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Structural studies of metal-dependent hydrolases: Histone Deacetylase 6 and Glutamate Carboxypeptidase II
Shukla, Shivam ; Bařinka, Cyril (advisor) ; Stříšovský, Kvido (referee) ; Kolenko, Petr (referee)
Zinc-dependent hydrolases are a class of metalloenzymes that require zinc ions to catalyse hydrolytic reactions. Structural studies of these enzymes shall provide detailed information about the processing of their natural substrates, domain organization, and overall structural fold. This thesis describes the structural properties of two different metallohydrolases 1) human histone deacetylase 6 (HDAC6) and 2) glutamate carboxypeptidase II (GCPII) by utilizing a different set of biophysical techniques. HDAC6 is a structurally unique multidomain enzyme comprised of unstructured and globular domains. It regulates the plethora of cellular processes by removing an acetyl group from lysine side chains of target proteins. It has been known to deacetylate non-histone substrates such as tubulin, Hsp90, cortactin, and peroxiredoxins. Given its structural complexity, complete structural information of full-length HDAC6 is missing and available information is limited to its globular domains only. Hence, the integrative approach was employed in combining experimental data from several orthogonal biophysical techniques to build an in-solution structural model of HDAC6. The study reports that HDAC6 adopts multiple conformations due to its unstructured regions and exists as an ensemble of conformers in solution....
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Preparation of the transcription factor FOXK1 DNA binding domain
Procházková, Valérie ; Novák, Petr (advisor) ; Košek, Dalibor (referee)
Transcription factors are proteins that regulate gene expression in different cell types. They play an important role in many cellular processes including regulation of cell cycle and cell differentiation. They possess DNA binding domains to recognize and bind specific DNA sequences. One type of DNA binding domain is the forkhead domain, which contains a region of 100-110 amino acid residues. This sequence is referred to DBD FOX and its spatial arrangement resembles a "winged helix". Proteins of the FOX family interact with double- stranded DNA via the α-helix H3, which represents highly conserved region within the proteins of this family. Other regions of the DBD further contribute in DNA binding, but as not significantly conserved, and their different properties are responsible for variable affinities of individual FOX proteins against binding motifs. Differences in three-dimensional structure may also alter biological functions of FOX proteins in the organism. FOX proteins are divided into 19 subfamilies, including the FOXK subfamily, consisting of two members, FOXK1 and FOXK2. FOXK proteins regulate aerobic glycolysis, cell proliferation and carcinogenesis. Their increased expression has been reported in cancer cells of skeletal tissue, stomach, colon, breast, lung, ovary, etc. However, the...
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Structural studies of LEDGF/p75 interactions
Těšina, Petr
3 ABSTRACT LEDGF/p75 protein is a human transcriptional co-activator and epigenetic reader associated with transcriptionally active chromatin. It is crucial for HIV integration and MLL1 fusion-driven leukemia development. Interactions of LEDGF/p75 with HIV integrase (HIV IN) and MLL1-menin complex are considered an attractive therapeutic target for drug development. LEDGF/p75 interacts with both HIV IN and MLL1-menin complex through its integrase binding domain (IBD). While the pathophysiological interactions of LEDGF/p75 IBD were intensively studied, little was known about the physiological ones. In addition to HIV IN and MLL1, the LEDGF/p75 IBD also interacts with JPO2, PogZ, ASK and MLL2. In search for specific inhibitors of LEDGF/p75 IBD interaction with HIV IN and MLL1, it is essential to obtain detailed information about its interactions with all binding partners. The IBD-MLL1-menin complex has been structurally characterized, but only partially. Using NMR spectroscopy, we identified and mapped a novel part of the IBD-MLL1 interface. This additional interface is able to maintain the interaction between LEDGF/p75 and MLL1 even without the presence of menin, which was considered necessary. Moreover, colony forming assays of primary leukemic blasts revealed that this additional interface is essential for...
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Structural studies of LEDGF/p75 interactions
Těšina, Petr
3 ABSTRACT LEDGF/p75 protein is a human transcriptional co-activator and epigenetic reader associated with transcriptionally active chromatin. It is crucial for HIV integration and MLL1 fusion-driven leukemia development. Interactions of LEDGF/p75 with HIV integrase (HIV IN) and MLL1-menin complex are considered an attractive therapeutic target for drug development. LEDGF/p75 interacts with both HIV IN and MLL1-menin complex through its integrase binding domain (IBD). While the pathophysiological interactions of LEDGF/p75 IBD were intensively studied, little was known about the physiological ones. In addition to HIV IN and MLL1, the LEDGF/p75 IBD also interacts with JPO2, PogZ, ASK and MLL2. In search for specific inhibitors of LEDGF/p75 IBD interaction with HIV IN and MLL1, it is essential to obtain detailed information about its interactions with all binding partners. The IBD-MLL1-menin complex has been structurally characterized, but only partially. Using NMR spectroscopy, we identified and mapped a novel part of the IBD-MLL1 interface. This additional interface is able to maintain the interaction between LEDGF/p75 and MLL1 even without the presence of menin, which was considered necessary. Moreover, colony forming assays of primary leukemic blasts revealed that this additional interface is essential for...
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Structural studies of LEDGF/p75 interactions
Těšina, Petr ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee) ; Spiwok, Vojtěch (referee)
3 ABSTRACT LEDGF/p75 protein is a human transcriptional co-activator and epigenetic reader associated with transcriptionally active chromatin. It is crucial for HIV integration and MLL1 fusion-driven leukemia development. Interactions of LEDGF/p75 with HIV integrase (HIV IN) and MLL1-menin complex are considered an attractive therapeutic target for drug development. LEDGF/p75 interacts with both HIV IN and MLL1-menin complex through its integrase binding domain (IBD). While the pathophysiological interactions of LEDGF/p75 IBD were intensively studied, little was known about the physiological ones. In addition to HIV IN and MLL1, the LEDGF/p75 IBD also interacts with JPO2, PogZ, ASK and MLL2. In search for specific inhibitors of LEDGF/p75 IBD interaction with HIV IN and MLL1, it is essential to obtain detailed information about its interactions with all binding partners. The IBD-MLL1-menin complex has been structurally characterized, but only partially. Using NMR spectroscopy, we identified and mapped a novel part of the IBD-MLL1 interface. This additional interface is able to maintain the interaction between LEDGF/p75 and MLL1 even without the presence of menin, which was considered necessary. Moreover, colony forming assays of primary leukemic blasts revealed that this additional interface is essential for...
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