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Structural-functional aspects of the Bordetella pertussis adenylate cyclase toxin
Březinová, Karolína ; Bumba, Ladislav (advisor) ; Brzobohatá, Hana (referee)
Whooping cough (pertussis) is a highly infectious respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. Even though the vaccination rate of the population is high, pertussis is one of the most widespread vaccine-preventable diseases. The bacterium produces a variety of virulence factors that facilitate the process of colonization of the ciliated epithelium and infection of the upper respiratory tract. Among the most important virulence factors is the adenylate cyclase toxin (CyaA). This toxin belongs to the so- called RTX (Repeat-In-ToXins) proteins, which are released from the bacterium using the Type 1 secretion apparatus (T1SS). CyaA is a multifunctional toxin, showing both hemolytic and cytotoxic activity. The cytotoxic activity is caused by the N-terminal adenylyl cyclase (AC) domain, which is translocated across the cytoplasmic membrane into the cell cytosol, where upon interaction with calmodulin catalyzes the uncontrolled conversion of adenosine triphosphate (ATP) to cyclic adenosine-3',5'-monophosphate (cAMP). CyaA is recognized by the integrin receptor CD11b/CD18 (also known as complement receptor type 3), which is primarily found on phagocytic cells of the host organism. This work focuses on the structural- functional aspects of the CyaA toxin and summarizes...
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Study of interactions between protein kinase CaMKK2 and calmodulin using fluorescence spectroscopy.
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Ca2+ /calmodulin-dependent kinases are members of CaMK family, which is involved in CaMK cascade. One of CaMK family members is Ca2+ /calmodulin-dependent kinase kinase 2 (CaMKK2), which is activated by Ca2+ /CaM-binding. There are some structural differences between CaMKK2 and other protein kinases, one of them is a structure near αE-helix and autoinhibitory domain. Due to the overlap of autoinhibitory domain and Ca2+ /CaM-binding domain it can be supposed that Ca2+ /CaM-binding induces structural changes near autoinhibitory do- main and thus can affect the accessibility of this region. CaMKK2 W445F mutant, which contains only one tryptophane residue Trp374 close to the αE-helix, was expressed and purified. Structural changes in this region were monitored using tryptophan fluorescence intensity quenching experiments, which can provide information about the accessibility of region surrounding tryptophan residue. The fluorescence of Trp374 was quenched using acrylamide. Comparison of fluorescence quenching experiments performed in the presence and absence of calmodulin suggests that the complex formation induces structural change in the region surrounding Trp374 . 1
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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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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 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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Study of interactions between protein kinase CaMKK2 and calmodulin using fluorescence spectroscopy.
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Ca2+ /calmodulin-dependent kinases are members of CaMK family, which is involved in CaMK cascade. One of CaMK family members is Ca2+ /calmodulin-dependent kinase kinase 2 (CaMKK2), which is activated by Ca2+ /CaM-binding. There are some structural differences between CaMKK2 and other protein kinases, one of them is a structure near αE-helix and autoinhibitory domain. Due to the overlap of autoinhibitory domain and Ca2+ /CaM-binding domain it can be supposed that Ca2+ /CaM-binding induces structural changes near autoinhibitory do- main and thus can affect the accessibility of this region. CaMKK2 W445F mutant, which contains only one tryptophane residue Trp374 close to the αE-helix, was expressed and purified. Structural changes in this region were monitored using tryptophan fluorescence intensity quenching experiments, which can provide information about the accessibility of region surrounding tryptophan residue. The fluorescence of Trp374 was quenched using acrylamide. Comparison of fluorescence quenching experiments performed in the presence and absence of calmodulin suggests that the complex formation induces structural change in the region surrounding Trp374 . 1
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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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