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Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2
Kalábová, Dana ; Obšilová, Veronika (advisor) ; Pavlíček, Jiří (referee) ; Žáková, Lenka (referee)
Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2 Abstract Caspase-2 is a protease standing apically in the cascade of reactions leading to apoptosis. Properly functional apoptosis eliminates damaged cells, autoreactive lymphocytes or redundant groups of cells in ontogeny. The process of caspase-2 activation must be precisely regulated. One of the described ways of caspase-2 regulation causing its inhibition is posttranslational modification phosphorylation with subsequent binding of the regulatory scaffold protein 14-3-3. The aim of this dissertation is to explain the molecular mechanism of this regulation. To understand the interaction between the proteins, it was necessary to first identify the phosphorylation sites in the caspase-2 molecule recognized by the 14-3-3 protein and then describe the detailed structure of the binding complex. The structure was characterized by a number of biochemical and biophysical methods, such as analytical ultracentrifugation, native electrophoresis in TBE buffer, polarization-fluorescence assay, hydrogen/deuterium exchange coupled to mass spectrometry, or crystallization; and the results led to stimulating conclusions. Activation of caspase-2 begins with its binding to adaptor proteins, cleavage and dimerization of the catalytic subunits. The...
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Structural studies of selected protein complexes involved in signal transduction
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Bouřa, Evžen (referee) ; Pavlíček, Jiří (referee)
Protein-protein interactions are critical for most physiological and pathophysiological processes. Detailed characterization of these interactions is therefore essential not only to understand the nature of these events, but also to design strategies to target these interactions. This work focuses on the study of the structure and interactions of several proteins and their complexes. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the p38/JNK protein kinase pathways, thereby directing cells toward an inflammatory response or apoptosis. ASK1 interacts with thioredoxin (TRX), a small dithiol oxidoreductase, which inhibits ASK1, but the mechanism of this inhibition has not been clarified. CaMKK1 and CaMKK2 are Ca2+ /calmodulin (CaM)-dependent protein kinases that regulate cellular energy balance, memory, and inflammation, among others. Both are inhibited by 14-3-3 proteins, but despite their domain and sequence similarities, the extent of 14-3-3 protein- mediated inhibition is different. Estrogen receptor alpha (ERα) is a nuclear receptor involved in breast cancer. Tamoxifen, an ERα antagonist, is used to treat this disease, but resistance often develops. 14-3-3 proteins interact with ERα and inhibit its transcriptional activity,...
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The effect of 14-3-3 protein on intradomain interaction of ubiquitin ligase Nedd4-2
Pohl, Pavel ; Obšilová, Veronika (advisor) ; Žáková, Lenka (referee) ; Pavlíček, Jiří (referee)
EN The human ubiquitin ligase Nedd4-2 (NEDD4L) ubiquitinates a wide range of membrane proteins and receptors, playing a key role in maintaining homeostasis. This enzyme is regulated by phosphorylation and subsequent interaction with 14-3-3 proteins, which primarily affects its ability to interact with various substrates. However, very little is known about the molecular basis of this protein-protein interaction. In this work, we focused on biophysical characterization of the role of individual phosphorylation sites and also on mapping the structural changes in the Nedd4- 2 protein induced by 14-3-3 protein binding. Our experiments using analytical ultracentrifugation methods revealed that two phosphorylation sites Ser342 and Ser448 are primarily required for stable binding of Nedd4-2 to 14-3-3 proteins. The crystal structure of the 14-3-3ηΔC:Nedd4-2335-455 T367A complex than revealed the simultaneous binding of both phosphorylated residues to the binding groove of 14-3-3 protein. Subsequent modeling based on small-angle X-ray scattering and chemical cross-linking data combined with mass spectrometry indicated extensive structural changes in the individual domains of the Nedd4-2 protein. Binding of 14-3-3η protein blocks the WW3 domain of Nedd4-2 in the central channel of 14-3-3 protein, while...
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Structural studies of 14-3-3 protein complexes and their stabilization by small molecule compounds
Lentini Santo, Domenico ; Obšil, Tomáš (advisor) ; Brynda, Jiří (referee) ; Pavlíček, Jiří (referee)
Protein-protein interactions (PPIs) play a crucial role in almost all biological processes. Many proteins require a number of dynamic interactions with other proteins and/or biomolecules to function. Proteomic studies have suggested that human protein-protein interactome consists of several hundred thousands of protein complexes. A detailed insight into these PPIs is essential for a complete understanding of the processes mediated by these protein complexes. Because many PPIs are involved in disease-related signaling pathways, such PPIs are important targets for pharmaceutical interventions, especially in situations where a more conventional target (e.g. the active site of an enzyme, the binding site of a receptor) cannot be used. This doctoral thesis focuses on 14-3-3 proteins, a family of eukaryotic adaptor and scaffolding proteins involved in the regulation of many signaling pathways. The 14-3-3 proteins function as interaction hubs and critical regulators of many enzymes, receptors and structural proteins. The main aim was to structurally characterize selected 14-3-3 protein complexes and investigate their stabilization by small molecule compounds. Using combination of protein crystallography, differential scanning fluorimetry, fluorescence polarization and analytical ultracentrifugation, the...
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Physiological role of Na+/H+ antiporters in yeast cells
Zahrádka, Jaromír
3 Abstract Yeast Saccharomyces cerevisiae belongs to important models for alkali-metal-cation homeostasis research. As other cells, certain intracellular content of K+ is necessary for S. cerevisiae, but Na+ or other alkali metal cations (Li+ , Rb+ ) are toxic for yeast cells. Uniporters Trk1 and Trk2 are responsible for K+ accumulation, while efflux of Na+ , Li+ , Rb+ and K+ is ensured by Ena ATPases, Na+ (K+ )/H+ antiporter Nha1 and K+ specific channel Tok1. Several regulators of K+ (Na+ ) transporters are already known, but reciprocal regulation between transporters and overall picture of the maintenance of alkali-metal-cation homeostasis is still unclear. In this work, K+ circulation (simultaneous uptake and export of K+ ) was shown to be important in alkali-metal-cation homeostasis maintenance. K+ circulation is maintained using reciprocal regulation and interactions between K+ exporters and importers. Though obtained results showed that the alkali-metal-cation homeostasis and associated physiological parameters (e.g. membrane potential, cell size, salt sensitivity) are strain specific, Nha1p was verified to be important for cell survival in ever-changing natural environment. Furthermore, two novel positive regulators of Nha1p activity were found, 14-3-3 proteins and Cka1 kinase. 14-3-3 proteins...
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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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Role of protein-protein interactions in regulation of signalling proteins and enzymes
Košek, Dalibor ; Obšil, Tomáš (advisor) ; Karpenko, Vladimír (referee) ; Pompach, Petr (referee)
EN Protein-protein interactions have an exceptional position among other mechanisms in the regulation of signal transduction. Their systematic investigation is very important and logical step in the process of understanding to the transduction and its mechanisms at a molecular level. During my Ph.D. I was particularly interested in three important processes. ASK1 kinase is well-known initiator of the apoptosis. Under physiological conditions it is maintained in an inactive state by its two interaction partners the 14-3-3 protein and TRX1. These two proteins dissociate in the presence of reactive oxygen species by unclear mechanism and the kinase is therefore activated. The next process is an interaction between the 14-3-3 protein and phosducin and investigation of their role in the G protein signalling especially important in the biochemistry of vision. The third process is an activation of protein Nth1 through the interaction with Bmh1, yeast analog of the 14-3-3 protein, and calcium cations. I employed various biophysical method, particularly analytical ultracentrifugation, in order to explain molecular mechanisms of described processes. These techniques were used to solve the low-resolution structures of complexes TRX1 and the 14-3-3 protein with corresponding binding domains of ASK1. These...
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Crosstalk of PKN3 and p130Cas/BCAR1 signaling
Dibus, Michal ; Rösel, Daniel (advisor) ; Voller, Jiří (referee)
Both p130Cas and PKN3 are important regulators of cellular signaling deregulation of which leads to malignant behavior of cancer cells. Recently we have found that SH3 domain of p130Cas mediates interaction with proline rich region of PKN3 suggesting their possible cooperation in regulation of these processes. In this work we have focused on the phosphorylation of p130Cas by PKN3 and identified serine 498 (S498) within the serine rich domain of p130Cas to be phosphorylated by PKN3 in vitro. Given that S498 is localized within the 14-3-3 binding motif and its phosphorylation is required for interaction of p130Cas with 14-3-3 proteins, we propose potential existence of novel PKN3/p130Cas/14-3-3 signaling axis. In the second part of the work we have studied this pathway in response to antiestrogen treatment in estrogen receptor positive breast cancer cell line MCF7. Although we have shown inactivation of PKN3 occurs as an early response to tamoxifen treatment, we do not rule out its possible role in further promotion of resistance to antiestrogens. Furthermore, understanding the signaling triggered by interaction of PKN3 with p130Cas and its possible downstream effects on promoting malignant growth of cancer cells would help in finding novel therapeutic targets.
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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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