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Capturing chromatin-associated proteins in nucleosomal context
Koutná, Eliška ; Veverka, Václav (advisor) ; Hlouchová, Klára (referee) ; Bařinka, Cyril (referee)
| 9 ABSTRACT Eukaryotic DNA is stored in the nucleus wrapped around histone octamers in the form of nucleosomes. These basic chromatin units can further associate with DNA-binding factors through DNA and the N-terminal histone tails that are subjected to various covalent posttranslational modifications that, in combination with DNA modifications, define the epigenetic code. Eukaryotic transcription is dependent on these specific histone modifications - their recognition by chromatin reader proteins triggers complex processes relying on the coordinated association of transcription regulatory factors. Although various modification states of a particular histone residue often lead to differential outcomes, it is not entirely clear how they are discriminated at a molecular level. Moreover, the contribution of intrinsically disordered regions outside of the specialized reader domains to nucleosome binding remains unexplored. In this thesis, the main focus is put on the transcription coactivator LEDGF/p75, capable of reading the H3K36me2/3 histone mark, and the pioneer transcription factor Sox2. Using structural biological and biophysical techniques, the interaction of LEDGF and Sox2 with nucleosomes is dissected, describing the peculiarities of intrinsically disordered DNA interacting motifs. Two...
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Roles of histone H3 lysine methylation in the gene expression regulation
Čizmazia, Viliam ; Veverka, Václav (advisor) ; Ormsby, Tereza (referee)
Viliam Čizmazia Roles of histone H3 lysine methylation in the gene expression regulation Abstract Histone post-translational modifications play a key role in epigenetic regulation of chromatin land- scape and various cellular functions. They can directly mediate interactions between DNA and histones, but also represent recognition signals for specific reader proteins. A particular type of these modifications, lysine methylation, marks a number of specific sites within the terminal as well or globular regions of histone proteins and encodes for various instructions for DNA-related processes such as gene transcription. The profiles of individual lysine methylations are regulated by specific methyltransferases (writers) and antagonizing demethylases (erasers). Their deregula- tion is often associated with diseases such as developmental abnormalities or cancer. For this rea- son, a number of histone-modifying enzymes are considered attractive therapeutic targets. This review is focused on key players and mechanisms underlying the deposition of the most important histone H3 lysine methylations, their genomic distribution and contextual roles in transcriptional events. In addition, it highlights the importance of structure-based approaches in exploring the molecular details behind the activity of histone...
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Modulation of the STING signaling pathway
Vaneková, Lenka ; Veverka, Václav (advisor) ; Hirsch, Ivan (referee) ; Dobeš, Jan (referee)
cGAS-STING signalling pathway plays the key role in the host immune defence in diverse pathologies including, autoimmune and autoinflammatory diseases, cancer, senescence and ageing, pathogen infection, i.e., bacterial, viral infection, such as hepatitis B (HBV). HBV infection can result in either an acute or a chronic type (CHB), both of wide range of immune invading mechanism potentially leading to liver cirrhosis, steatosis, or hepatocellular carcinoma. Currently, two available CHB therapies are approved, both of which rarely result in the complete cure and often require life-long application. The development and validation of novel CHB therapeutics relies on suitable CHB animal models. The main goal of this thesis was to develop a mouse model reflecting CHB based on hydrodynamic injection suitable for robust preclinical testing of novel CHB therapeutics. Two delivery systems were compared, adeno-associated plasmid vector (pAAV) and minicircle construct, encoding HBV genomes of two genotypes (A or D) with introduced point mutation in the START codon of the polymerase in two immunocompetent mouse strains, C57Bl/6 and C3H/HeN. Persisting expression of viral antigens was observed only in the C3H/HeN mice when using pAAV construct encoding HBV genome of genotype A with introduced T2308C point...
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Structural characterization of interactions between subunits of the GBAF chromatin remodeling complex
Naušová, Karolína ; Veverka, Václav (advisor) ; Rozbeský, Daniel (referee)
Epigenetics investigates heritable phenotypic changes that are not caused by alterations in DNA sequence. One of the major epigenetic tools is chromatin remodeling mediated by ATP-dependent chromatin remodeling complexes, which fundamentally affect gene expression and thus cellular fate. A mammalian variant of the ATP-dependent chromatin remodeling SWI/SNF complex is the BAF complex, which is involved in both activation and repression of gene expression. There have been identified three major variants of BAF complex one of which is non-canonical BAF, also known as GLTSCR1 containing BAF, GBAF. Each complex consists of up to 15 subunits, some of which are specific only to one of the complex variants. Mutations in genes coding subunits of BAF lead to several genetic disorders or to the cancer development. The GBAF complex contains specific subunits, BRD9 (Bromodomain containing protein 9), GLTSCR1 (Glioma tumor suppressor candidate region 1) and GLTSCR1L (GLTSCR1-like), which are essential for its formation. Although the exact function of the GBAF complex has not been elucidated yet, it is often associated with synovial sarcoma and rhabdoid tumors. Two of the three complexes are impaired in those tumors and gene expression is maintained only due to the GBAF complex. If GBAF would additionally loose...
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Detailed characterization of the interaction between LEDGF/p75 and MeCP2
Naušová, Karolína ; Veverka, Václav (advisor) ; Hrabal, Richard (referee)
Epigenetics investigates heritable phenotype changes that are not caused by alternations in DNA sequence. Major epigenetic mechanisms include covalent DNA modifications (particularly methylation), histone and chromatin modifications and RNA interference. These mechanisms are involved in number of processes from transcription to translation. Lens epithelium-derived growth factor (LEDGF/p75) is ubiquitously expressed in human body and it is considered to be a transcriptional coactivator upregulated upon stress conditions. LEDGF/p75 consists of several domains. The N-terminal PWWP domain plays very important role from epigenetic point of view as it is able to bind di- and trimethylated lysine 36 of histone 3, which is considered as an epigenetic marker of transcriptionally active chromatin. LEDGF/p75 interaction partners include e.g. HIV integrase, MLL1-MENIN complex or MeCP2. A shorter isoform of LEDGF/p75 called LEDGF/p52 shares with LEDGF/p75 its N- terminal part that is responsible for interaction with DNA and chromatin. Methyl-CpG-binding protein 2 (MeCP2) is present everywhere in human body with the highest abundance in brain. MeCP2 is a transcriptional modulator remodelling chromatin, therefore its function is to activate or repress gene depending on the molecular and cellular context. Among...
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Structural NMR studies of protein complexes
Hexnerová, Rozálie ; Veverka, Václav (advisor) ; Hrabal, Richard (referee) ; Hlouchová, Klára (referee)
Protein-protein interactions are involved in various biological processes and detailed characterization of their structural basis by the means of structural biology is often instrumental for rigorous understanding of underlying molecular mechanisms. This information is important not only for fundamental biology but also plays an important role in search for sites amenable for therapeutic intervention. Nuclear magnetic resonance spectroscopy is alongside X-ray crystallography and single-particle cryo-electron microscopy one of the key high-resolution techniques in structural biology. Although its applicability to larger systems has a well-known physical limit, it offers unique capabilities in addressing highly dynamic or inherently heterogeneous systems. In this doctoral thesis, the solution-based NMR approach was used for detailed structural characterization of selected biologically important proteins and their complexes that provided important insights into their biological roles. In three distinct projects, I (i) studied the relationship between the structural effects of particular modifications in the insulin-like growth factor II (IGF-II) and their selectivity to the insulin axis receptors; (ii) the specific binding mechanism of the SH3 domain from the Crk-associated substrate (CAS); (iii) and...
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Protein domains utilizable for development of binding molecules
Dobešová, Petra ; Malý, Petr (advisor) ; Veverka, Václav (referee)
Small protein domains represent basic building blocks of naturally occurring proteins. Many of them exhibit excellent stability, lack disulfide bonds and their structures, therefore, represent attractive tools for generation of artificial binding molecules. First step in the production of novel binding proteins is the definition of a basic domain structure, called "scaffold", which is identified using in silico approaches, resulting in discovery of mutable amino acid residues. Then, randomization of such residues leads to design of a highly complex combinatorial library as a key tool for targeted selection of protein variants. Based on chosen selection approach, the particular protein variants can be tested for their ability to recognize the target molecule with high specificity and binding affinity. Small binding proteins lack post-translation modifications, exhibit thermal stability, are resistant to many organic solvents and can be produced on a mass scale in bacterial host cells. In addition, they can be easily modified and used in vivo with excellent tissue penetration. Due to these beneficial properties, small artificial binding proteins are extraordinary useful biotechnological tools and represent a promising alternative to monoclonal antibodies. The aim of this work is to summarize our knowledge on...
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