homeostasis within the cell. In particular, p53 was shown to interact with a molecule of heme, and this interaction ultimately disrupts the DNA-binding ability of p53 and promotes its proteasomal degra- dation. This work focuses on a detailed description of heme binding to the p53. For this purpose, we isolated two forms of p53, heme-free and heme-bound. We discovered that conformational dynamics of heme-free and heme-bound p53 differ, with the latter exhibi- ting a higher degree of flexibility. We also confirmed previous reports that heme indeed interacts with a cysteine residue in a specific manner. However, heme binding does not disrupt the oligomeric state of p53 or its native zinc binding ability. Finally, we showed that heme-bound p53 exhibits severely impaired DNA-binding ability as opposed to the heme-free form. Keywords: heme, sensor proteins, p53 protein, transcription factor, intrinsically disor- dered proteins
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Preparation and preliminary characterization of the eukaryotic initiation factor 2α and its heme regulated kinase
Ovad, Tomáš ; Martínková, Markéta (advisor) ; Stráňava, Martin (referee)
Heme sensor proteins perform a variety of important functions in both prokaryotic and eukaryotic organisms. Heme-regulated inhibitor (HRI) is an example of a eukaryotic heme-sensor protein, which catalyzes the phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α). In this bachelor thesis, the pET-21c(+)/eIF2α plasmid was amplified and its authenticity for the eIF2α expression was verified with the use of two independent methods. Next, HRI and eIF2α were produced using the recombinant expression in E. coli BL-21(DE3) cells transformed with the pET- 21c(+)/eIF2α and pET-21c(+)/HRI plasmid, respectively. Both proteins were then isolated from the cells and purified with the use of affinity chromatography and gel permeation chromatography. eIF2α was obtained in sufficient yield (560 μg out of 1 l of TB medium) and purity (90%). A lower yield (25 μg out of 1 l of TB medium) and purity (20%) was reached in the case of HRI. On the other hand, the authenticity of the HRI product was confirmed using spectrophotometric characterization and its enzyme activity was verified as well. Pilot experiments showed that GTP may replace ATP in the process of eIF2α phosphorylation, while UTP and CTP may not.
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