National Repository of Grey Literature 92 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Structural studies of selected signaling protein complexes.
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee) ; Maloy Řezáčová, Pavlína (referee)
The ability of proteins to bind other molecules in response to various stimuli in their microenvironment serves as a platform for extensive regulatory networks coordinating downstream cell actions. The correct function of these signaling pathways depends mostly on noncovalent interactions often affecting the structure of proteins and protein complexes. Understanding the molecular mechanism of a protein function in cell signaling therefore often depends on our knowledge of a three-dimensional structure. In this doctoral thesis, I present the work that led to the understanding of several protein-protein and protein-ligand interactions implicated in cell signaling at the molecular level. I applied nuclear magnetic resonance spectroscopy, small angle X-ray scattering and other biophysical methods to determine the molecular basis of inhibition of four signaling proteins: Calcium/Calmodulin (Ca2+ /CaM)-dependent protein kinase kinase 2 (CaMKK2); protease Caspase-2; Forkhead transcription factor FOXO3, and Apoptosis signal-regulating protein kinase 1 (ASK1). In particular, I investigated the distinct roles of 14-3-3 and Ca2+ /CaM in the regulation of CaMKK2 activity. I also studied in detail the mechanism how 14-3-3 interferes with the caspase-2 oligomerization and its nuclear localization as well as...
Glutamate Carboxypeptidase II - Structural and Biochemical Characterization and Structure-Assisted Drug Design
Ptáček, Jakub ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Brynda, Jiří (referee)
Glutamate carboxypeptidase II (GCPII) is a human membrane-bound metallopeptidase discovered more than 30 years ago. It has attracted attention of biomedical scientists thanks to its diverse tissue expression profile and different biological functions. GCPII is detected on the surface of astrocytes in both central and peripheral nervous systems where it is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. Glutamate, one of the hydrolytic products, is a potent excitatory neurotransmitter and its overproduction has been shown to be responsible for cell death in various neurological disorders by a so-called glutamate excitotoxicity mechanism. Together with the fact that NAAG acts neuroprotectively it has been postulated (and later confirmed) that GCPII inhibition has a therapeutic potential in such disorders. Prostate cancer (PCa) is the second most prevalent cancer in men and despite its slow progression it is prone to metastasize thus posing a life threat. GCPII has been found to be overexpressed in prostate tumor cells compared to the healthy tissue (therefore it is also termed prostate-specific membrane antigen - PSMA) thus representing an excellent biomarker of PCa validated by many publications and clinical studies....
Development of analytical tools for quantification and screening for inhibitors of glutamate carboxypeptidases II and III
Navrátil, Václav ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Šedo, Aleksi (referee)
Glutamate carboxypeptidase II (GCPII) usually called prostate specific membrane antigen (PSMA) is membrane bound metallopeptidase expressed mainly in prostate carcinoma (PCa). Agents targeting GCPII suitable for both imaging and treatment of PCa are in development and they show promising results in advanced clinical trials. Some studies showed that GCPII may serve also as PCa blood serum marker, but this has not been validated due to the lack of methods suitable for accurate detection of GCPII in human blood. Moreover, GCPII is also expressed in brain, where it cleaves inhibitory N-acetyl-α-L- aspartyl-L-glutamate (NAAG) to release excitatory L-glutamate and GCPII inhibition has been shown to be neuroprotective in animal models of several neuropathies. Tight binding inhibitors of GCPII have been identified by rational design, but all have poor bioavailability and thus cannot be used in clinics. Identifying new scaffolds by 'brute force' screening methods is thus essential; however, no such method for GCPII has been developed so far. Glutamate carboxypeptidase III (GCPIII) is also expressed in brain and cleaves NAAG. It is thus an important protein for understanding of GCPII function as well as GCPII targeting in medicine. Here, we focused on development of novel methods for quantification of both...
Study of regulatory mechanisms of selected protein kinases
Petrvalská, Olívia ; Obšil, Tomáš (advisor) ; Jiráček, Jiří (referee) ; Schneider, Bohdan (referee)
Through binding interactions with more than 300 binding partners, 14-3-3 proteins regulate large amount of biologically relevant processes, such as apoptosis, cell cycle progression, signal transduction or metabolic pathways. The research discussed in this dissertation thesis was focussed on investigating the role of 14-3-3 proteins in the regulation of two selected protein kinases ASK1 and CaMKK2. The main goal was to elucidate the mechanisms by which phosphorylation and 14-3-3 binding regulate functions of these protein kinases using various biochemical and biophysical methods, such as site-directed mutagenesis, enzyme activity measurements, analytical ultracentrifugation, small-angle X-ray scattering, chemical crosslinking, nuclear magnetic resonance and fluorescence spectroscopy. A structural model of the complex between the catalytic domain of protein kinase ASK1 with 14-3-3ζ, which was calculated using the small-angle X-ray scattering and chemical crosslinking data, suggested that this complex is conformationally heterogeneous in solution. This structural model together with data from time-resolved fluorescence and nuclear magnetic resonance suggested that the 14-3-3ζ protein interacts with the catalytic domain of ASK1 in the close vicinity of its active site, thus indicating that the complex...
Development of high-throughput screening assay for the identification of inhibitors targeting influenza A polymerase
Karlukova, Elena ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus A circulates in birds and mammals and causes severe infectious disease that affects from 3 to 5 million people each year. There are two classes of anti-influenza drugs currently available: neuraminidase and M2 channel inhibitors. However, increasing resistance against these two types of inhibitors along with the potential emergence of new viral strains and unpredictability of pandemic outbreaks emphasize an unmet need for new types of inhibitors. RNA-dependent influenza polymerase serves as a novel promising target for the development of anti-influenza medications. The aim of this master thesis is to develop in vitro high-throughput assays for screening of compounds targeting influenza RNA polymerase, particularly, its cap binding and endonuclease domains. For cap-binding domain the screening is based on DIANA (DNA-linked Inhibitor ANtibody Assay) method that was recently developed in our laboratory; for endonuclease domain, the method is based on AlphaScreen technology. For the purposes of the methods development, recombinant cap binding domain of PB2 subunit and N-terminal endonuclease domain of PA subunit of influenza polymerase were expressed with appropriate fusion tags and purified using affinity and gel permeation chromatography. The probes for the screening assays were...
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
Study of interactions between low-molecular mass compounds and the DNA-binding domain of forkhead transcription factor FOXO3
Kohoutová, Klára ; Obšil, Tomáš (advisor) ; Žáková, Lenka (referee)
This bachelor thesis is part of a project focused on studying low-molecular mass compounds able to inhibit the interaction between DNA-binding domain of human forkhead transcription factor FOXO3 and the target DNA. FOXO3 is one of four members of FOXO class transcription factors which belong to forkhead family of transcription factors. Forkhead transcription factors are evolutionary conserved proteins playing important roles in numerous cellular processes. These include cell-cycle regulation, oxidative stress response, control of cellular metabolism and apoptosis. FOXO3 plays an important role in cancer cells where it acts not only as a tumor suppressor but also can enhance their resistance to chemotherapy. Considering its biological functions, the study of small-molecule inhibitors of FOXO3 transcription factor is of particular importance. This bachelor thesis was focused on compound S9 oxalate as a potential inhibitor of FOXO3-DNA interaction. Main goals of this thesis were: (I) preparation of both unlabeled and 15 N labeled DNA- binding domain of FOXO3 transcription factor, (II) characterization of interactions between FOXO3 DBD and compound S9 oxalate using NMR and electrophoretic mobility shift analysis (EMSA), and (III) prediction of binding conformation and interactions between FOXO3 DBD and...
Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Novák, Petr (referee)
Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...

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