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Study of interaction between ASK1 kinase and thioredoxin.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
MAP kinase signaling cascade plays an important role in the cellular response to various stress stimuli from the external environment. This signaling cascade is divided into three levels: MAP kinase kinase kinases (MAP3K) phosphorylate and thus activate MAP kinase kinases (MAP2K) and those subsequently phosphorylate and thus activate MAP kinase (MAPK) pathway, which regulates many cellular functions such as apoptosis, cell differentiation and morphogenesis. One of the important MAP3K is protein kinase ASK1 (Apoptosis signal-regulating kinase 1), which is an important regulator of cellular immune and stress responses. Given that the increased activity of ASK1 is related to the development of serious diseases such as cancer, cardiovascular and neurodegenerative diseases, ASK1 is an interesting target in the pharmacy in the development of new drugs. Human ASK1 consists of 1374 amino acids and is divided into three domains: a central Ser/Thr catalytic domain and two coiled-coil domains, of which the first is located at the N- and the second at the C-terminus of the molecule of this protein kinase. ASK1 is regulated by its binding partners, which include a small cellular redox protein thioredoxin (Trx-1), which binds to the N-terminal part of ASK1. Trx-1 is a potent antioxidant and so it protects cells...
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Preparation of expression constructs of phosducine binding partners.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
Key words: SUG1, phosducin, G protein signaling, expression, pGEX-4T-1, pST39 Phosducin is an acidic protein found in cytosol and is involved in G protein signaling within rods of photoreceptors. Phosducin also influences stress-dependent hypertension. One of phosducin's important binding partners is SUG1, a 26S proteasome subunit, which belongs to a family of proteins that have an ATPase activity. This bachelor thesis takes part in a broader research, which tries to clarify physiological functions of the interaction of SUG1 with phosducin. Main goal of this study was to prepare constructs of SUG1 (human isoform 1) suitable for the recombinant protein production in an expression system E. coli BL21 (Rosetta). Gene that encodes SUG1 was inserted into two different plasmid DNAs that allow expression of SUG1 as two different fusion proteins in a prokaryotic expression system. Nucleotide sequences of these constructs were verified using sequencing. Next, expression tests revealed sufficient production of both recombinant fusion proteins. The main result of this bachelor thesis is the successful expression of human SUG1 in E. coli BL21 (Rosetta) cells. This result will enable the development of the purification protocol for the large scale expression of both recombinant fusion proteins as well as the...
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Study of interaction between ASK1 kinase and thioredoxin.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
MAP kinase signaling cascade plays an important role in the cellular response to various stress stimuli from the external environment. This signaling cascade is divided into three levels: MAP kinase kinase kinases (MAP3K) phosphorylate and thus activate MAP kinase kinases (MAP2K) and those subsequently phosphorylate and thus activate MAP kinase (MAPK) pathway, which regulates many cellular functions such as apoptosis, cell differentiation and morphogenesis. One of the important MAP3K is protein kinase ASK1 (Apoptosis signal-regulating kinase 1), which is an important regulator of cellular immune and stress responses. Given that the increased activity of ASK1 is related to the development of serious diseases such as cancer, cardiovascular and neurodegenerative diseases, ASK1 is an interesting target in the pharmacy in the development of new drugs. Human ASK1 consists of 1374 amino acids and is divided into three domains: a central Ser/Thr catalytic domain and two coiled-coil domains, of which the first is located at the N- and the second at the C-terminus of the molecule of this protein kinase. ASK1 is regulated by its binding partners, which include a small cellular redox protein thioredoxin (Trx-1), which binds to the N-terminal part of ASK1. Trx-1 is a potent antioxidant and so it protects cells...
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Preparation of expression constructs of phosducine binding partners.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
Key words: SUG1, phosducin, G protein signaling, expression, pGEX-4T-1, pST39 Phosducin is an acidic protein found in cytosol and is involved in G protein signaling within rods of photoreceptors. Phosducin also influences stress-dependent hypertension. One of phosducin's important binding partners is SUG1, a 26S proteasome subunit, which belongs to a family of proteins that have an ATPase activity. This bachelor thesis takes part in a broader research, which tries to clarify physiological functions of the interaction of SUG1 with phosducin. Main goal of this study was to prepare constructs of SUG1 (human isoform 1) suitable for the recombinant protein production in an expression system E. coli BL21 (Rosetta). Gene that encodes SUG1 was inserted into two different plasmid DNAs that allow expression of SUG1 as two different fusion proteins in a prokaryotic expression system. Nucleotide sequences of these constructs were verified using sequencing. Next, expression tests revealed sufficient production of both recombinant fusion proteins. The main result of this bachelor thesis is the successful expression of human SUG1 in E. coli BL21 (Rosetta) cells. This result will enable the development of the purification protocol for the large scale expression of both recombinant fusion proteins as well as the...
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