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Structural and functional characterization of the E3 ligase BIRC6
GRATZL, Sascha
The aim of the thesis was to characterize the ubiquitin ligase BIRC6 in several contexts. The goal was to expand the knowledge on the selectivity of E1-E2 recognition, to increase the known substrate range of BIRC6, and to elucidate the molecular interactions fulfilled by BIRC6 during autophagy via interaction with GABARAP. In addition, it was aimed to gain a better understanding on the general ubiquitination mechanism in E2/E3 hybrid enzymes, to identify the ubiquitination sites of known apoptotic and autophagic substrates and to enlighten substrate recognition by BIRC6.
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Structure-assisted design of inhibitors targeting medicinally relevant enzymes
Djukic, Stefan ; Maloy Řezáčová, Pavlína (advisor) ; Kutá-Smatanová, Ivana (referee) ; Kolenko, Petr (referee)
Structure-assisted drug discovery is a powerful approach that utilizes detailed knowledge on 3D structure to design and optimize new inhibitors targeting medically relevant enzymes. X-ray crystallography is a widely used structural biology technique since it provides detailed snapshot of protein-inhibitor complex, which is used to analyze protein- inhibitor interactions. PNP plays an important role in salvage pathway of purine metabolism, it is a target in treatment of T-cell malignancies and/or parasitic infections. Our effort focused on human and M. tuberculosis PNP, and our aim was to develop new inhibitors with high selectivity and specificity. Our inhibitors are acyclic nucleoside phosphates with 9- deazahypoxanthine nucleobase that contain three moieties binding to all three regions of the active site: purine, phenyl and phosphonate moieties. The best inhibitors have IC50 values as low as 19 nM (human) and 4 nM (M. tuberculosis). The presence of short substituents at central phenyl moiety, such as methoxy and bromide group, decreases inhibitor's affinity towards human PNP, but does not affect affinity towards mycobacterial PNP. At the same time, bulky substituents, such as fluorinated phenyl ring, decrease inhibitor's affinity towards human PNP but increase affinity towards mycobacterial...
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Structural and functional studies of MICAL signalling in cytoskeletal dynamics
Rožová, Dominika ; Rozbeský, Daniel (advisor) ; Sulimenko, Vadym (referee)
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis...
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Regulation of purine nucleotide metabolism as a pharmacological target
Brinsa, Vítězslav ; Maloy Řezáčová, Pavlína (advisor) ; Hlouchová, Klára (referee)
Purine nucleotides are essential basic building blocks for DNA and RNA synthesis. They can also serve as energy storage and transfer unit and play an important role in cell signalling and regulation of variety of biochemical processes. It is crucial for the cells to maintain a sufficient supply of purine nucleotides in order to secure its survival and cell division. Level of purine nucleotide pool in the human body is regulated via purine nucleotide metabolism, which consists of three coordinated processes: de novo synthesis pathway, salvage pathway and degradation pathway of purine nucleotides. Regulation of those three pathways is under control of various mechanisms including regulation on the level of enzyme expression, allosteric regulation of enzyme activity or forming a multienzime complexes, i. e. purinosomes in the de novo synthesis pathway. Phosphoribosyl pyrophosphate synthetase I (PRS-I) and cytosolic purine 5'-nucleotidase (cN-II) play an important role in purine nucleotide metabolism. These enzymes contribute significantly to the purine nucleotide pool regulation by means of their allostericaly regulated activity. Malfunctions of their catalytic activity are connected with various pathologies such as gout, hyperuricosuria, neurological dysfunctions and acute lymphoblastic leukaemia...
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Structural studies of transcription factors implicated in regulation of metabolism of pancreatic beta cells
Duchoslav, Vojtěch ; Maloy Řezáčová, Pavlína (advisor) ; Vaněk, Ondřej (referee)
Nkx6.1 is a homeodomain protein (37.8 kDa) and an important transcription factor, which regulates transcription of key genes in pancreatic ß-cells. Insufficient expression of this protein leads to reduced glucose uptake from blood as a consequence of suppressed transcription of the glucose transporter Glut2 and impaired glucose metabolism. Furthermore, the proliferation of pancreatic ß-cells is suppressed due to insufficient transcription of Cyclin D2, a protein regulating the mitosis. Moreover, the biosynthesis of insulin is impaired duet he diminished transcription of the genes coding for Ero1lb a Slc30a8, which as a consequence leads to reduced production of the mature insulin. Nkx6.1 could play a role in the pathogenesis of the type 2 diabetes , where ß-cells show diminished ability to compensate high demand for insulin. This malfunction is the cause of an insufficient ability to secrete insulin and death of pancreatic cells. Perhaps driven by misregulation of transcription of the genes that are involved in the mentioned processes. Nkx6.1 recognizes a strictly conserved 8-base pair DNA sequence (TTAATTAC). Its binding to DNA is regulated by an acidic domain at the C-terminus. Within the bachelor thesis, the resonances were assigned to the backbone atoms of the Nkx6.1 protein using nuclear...
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Biophysical characterization of the CAR/RXR protein complex with its binding partners
Duchoslav, Vojtěch ; Bouřa, Evžen (advisor) ; Pavlíček, Jiří (referee)
The constitutive androstane receptor (CAR) plays an important role as a xenosensor in the organism and is therefore widely expressed in the kidney, liver, gallbladder and in the small intestine epithelium, where the biotransformation of xenobiotic occurs. CAR is also an important factor in the elimination of bile acids and bilirubin. CAR has also been shown to have an important role in regulation of glucose and lipid metabolism. Impairment of lipid and glucose metabolism is a common cause of cardiovascular and metabolic diseases such as atherosclerosis, type 2 diabetes, obesity and insulin resistance. These diseases are called metabolic syndrome and result in severe organ damage. CAR respective its complex with RXRα (retinoid X receptor α) has become a promising biological target for drug discovery for metabolic syndrome. The major aim of this study was a structural characterization of the CAR/RXRα protein complex together with the agonist IV676*HCl, which would describe in detail the interaction of this small molecule with the receptor. Obtained structural information would be used to design improved agonists.
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Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes
Zeman, Jakub
In eukaryotic translation, eukaryotic initiation factors (eIFs) are at least as important as the ribosome itself. Some of these factors play different roles throughout the entire process to ensure proper assembly of the preinitiation complex on mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most important factor integrating signals from others and coordinating their functions on the ribosome is eIF3. In Saccharomyces cerevisiae, eIF3 is formed by five subunits. All these subunits contain structural motifs responsible for contact with ribosomal proteins and RNAs. In addition to these highly structured parts, the rest of eIF3 is unstructured and very flexible. Therefore, despite the recent progress thanks to the use of a cryo-electron microscopy, a precise structure and position of eIF3 on the 40S ribosomal subunit are still not known. Also, the presence of eIF3 on 80S during early elongation and its role in reinitiation and readthrough are not fully understood. In order to crack mysteries of yeast eIF3, we used x-ray crystallography, chemical cross- linking coupled to mass spectrometry, and various biochemical and genetic assays. We demonstrated that eIF3 is very compactly packed when free in solution. This...
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Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes
Zeman, Jakub
In eukaryotic translation, eukaryotic initiation factors (eIFs) are at least as important as the ribosome itself. Some of these factors play different roles throughout the entire process to ensure proper assembly of the preinitiation complex on mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most important factor integrating signals from others and coordinating their functions on the ribosome is eIF3. In Saccharomyces cerevisiae, eIF3 is formed by five subunits. All these subunits contain structural motifs responsible for contact with ribosomal proteins and RNAs. In addition to these highly structured parts, the rest of eIF3 is unstructured and very flexible. Therefore, despite the recent progress thanks to the use of a cryo-electron microscopy, a precise structure and position of eIF3 on the 40S ribosomal subunit are still not known. Also, the presence of eIF3 on 80S during early elongation and its role in reinitiation and readthrough are not fully understood. In order to crack mysteries of yeast eIF3, we used x-ray crystallography, chemical cross- linking coupled to mass spectrometry, and various biochemical and genetic assays. We demonstrated that eIF3 is very compactly packed when free in solution. This...
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Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes
Zeman, Jakub ; Valášek, Leoš (advisor) ; Štefl, Richard (referee) ; Man, Petr (referee)
In eukaryotic translation, eukaryotic initiation factors (eIFs) are at least as important as the ribosome itself. Some of these factors play different roles throughout the entire process to ensure proper assembly of the preinitiation complex on mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most important factor integrating signals from others and coordinating their functions on the ribosome is eIF3. In Saccharomyces cerevisiae, eIF3 is formed by five subunits. All these subunits contain structural motifs responsible for contact with ribosomal proteins and RNAs. In addition to these highly structured parts, the rest of eIF3 is unstructured and very flexible. Therefore, despite the recent progress thanks to the use of a cryo-electron microscopy, a precise structure and position of eIF3 on the 40S ribosomal subunit are still not known. Also, the presence of eIF3 on 80S during early elongation and its role in reinitiation and readthrough are not fully understood. In order to crack mysteries of yeast eIF3, we used x-ray crystallography, chemical cross- linking coupled to mass spectrometry, and various biochemical and genetic assays. We demonstrated that eIF3 is very compactly packed when free in solution. This...
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Structural studies of transcription factors implicated in regulation of metabolism of pancreatic beta cells
Duchoslav, Vojtěch ; Maloy Řezáčová, Pavlína (advisor) ; Vaněk, Ondřej (referee)
Nkx6.1 is a homeodomain protein (37.8 kDa) and an important transcription factor, which regulates transcription of key genes in pancreatic ß-cells. Insufficient expression of this protein leads to reduced glucose uptake from blood as a consequence of suppressed transcription of the glucose transporter Glut2 and impaired glucose metabolism. Furthermore, the proliferation of pancreatic ß-cells is suppressed due to insufficient transcription of Cyclin D2, a protein regulating the mitosis. Moreover, the biosynthesis of insulin is impaired duet he diminished transcription of the genes coding for Ero1lb a Slc30a8, which as a consequence leads to reduced production of the mature insulin. Nkx6.1 could play a role in the pathogenesis of the type 2 diabetes , where ß-cells show diminished ability to compensate high demand for insulin. This malfunction is the cause of an insufficient ability to secrete insulin and death of pancreatic cells. Perhaps driven by misregulation of transcription of the genes that are involved in the mentioned processes. Nkx6.1 recognizes a strictly conserved 8-base pair DNA sequence (TTAATTAC). Its binding to DNA is regulated by an acidic domain at the C-terminus. Within the bachelor thesis, the resonances were assigned to the backbone atoms of the Nkx6.1 protein using nuclear...
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