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Structure determination of helical domain of DNA damage-inducible protein 2
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human Ddi2 and his homologues are scarcely explored family of ubiquitin- like/ubiquitin-associated domain proteins (UBL/UBA proteins), containing domain which is structurally related to dimeric aspartyl proteases from retroviruses. The most studied of this family is Ddi1 protein from Saccharomyces cerevisiae, which functions within the ubiquitin- proteasome system. This key cellular system exploits tightly regulated enzymatic apparatus for highly selective posttranslational modifications of proteins with molecules of ubiquitin, which serves as intracellular signal determining the proteins fate, importantly its degradation in many cases. Ddi1 plays a role of proteasome adaptor within this context, helping the recognition of ubiquitylated proteins by the proteasome. Even though the function as a proteasome receptor is possible for human Ddi2 protein as well, its cellular role might have been altered during the evolution. One of the important steps in decoding its purpose in cell is exploration of function of its individual domains. This work focuses on structural study of neighbouring region of this protease domain, where the helix-rich region called HDD domain is located. This region of Ddi2 was cloned, expressed and subjected to the NMR measurements. Structural information based on the NMR data was...
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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 effect of amino acid repertoire on protein structure and function
Treťjačenko, Vjačeslav ; Hlouchová, Klára (advisor) ; Obšil, Tomáš (referee) ; Pačes, Jan (referee)
To understand protein structure emergence is to comprehend the evolutionary transition from messy chemistry to the first heritable molecular systems. Early proteins were probably flexible in structure, promiscuous in activity and ambiguous in sequence. Moreover, first sequences were presumably composed of prebiotically plausible amino acids from endogenous and exogenous sources which form only a subset of the extant protein alphabet. Here we investigate the effect of most recent additions to the amino acid alphabet on protein structure/function relationship and the properties of random proteins as the evolutionary point-zero for the earliest sequences as well as for proteins emerging de novo from the non-coding parts of the genome. Random or never born proteins are of a special interest for the contemporary biology as they unveil the unexposed side of the protein sequence space. We constructed an in silico library of random proteins with the natural amino acid alphabet, analyzed its structure/disorder/aggregation content and selected 45 sequences for subsequent experimental preparation and biophysical characterization. We observed that structure content in random sequence space does not differ significantly from the natural proteins. However, the analyses of the aggregation propensity showed a...
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Study of structural differences among 14-3-3 protein isoforms.
Macáková, Eva ; Obšil, Tomáš (advisor) ; Gryčová, Lenka (referee)
The 14-3-3 proteins are a family of important regulatory proteins, found in all eukaryotes, which are involved in many cellular processes. In this diploma thesis, we studied structure/function relationships of 14-3-3 proteins, in this case it was the influence of the structure of H8-H9 loop on the binding affinity in barley isoform hv 14-3-3A and human isoform 14-3-3ζ. According to former results, hv 14-3-3A binds to a ligand with lowest affinity, which could be caused by present of a glycin in H8-H9 loop, while in other isoforms there is a serin on the same position. We measured the binding affinity in protein hv 14-3-3A WT and its mutant, which contained the serin instead of the glycin in H8-H9 loop. For comparison, we also measured the binding affinity of human isoform 14-3-3ζ containing the serin in H8-H9 loop and its mutant, where the the serin was replaced by the glycin. Proteins were expressed in E. coli cells strain BL21(DE3) and then purified. The dissociation constant for the binding of peptide pRaf-259 labeled with fluorophores FITC and ATTO was measured using both the fluorescence correlated spectroscopy and the steady-state fluorescence intensity. Our results showed that in both isoforms the mutation of H8-H9 loop causes decrease in the binding affinity.
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Molecular dynamics simulations of complexes consisting of proteins and nucleic acids
Hammer, Jiří ; Barvík, Ivan (advisor) ; Obšil, Tomáš (referee)
The goal of this diploma thesis was to study interactions of Argonaute (Ago) protein in a complex with nucleic acids. Based on the available crystal structures of full length Argonaute (from A. aeolicus, Aa-Ago) and/or its domains (human PAZ domain, Hs-PAZ), twelve different simulations were computed. Two initial simulations used model of Aa-Ago with either a duplex of DNA/RNA or RNA/RNA. Major difference was in behavior of the PAZ domain (especially its arginine residues), which tolerated the guide DNA in one simulation, but was disturbing the RNA guide strand in the second. Such an interaction could serve as a mechanism of the substrate recognition. In additional simulations (3-9) employing the Hs-PAZ domain, where no disturbance was found in the DNA/RNA hetero-duplex. Different arrangements of the active site geometry as well as empirical parameterizations of Mg2+ ion were probed and analyzed. The DD-catalytic motif plus D683 in Aa-Ago (equivalent to H807 in human Argonaute2) was observed to coordinate the Mg2+ ion in one and two metal ion dependent catalysis models. Highly conserved R570 and E578 created mutual hydrogen bonds and hence stabilized the active site. To make the cleavage irreversible, a role for the first (unpaired) nucleotide from 5'-end of the guide strand was suggested. It lies in a...
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Harnessing soluble forms of NK cell receptors and their ligands for cancer immunotherapy
Kalousková, Barbora ; Vaněk, Ondřej (advisor) ; Obšil, Tomáš (referee) ; Maloy Řezáčová, Pavlína (referee)
Natural killer cells (NK cells) are a family of lymphocytes with a natural ability to kill infected, harmed, or malignantly transformed cells. As these cells are part of the innate immunity, the cytotoxic mechanisms are activated upon recognizing specific patterns without prior antigen sensitization. This recognition is crucial for NK cell function in the maintenance of homeostasis and immunosurveillance. NK cells not only act directly towards malignant cells but also participate in the complex immune response by producing cytokines or crosstalk with other immune cells. Cancer may be seen as a break of all immune defenses when malignant cells escape the immunity and invade surrounding tissues creating a microenvironment supporting tumor progression. This process may be reverted by interventions into immune response shaping with immunotherapy, which may lead to restoration of immune recognition. NK cells are also effector cells important for immunotherapy, may be used for adoptive transfer, genetically modified with chimeric antigen receptors, or triggered with appropriate antibodies. NK cell receptors, responsible for target recognition and activation of cytotoxic machinery, may also be targeted in immunotherapy. However, this kind of immunoactive therapeutics may be designed only with the deep...
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Conformation of the adenylate cyclase toxin of Bordetella pertussis.
Motlová, Lucia ; Fišer, Radovan (advisor) ; Obšil, Tomáš (referee) ; Holoubek, Aleš (referee)
This work is focused on the RTX (Repeats in ToXin) domains structure of selected RTX toxins and its impact on secretion and protein folding. The structural analysis included RTX domains of ApxI (Actinobacillus pleuropneumoniae-RTX-toxin I) from Actinobacillus pleuropneumoniae, HlyA (Alfa-hemolysin) from Escherichia coli and LtxA (Leukotoxin A) from Aggregatibacter actinomycetemcomitans and blocs 4 a 5 RTX domain CyaA (adenylate cyclase toxin) from Bordetella pertussis. The structures of LtxA RTX domain and CyaA RTX blocs 4 and 5 were obtained and characterized. Two models of CyaA RTX domain were built based on SAXS (Small Angle X-ray Scattering) model, previously solved RTX structures and RTX structures presented here.
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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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Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
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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...
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