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Study of the structure of 14-3-3:phosducin complex
Kacířová, Miroslava ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
The aim of this diploma thesis is a biophysical characterization of the protein complex that consists of two regulatory proteins, the phosducin (Pd) and the 14-3-3 protein. These proteins are involved in the regulation of a signal cascade in vertebrate eye's retina. Pd is a 33kDa protein located in photoreceptor cells in retina, but it has been found in other tissues as well. In retina, phosducin affects transfer of light signaling from eye to brain, by binding Gtβγ subunit of transducin that is the main part of G-protein signaling. In light-adapted retina, unphosphorylated phosducin down-regulates the light response by binding to Gtβγ. This process is important for protecting retina in eyes in response to very intense light. It has also been found that phosducin affects hypertension. Phosducin reduces blood pressure of human and mice, especially during sleep. The function of phosducin is regulated in dark-adapted retina by 14-3-3. 14-3-3 is a 28kDa protein that has been found in many eukaryotic tissues, e.g. brain, and is involved in many processes, e.g. apoptosis. The 14-3-3 protein binds phosphorylated Pd and keeps him in a rod inner segment. For 14-3-3 to Pd binding, two sites on Pd must be phosphorylated, Ser54 and Ser73. This interaction, hinders Pd binding to Gtβγ, and hence enables the...
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The mechanism of the regulation of phosducin function
Kacířová, Miroslava
This dissertation is focused on 30 kDa protein phosducin (Pdc) and on the regulation of its function through the interaction with 28 kDa adaptor protein 14-3-3. These two proteins participate in G-protein signal transduction pathways, especially in the process of light signal transduction. It is assumed that Pdc binds to the Gtβγ complex of G-protein called transducin and through this interaction it inhibits the reassociation of Gtβγ with Gtα thus reducing the visual signal transfer. This process is thought to participate in a long- term light adaptation. The regulation of Pdc function is further regulated by its phosphorylation and subsequent binding to the 14-3-3 protein. It has been speculated that the 14-3-3 binding plays a key role in the inhibition of the interaction between phosphorylated Pdc (Pdc-PP) and Gtβγ. The formation of the 14-3-3/Pdc-PP complex leads to the reassociation of Gtβγ with Gtα and consequently to the amplification of visual signal transfer. Nevertheless, the mechanism by which the 14-3-3 protein binding inhibits the interaction between Pdc and Gtβγ remains elusive. The main aims of this dissertation were: (i) to investigate the structure of Pdc in its apo-state (in the absence of the binding partner) and in the complex with 14-3-3, and (ii) to suggest the mechanism of the...
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The mechanism of the regulation of phosducin function
Kacířová, Miroslava
This dissertation is focused on 30 kDa protein phosducin (Pdc) and on the regulation of its function through the interaction with 28 kDa adaptor protein 14-3-3. These two proteins participate in G-protein signal transduction pathways, especially in the process of light signal transduction. It is assumed that Pdc binds to the Gtβγ complex of G-protein called transducin and through this interaction it inhibits the reassociation of Gtβγ with Gtα thus reducing the visual signal transfer. This process is thought to participate in a long- term light adaptation. The regulation of Pdc function is further regulated by its phosphorylation and subsequent binding to the 14-3-3 protein. It has been speculated that the 14-3-3 binding plays a key role in the inhibition of the interaction between phosphorylated Pdc (Pdc-PP) and Gtβγ. The formation of the 14-3-3/Pdc-PP complex leads to the reassociation of Gtβγ with Gtα and consequently to the amplification of visual signal transfer. Nevertheless, the mechanism by which the 14-3-3 protein binding inhibits the interaction between Pdc and Gtβγ remains elusive. The main aims of this dissertation were: (i) to investigate the structure of Pdc in its apo-state (in the absence of the binding partner) and in the complex with 14-3-3, and (ii) to suggest the mechanism of the...
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The mechanism of the regulation of phosducin function
Kacířová, Miroslava ; Obšil, Tomáš (advisor) ; Krůšek, Jan (referee) ; Pavlíček, Jiří (referee)
This dissertation is focused on 30 kDa protein phosducin (Pdc) and on the regulation of its function through the interaction with 28 kDa adaptor protein 14-3-3. These two proteins participate in G-protein signal transduction pathways, especially in the process of light signal transduction. It is assumed that Pdc binds to the Gtβγ complex of G-protein called transducin and through this interaction it inhibits the reassociation of Gtβγ with Gtα thus reducing the visual signal transfer. This process is thought to participate in a long- term light adaptation. The regulation of Pdc function is further regulated by its phosphorylation and subsequent binding to the 14-3-3 protein. It has been speculated that the 14-3-3 binding plays a key role in the inhibition of the interaction between phosphorylated Pdc (Pdc-PP) and Gtβγ. The formation of the 14-3-3/Pdc-PP complex leads to the reassociation of Gtβγ with Gtα and consequently to the amplification of visual signal transfer. Nevertheless, the mechanism by which the 14-3-3 protein binding inhibits the interaction between Pdc and Gtβγ remains elusive. The main aims of this dissertation were: (i) to investigate the structure of Pdc in its apo-state (in the absence of the binding partner) and in the complex with 14-3-3, and (ii) to suggest the mechanism of the...
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Stability of the catalytic domain of neutral trehalase studied by differential scanning fluorimetry
Šmídová, Aneta ; Obšilová, Veronika (advisor) ; Kacířová, Miroslava (referee)
This bachelor thesis is part of a project aiming for the crystallization of trehalase domain of neutral trehalase Nth1 from Saccharomyces cerevisiae. The main goal of this thesis is to optimize the purification protocol of two different constructs of yeast Nth1 and determine the optimal buffer for crystallography of these constructs using the differential scanning fluorimetry DSF. Trehalases are important, highly conserved enzymes found in many organisms. These enzymes belong to the class of hydrolases, subgroup of glycosidases and their common feature is hydrolytic cleavage of trehalose molecule into two glucose subunits. Trehalose is a naturally occurring non-reducing disaccharide which serve in yeast cells as storage carbohydrate and energy and stress metabolite. Trehalose and its hydrolysis is very important in the life cycle of the insect, as it is present as the main sugar component of insect hemolymph, so some trehalase inhibitors could be used as insecticides. By this time, however, only one structure of trehalase from a prokaryotic organism Escherichia coli was solved. For this reason it is very important to solve the structure of trehalase from a eukaryotic organism. Keywords: Nth1, expresion, purification, DSF Key subjects: Protein biochemistry, biophysical chemistry
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Study of the structure of 14-3-3:phosducin complex
Kacířová, Miroslava ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
The aim of this diploma thesis is a biophysical characterization of the protein complex that consists of two regulatory proteins, the phosducin (Pd) and the 14-3-3 protein. These proteins are involved in the regulation of a signal cascade in vertebrate eye's retina. Pd is a 33kDa protein located in photoreceptor cells in retina, but it has been found in other tissues as well. In retina, phosducin affects transfer of light signaling from eye to brain, by binding Gtβγ subunit of transducin that is the main part of G-protein signaling. In light-adapted retina, unphosphorylated phosducin down-regulates the light response by binding to Gtβγ. This process is important for protecting retina in eyes in response to very intense light. It has also been found that phosducin affects hypertension. Phosducin reduces blood pressure of human and mice, especially during sleep. The function of phosducin is regulated in dark-adapted retina by 14-3-3. 14-3-3 is a 28kDa protein that has been found in many eukaryotic tissues, e.g. brain, and is involved in many processes, e.g. apoptosis. The 14-3-3 protein binds phosphorylated Pd and keeps him in a rod inner segment. For 14-3-3 to Pd binding, two sites on Pd must be phosphorylated, Ser54 and Ser73. This interaction, hinders Pd binding to Gtβγ, and hence enables the...
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