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Study of protein-protein interactions of human E3 ubiquitin ligase Nedd4-2.
Joshi, Rohit Ashok ; Obšilová, Veronika (advisor) ; Krůšek, Jan (referee) ; Holoubek, Aleš (referee)
Posttranslační modifikace prostřednictvím ubikvitinace hrají klíčovou roli v regulaci membránových proteinů. Nedd4-2, lidská HECT E3 ubikvitin ligáza, je poslední komponentou ubikvitinační kaskády, která přenáší molekuly ubikvitinu a spouští endocytózu svých následných cílových molekul. Dysregulace Nedd4-2 může způsobovat různé poruchy, včetně epilepsie, respirační úzkosti a Liddleova syndromu. Přestože se na regulaci Nedd4-2 podílejí různé adaptorové proteiny, v tomto výzkumu jsme se zaměřili na konzervované proteiny 14-3- 3, známé negativní regulátory Nedd4-2. V této studii jsme provedli biofyzikální charakterizaci konstruktů Nedd4-2190-581 a Nedd4-2186-975 v komplexu s 14-3-3, abychom získali další náhled do dynamiky této interakce. Naše výsledky časově rozlišené fluorescenční spektroskopie odhalily, že vazba 14-3-3 ovlivňuje emisní vlastnosti a pohyblivost specifických WW domén (WW3 a WW4) Nedd4-2, zatímco ostatní (WW1) šetří. Zajímavé je, že katalytická doména HECT prochází při tvorbě komplexu konformačními změnami a zvýšenou exponovaností rozpouštědlu. Předpokládáme, že sterická inhibice domén WW3 a WW4 v kombinaci s konformačními změnami v katalytické doméně může být základem regulačního mechanismu zprostředkovaného vazbou proteinem 14-3-3. Chemické zesítění spolu s hmotnostní spektrometrií...
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Viral proteins that facilitate RNA replication
Černý, Prokop ; Bouřa, Evžen (advisor) ; Faltová, Lenka (referee)
In recent years, the pathogenic human coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a global pandemic of highly infectious disease COVID--19 (coronavirus disease 2019). According to the World Health Organization, as of August 20, 2023, there have been nearly 770 million confirmed cases and nearly 7 million deaths associated with the disease. However, only a small number of effective antiviral drugs are currently known to help treat the disease in already infected patients. Therefore, in this work, the interaction of host 14-3-3 proteins with the nucleocapsid protein of SARS-CoV-2 was studied as a potential biological target for the rational design of new antiviral drugs. The importance of this interaction in the virus life cycle has not been fully elucidated, but it probably has an important regulatory function affecting multiple processes in the infected cell. The SARS-CoV-2 N protein contains several binding motifs for 14-3-3 proteins in its sequence, two of which (at positions S197 and T205), most likely responsible for the protein- protein interaction, were studied in this work. Microscale thermophoresis was performed to determine the affinity of each of these phosphorylation sites on the nucleoprotein for the 14-3-3ζ protein. The key phosphorylated residue...
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Structural study of the complex between the 14-3-3 protein, CaMKK1 and CaMKK1:Ca2+/CaM
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
The Ca2+ -signaling pathway is an important mechanism of cell signaling. Ca2+ /Cal- modulin (CaM)-dependent protein kinases (CaMKs) are members of Ser/Thr protein kinase family. CaMKs are regulated by Ca2+ /CaM binding in response to increase in intracellular level of Ca2+ . An important member of this protein family is Ca2+ /CaM- dependent protein kinase kinase (CaMKK), which is an upstream activator of CaMKI and CaMKIV. There are two isoforms of CaMKK, CaMKK1 and CaMKK2. CaMKK1 is regulated not only by Ca2+ /CaM-binding, but also by phosphorylation by cAMP-dependent protein kinase A (PKA). PKA phosphorylation induces inter- action with the 14-3-3 proteins. Previous studies of interaction between CaMKK1 and 14-3-3 proteins suggested, that the interaction with 14-3-3 proteins keeps CaMKK1 in the PKA-induced inhibited state and blocks its active site. However, the exact mecha- nism of this inhibition is still unclear mainly due to the absence of structural data. Main aim of this diploma thesis was to characterize the protein complexes between CaMKK1, Ca2+ /CaM and 14-3-3γ using analytical ultracentrifugation, small angle X-ray scattering, and chemical cross-linking coupled to mass spectrometry. Analytical ultracentrifugation revealed concentration-dependent dimerization of CaMKK1, which is...
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Properties and function of middle T antigen of the murine polyomavirus
Fabiánová, Anna ; Forstová, Jitka (advisor) ; Čáp, Michal (referee)
Polyomaviruses are small DNA viruses, which are able to induce a broad variety of tumors. The main oncoprotein of the mouse polyomavirus (MPyV) is middle T antigen (MT antigen) which is able to transform cells. MT antigen has not an enzymatic activity of its own. It is able to activate signal transduction of host cells through its interactions with certain cellular proteins. These proteins include protein phosphatase 2A (PP2A), Src kinase, phosphatidylinositol 3 kinase (PI3K), Shc protein, 14-3-3 protein and phospholipase Cγ1 (PLCγ1). This work is focused on interaction between MT antigen and cellular proteins and on the impact of this interaction on cell transformation. Since MT antigen is a potent oncogene, the work also deals with the character of transformed cells and tumor development in mouse mammary epithelium. Keywords: polyomaviruses, MT antigen, PP2A, PI3K, PLCγ1, Shc protein, 14-3-3 protein
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Structural characterization of human protein kinase CaMKK2 and its interactions with binding partners
Koupilová, Nicola ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...
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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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Structural study of the complex between the 14-3-3 protein, CaMKK1 and CaMKK1:Ca2+/CaM
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
The Ca2+ -signaling pathway is an important mechanism of cell signaling. Ca2+ /Cal- modulin (CaM)-dependent protein kinases (CaMKs) are members of Ser/Thr protein kinase family. CaMKs are regulated by Ca2+ /CaM binding in response to increase in intracellular level of Ca2+ . An important member of this protein family is Ca2+ /CaM- dependent protein kinase kinase (CaMKK), which is an upstream activator of CaMKI and CaMKIV. There are two isoforms of CaMKK, CaMKK1 and CaMKK2. CaMKK1 is regulated not only by Ca2+ /CaM-binding, but also by phosphorylation by cAMP-dependent protein kinase A (PKA). PKA phosphorylation induces inter- action with the 14-3-3 proteins. Previous studies of interaction between CaMKK1 and 14-3-3 proteins suggested, that the interaction with 14-3-3 proteins keeps CaMKK1 in the PKA-induced inhibited state and blocks its active site. However, the exact mecha- nism of this inhibition is still unclear mainly due to the absence of structural data. Main aim of this diploma thesis was to characterize the protein complexes between CaMKK1, Ca2+ /CaM and 14-3-3γ using analytical ultracentrifugation, small angle X-ray scattering, and chemical cross-linking coupled to mass spectrometry. Analytical ultracentrifugation revealed concentration-dependent dimerization of CaMKK1, which is...
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Preparation and characterization of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2).
Jarosilová, Kateřina ; Obšil, Tomáš (advisor) ; Vondrášek, Jiří (referee)
Calmodulin kinase cascade is a signaling pathway which is involved in the response to the increasing intracellular calcium levels. Ca2+ is a ubiquitous second messenger which promotes wide-range of cellular signaling events. Many of these signaling pathways start with the binding of Ca2+ to its primary intracellular receptor calmodulin. Calmodulin in turn binds to its downstream targets in the Ca2+ /calmodulin signaling cascade. One of the most important enzymes of this cascade is a Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). CaMKK2 is a serine/threonine protein kinase which regulates for example gene transcription or energy homeostasis by phosphorylation of its downstream targets. Catalytic domain (which provides kinase activity) is located in the middle part of the protein and possesses structure typical for kinases. CaMKK2 consists of 588 amino acids but the secondary structure is known only for the region of the kinase domain (298 residues). The rest of the protein is assumed to be unstructured as long as CaMKK2 is not bound to any interaction partner. The aim of this study was to prepare several constructs of human isoform of CaMKK2 for the further structural and activity studies. It is believed that CaMKK2 is regulated by site-specific phosphorylation. Phosphorylation of some...
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Study of interaction between phosducin and the 14-3-3 protein
Šimůnek, Jiří ; Obšil, Tomáš (advisor) ; Alblová, Miroslava (referee)
The main goal of this bachelor thesis is to study the interaction between phosducin and the 14-3-3 protein which are involved in the regulation of visual signal transduction in vertebrate retina. This process is mediated by G-protein signaling pathway and is regulated by several proteins including phosducin depending on light intensity. Phosducin, a 33kDa protein, is expressed in many tissues mainly in photoreceptor cells of the retina. The visual signal transduction and its amplification are regulated through inhibition of the G-protein (transducin) function. The signal transduction involves a dissociation of heterotrimeric transducin (Gtαβγ) to the α subunit (Gtα) and the βγ complex (Gtβγ). The signalling is terminated by their reassociation back to the Gtαβγ complex. The phosducin binds to Gtβγ and thus prevents the formation of the Gtαβγ heterotrimer. This decreases the amount of functional Gtαβγ complexes and suppresses the signal transduction. If the signal transduction needs to be amplified (e.g. during the night) then phosducin is phosphorylated and this inhibits its interaction with Gtβγ. The phosphorylated phosducin is bound to the 14-3-3 protein. However, the role of the 14-3-3 protein in the regulation of phosducin is still unclear. The 14-3-3 protein is a 28kDa protein which is...
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Properties and function of middle T antigen of the murine polyomavirus
Fabiánová, Anna ; Forstová, Jitka (advisor) ; Čáp, Michal (referee)
Polyomaviruses are small DNA viruses, which are able to induce a broad variety of tumors. The main oncoprotein of the mouse polyomavirus (MPyV) is middle T antigen (MT antigen) which is able to transform cells. MT antigen has not an enzymatic activity of its own. It is able to activate signal transduction of host cells through its interactions with certain cellular proteins. These proteins include protein phosphatase 2A (PP2A), Src kinase, phosphatidylinositol 3 kinase (PI3K), Shc protein, 14-3-3 protein and phospholipase Cγ1 (PLCγ1). This work is focused on interaction between MT antigen and cellular proteins and on the impact of this interaction on cell transformation. Since MT antigen is a potent oncogene, the work also deals with the character of transformed cells and tumor development in mouse mammary epithelium. Keywords: polyomaviruses, MT antigen, PP2A, PI3K, PLCγ1, Shc protein, 14-3-3 protein
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