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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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Identification of small compounds disrupting protein-protein interaction in influenza A polymerase.
Hejdánek, Jakub ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...
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Interaction of a surface marker of immune cells with low-molecular weight ligands and their polymer conjugates
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...
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BIOPHYSICAL AND FUNCTIONAL CHARACTERIZATION OF DDI1-LIKE ASPARTIC PROTEASES INVOLVED IN REPLICATION STRESS RESPONSE
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Čermák, Lukáš (referee)
Accurate, timely replication of a DNA molecule is a pivotal moment in the life cycle of every living organism. Any temporal or spatial defect putting the fine-tuned replication machinery off balance causes the so-called replication stress. As the replication machinery consists mainly of enzymes and other proteins, it is not surprising that many of the obstacles most severely blocking the replication machinery progress are of protein origin. Therefore, specialized proteases responsible for relieving replication stress matured during evolution. However, neither the full repertoire of proteolytic enzymes and their particular substrates taking place in countering the DNA replication stress nor detailed molecular mechanisms involved remain unknown. This thesis describes how conserved putative aspartic proteases of the Ddi1-like family engage in countering DNA replication stress via a proteolysis dependent mechanism. We structurally and biophysically characterized yeast and human members of the Ddi1-like family, explored their interactions with ubiquitin and polyubiquitin chains, and identified hypersensitivity to DNA replication inhibitor hydroxyurea in a yeast strain double deleted for DDI1 gene together with a DNA dependent metalloprotease WSS1. Detailed analysis of the DDI1 role in hydroxyurea...
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Structure and interactions of selected forkhead transcription factors
Kohoutová, Klára ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee)
This diploma thesis is a part of a project aiming to develop and study specific inhibitors of FOXO3 transcriptional activity. FOXO3 belongs together with FOXO1, FOXO4 and FOXO6 to FOXO subfamily of forkhead family transcription factors. FOXO transcription factors are evolutionary conserved proteins playing important roles in numerous cellular processes, such as apoptosis, cell cycle regulation and metabolism. Due to their ability to induce apoptosis and to block the cell cycle they are considered tumor suppresors. However, it has been shown that increased activity of FOXO proteins is connected with many kinds of cancer. In such cases FOXO proteins function to maintain cell homeostasis. They promote tumor resistance against chemotherapy as well as they speed up its growth. The aim of this project is to develop specific inhibitors able to bind to FOXO3 DNA-binding domain (DBD, residues 156-269) and to block its interaction with target DNA. Development of specific inhibitors of FOXO3 transcriptional programme requires knowledge of solution structure of all FOXO DBDs and detailed insight into their interaction with target DNA. So far crystal structures of complexes of FOXO1, FOXO3 and FOXO4 with target DNA and solution NMR structures of apo DBDs of FOXO3 and FOXO4 have been solved. One of the goals of...
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Substrate specificity of histone deacetylases
Ustinova, Kseniya ; Bařinka, Cyril (advisor) ; Bumba, Ladislav (referee) ; Obšil, Tomáš (referee)
In the cell, tubulin undergoes post-translational modifications that create functionally distinct microtubules and mark them for specialized functions. Acetylation of Lys40 of α-tubulin is one of such post-translational modifications controlled by the activity of histone deacetylase 6 (HDAC6). The Lys40 acetylation is a hallmark of stable microtubules, it protects them from mechanical aging, influences cell motility as well as axonal branching and maintenance of neuronal processes. Tubulin stands out as the most prominent physiological substrate for HDAC6. Being a multidomain cytosolic protein, HDAC6 is involved in the myriad of cellular processes and is a promising target for the treatment of cancer and neurodegenerative diseases. The understanding of the mechanisms of HDAC6 interactions with its substrates, especially with tubulin, can open avenues for the development of new treatment strategies exploiting highly selective HDAC6 inhibitors. In this thesis, we have investigated the molecular basis of tubulin recognition by HDAC6. We provided a detailed kinetic analysis showing the HDAC6 deacetylation rate of free tubulin is 1500-fold faster than microtubules. Additionally, we have shown that amino acids of the flexible Lys40 loop (except P1 and P-1) make a minor contribution to the substrate...
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Study of interactions of forkhead box O (FOXO) transcription factors with DNA
Hofmanová, Adéla ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
This work studies the "O" subgroup of FOX transcription factors, which consists of four members (FOXO1, FOXO3, FOXO4 and FOXO6). They are important regulatory molecules that play a critical role in a number of physiological and pathological processes such as cell cycle control, the body's response to stress, differentiation and apoptosis. Due to their ability to induce cell death, they are generally considered to be tumor suppressors. However, recent studies have shown that they can also induce an opposite effect, i.e. to promote tumor progression or induce resistance to drugs used in the therapy of certain types of tumors. Despite intensive research, a number of questions regarding the function of FOXO proteins still remain unanswered. One question is whether the small structural differences observed in the highly conserved DNA-binding domains (DBD) of FOXO transcription factors affect their DNA- binding affinities. Furthermore, it is unclear whether the recently described protein-protein interaction of FOXO-DBD with the transcription factor p53 affects their DNA-binding affinity. Moreover, the role of the binding site for Mg2+ ion which was found in the crystal structure of FOXO4-DBD:DNA, is also still not understood. To clarify these questions, the DNA-binding domains of the human transcription...
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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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The preparation and characterisation of analogues of insulin and IGF-2 selective for both isoform of insulin receptor and IGF-1 receptor
Mlčochová, Květoslava ; Žáková, Lenka (advisor) ; Obšil, Tomáš (referee) ; Šulc, Miroslav (referee)
Insulin and insulin-like growth factor 1 (IGF-1) and 2 (IGF-2) are related protein hormones with different but overlapping biological functions. All the hormones interact with a receptor within the insulin-IGF system (insulin receptor A and B, IGF-1 receptor), however with different affinity. The different interaction with individual receptors is just one of the main tools for regulation of the system that is essential for the proper functioning of the organism. Although the residues directly interacting with receptors are mainly located in A and B domains, the C and D domains probably play a role in receptor specificity. Here, we firstly focused on the impact of D domains of IGF-1 and 2 (D1 and D2 domains) and C domain of IGF- 2 (C2 domain). To probe the impact of C and D domains, we prepared insulin analogues containing a part of or an entire domain following a pattern seen in IGFs. The receptor-binding affinities of these analogues and their receptor autophosphorylation potentials were characterised. Our results revealed that the initial part of D1 domain has a detrimental effect on IR affinity that is only slightly enhanced by the rest of the D1 domain. D2 domain has rather neutral effect on IR affinity. We further showed that the addition of amino acids derived from the C2 domain to 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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