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Recombinant preparation of DNA binding domain of transcription factor TEAD1
Kúdelová, Veronika ; Novák, Petr (advisor) ; Dračínská, Helena (referee)
TEAD proteins belong to a significant family of transcription factors that contribute to the regulation of organism growth and cell differentiation during its development by activating the expression of a wide variety of genes. This family shares two highly conserved sites, the TEA DNA binding domain, after which the proteins have been named, and the domain by which transcription factors bind other coactivators. Because TEAD proteins are not able to activate transcription themselves, they interact with a number of coactivators. These coactivators allow the transcription of the gene of interest to be regulated. Failure of TEAD protein activity regulation can lead to cancer. Therefore, TEAD family proteins nowadays play an important role in the development of new anticancer drugs. One way of inhibiting these proteins is to block the active site in their DNA binding domain, thus, to block their binding to DNA. This bachelor thesis deals with recombinant expression of said DNA binding domain of transcription factor TEAD1, which is extended by amino acids in unstructured regions. After finding suitable conditions of protein production, we proceeded to large volume production which was followed by purification and protein identity verification. Finally, the ability of the produced protein to interact...
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Study of expression of the nuclear receptor nhr-97 in Caenorhabditis elegans
Boušová, Kristýna ; Stiborová, Marie (advisor) ; Vaněk, Ondřej (referee)
Nuclear hormone receptors (NHR) are important transcription factors that regulate development and metabolism in the large group of animals. Caenorhabditis elegans contains 284 nuclear receptors, which is unusually large amount compared to receptors of Drosophila melanogaster (18) and humans (48). 15 receptors of the C. elegans have homologous receptor structure with receptors of D. melanogaster and mammals. The remaining 269 NHR are specific to nematodes and belong to the group of supplementary nuclear receptors (SupNRs), the evolutionary precursor of the HNF4 - an important transcription factor in humans. In this work we describe the nuclear hormone receptor nhr-97 C. elegans, whose expression and function have not yet been studied. The gene is encoded in the genome of C. elegans and is among SupNRs. Nhr-97 consists of two isoforms A and B, whose expression in C. elegans tissues is different. Localization of gene expression in vivo was determined using lines expressing nhr-97:: GFP. For the A isoform expression of nhr-97::GFP was localized in neurons in the pharynx and the tail, in the intestine and hypodermis, in isoform B in the pharynx, in neurons around the corpus of pharynx, the head mesodermal cell and in anal sphincter. Nhr-97 expression during development of C. elegans was determined by...
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Preparation of the transcription factor FOXK1 DNA binding domain
Procházková, Valérie ; Novák, Petr (advisor) ; Košek, Dalibor (referee)
Transcription factors are proteins that regulate gene expression in different cell types. They play an important role in many cellular processes including regulation of cell cycle and cell differentiation. They possess DNA binding domains to recognize and bind specific DNA sequences. One type of DNA binding domain is the forkhead domain, which contains a region of 100-110 amino acid residues. This sequence is referred to DBD FOX and its spatial arrangement resembles a "winged helix". Proteins of the FOX family interact with double- stranded DNA via the α-helix H3, which represents highly conserved region within the proteins of this family. Other regions of the DBD further contribute in DNA binding, but as not significantly conserved, and their different properties are responsible for variable affinities of individual FOX proteins against binding motifs. Differences in three-dimensional structure may also alter biological functions of FOX proteins in the organism. FOX proteins are divided into 19 subfamilies, including the FOXK subfamily, consisting of two members, FOXK1 and FOXK2. FOXK proteins regulate aerobic glycolysis, cell proliferation and carcinogenesis. Their increased expression has been reported in cancer cells of skeletal tissue, stomach, colon, breast, lung, ovary, etc. However, the...
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Preparation of transcription factor FOXK2 - DNA binding domain
Kobrle, Lukáš ; Kukačka, Zdeněk (advisor) ; Bělonožníková, Kateřina (referee)
Transcription factors are specific proteins, which are able to regulate transcription and which are divided into individual families based on structural motifs. One of these families is the FOX family, which also includes the transcription factor FOXK2, which was the studying subject of this work. The FOXK2 protein regulates many cellular processes, including cell proliferation and metabolism and plays an important role in carcinogenesis, as its high expression has been reported in cases of lung, liver and kidney cancer. In this work, the DNA binding domain of the transcription factor FOXK2 was prepared by recombinant expression of Escherichia coli cells. The obtained protein was afterwards purified and its interaction with DNA was studied by native mass spectrometry.
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Recombinant preparation of DNA binding domain of transcription factor TEAD1
Kúdelová, Veronika ; Novák, Petr (advisor) ; Dračínská, Helena (referee)
TEAD proteins belong to a significant family of transcription factors that contribute to the regulation of organism growth and cell differentiation during its development by activating the expression of a wide variety of genes. This family shares two highly conserved sites, the TEA DNA binding domain, after which the proteins have been named, and the domain by which transcription factors bind other coactivators. Because TEAD proteins are not able to activate transcription themselves, they interact with a number of coactivators. These coactivators allow the transcription of the gene of interest to be regulated. Failure of TEAD protein activity regulation can lead to cancer. Therefore, TEAD family proteins nowadays play an important role in the development of new anticancer drugs. One way of inhibiting these proteins is to block the active site in their DNA binding domain, thus, to block their binding to DNA. This bachelor thesis deals with recombinant expression of said DNA binding domain of transcription factor TEAD1, which is extended by amino acids in unstructured regions. After finding suitable conditions of protein production, we proceeded to large volume production which was followed by purification and protein identity verification. Finally, the ability of the produced protein to interact...
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Study of expression of the nuclear receptor nhr-97 in Caenorhabditis elegans
Boušová, Kristýna ; Stiborová, Marie (advisor) ; Vaněk, Ondřej (referee)
Nuclear hormone receptors (NHR) are important transcription factors that regulate development and metabolism in the large group of animals. Caenorhabditis elegans contains 284 nuclear receptors, which is unusually large amount compared to receptors of Drosophila melanogaster (18) and humans (48). 15 receptors of the C. elegans have homologous receptor structure with receptors of D. melanogaster and mammals. The remaining 269 NHR are specific to nematodes and belong to the group of supplementary nuclear receptors (SupNRs), the evolutionary precursor of the HNF4 - an important transcription factor in humans. In this work we describe the nuclear hormone receptor nhr-97 C. elegans, whose expression and function have not yet been studied. The gene is encoded in the genome of C. elegans and is among SupNRs. Nhr-97 consists of two isoforms A and B, whose expression in C. elegans tissues is different. Localization of gene expression in vivo was determined using lines expressing nhr-97:: GFP. For the A isoform expression of nhr-97::GFP was localized in neurons in the pharynx and the tail, in the intestine and hypodermis, in isoform B in the pharynx, in neurons around the corpus of pharynx, the head mesodermal cell and in anal sphincter. Nhr-97 expression during development of C. elegans was determined by...
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