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Physiological and pathophysiological aspects of hydrogen sulfide metabolism in humans
Neminářová, Kristýna ; Kožich, Viktor (advisor) ; Tolde, Ondřej (referee)
Hydrogen sulfide (H2S), known as a toxic gas for a long time, was recently shown to be an important signaling molecule. Hydrogen sulfide is produced in small concentration in organism and exhibits a physiological role in many tissues (brain, blood vessels, lungs). Hydrogen sulfide is mostly formed enzymatically from L-cysteine by two enzymes - cystathionin β-synthase (CBS, EC 4.2.1.22, L- serine hydro-lyase) occurring especially in the brain and cystathionine γ-lyase (CSE, EC 4.4.1.1, L- cystathionine cysteine-lyase) generating hydrogen sulfide mainly in the small intestine, portal vein and thoracic aorta. In vessels hydrogen sulfide acts as a vasorelaxant factor and reduces blood pressure while in the brain it is involved in neuronal transmission. In addition to these effects, hydrogen sulfide plays a role in inflammatory processes as well as in the transmission of pain. Hydrogen sulfide acts through activation of KATP channel (in blood vessels, digestive tract, and in inflammation), activation of NMDA receptors (brain), reduces molecules causing oxidative stress (lungs, brain) and affects influx of Ca2+ ions into the cells (retina). Based on these findings it is apparent that the modulations of metabolism of hydrogen sulfide may have a therapeutic potential, e.g. in vascular disease or in...
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Molecular mechanisms in homocystinuria: spatial arrangement of human cystathionine β-synthase
Hnízda, Aleš ; Kožich, Viktor (advisor) ; Holada, Karel (referee) ; Jiráček, Jiří (referee)
Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...
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Regulatory mechanisms of ornithin transcarbamylase and beta-glucocerebrosidase gene expression and their relevance to diagnostics
Lukšan, Ondřej ; Jirsa, Milan (advisor) ; Kožich, Viktor (referee) ; Kříž, Vítězslav (referee)
5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...
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Physiological and pathophysiological aspects of hydrogen sulfide metabolism in humans
Neminářová, Kristýna ; Kožich, Viktor (advisor) ; Tolde, Ondřej (referee)
Hydrogen sulfide (H2S), known as a toxic gas for a long time, was recently shown to be an important signaling molecule. Hydrogen sulfide is produced in small concentration in organism and exhibits a physiological role in many tissues (brain, blood vessels, lungs). Hydrogen sulfide is mostly formed enzymatically from L-cysteine by two enzymes - cystathionin β-synthase (CBS, EC 4.2.1.22, L- serine hydro-lyase) occurring especially in the brain and cystathionine γ-lyase (CSE, EC 4.4.1.1, L- cystathionine cysteine-lyase) generating hydrogen sulfide mainly in the small intestine, portal vein and thoracic aorta. In vessels hydrogen sulfide acts as a vasorelaxant factor and reduces blood pressure while in the brain it is involved in neuronal transmission. In addition to these effects, hydrogen sulfide plays a role in inflammatory processes as well as in the transmission of pain. Hydrogen sulfide acts through activation of KATP channel (in blood vessels, digestive tract, and in inflammation), activation of NMDA receptors (brain), reduces molecules causing oxidative stress (lungs, brain) and affects influx of Ca2+ ions into the cells (retina). Based on these findings it is apparent that the modulations of metabolism of hydrogen sulfide may have a therapeutic potential, e.g. in vascular disease or in...
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Regulatory mechanisms of ornithin transcarbamylase and beta-glucocerebrosidase gene expression and their relevance to diagnostics
Lukšan, Ondřej ; Jirsa, Milan (advisor) ; Kožich, Viktor (referee) ; Kříž, Vítězslav (referee)
5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...
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The metabolism and signaling of hydrogen sulfide: the role of CBS-related proteins in Caenorhabditis elegans
Vozdek, Roman ; Kožich, Viktor (advisor) ; Macůrková, Marie (referee) ; Jiráček, Jiří (referee)
Hydrogen sulfide (H2S) is a toxic gas that causes respiratory failure and death at high concentrations, but at low concentrations, it functions as a signaling molecule in vasodilation and neuromodulation, and it protects cells and tissues from reperfusion injury, hypoxia, hyperglycemia and endothelial dysfunction. Several model organisms have been used to study the physiological roles and signaling pathways of H2S. The roundworm Caenorhabditis elegans is a remarkable model for studying the physiology, developmental biology and signaling of H2S; however, the metabolism of H2S in this animal is largely unknown. Cystathionine beta-synthase (CBS) is one of three H2S-producing enzymes in mammals. Notably, C. elegans possesses 6 genes that encode proteins homologous to CBS, namely cbs- 1, cbs-2, cysl-1, cysl-2, cysl-3 and cysl-4. In this thesis we studied the roles of these genes in H2S metabolism and signaling. First, we identified cbs-1 as the gene encoding CBS in C. elegans; the recombinant purified CBS-1 protein exhibited canonical CBS activity, and RNA interference-mediated silencing of cbs-1 resulted in decreased CBS activity and increased homocysteine levels in worm extracts, recapitulating the phenotypes of CBS deficiency in mammals. Notably, the nematode and human enzymes differ in their domain...
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Study of physiological functions of betaine homocysteine S-methyltransferase and betaine homocysteine S-methyltransferase 2
Mládková, Jana ; Jiráček, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kožich, Viktor (referee)
Betaine homocysteine S-methyltransferase (BHMT) and betaine homocysteine S-methyltransferase 2 (BHMT-2) are mammalian cytosolic metalloenzymes. They both participate in the metabolism of homocysteine (Hcy), specifically Hcy remethylation, mainly in liver and kidney cells. BHMT catalyzes the transfer of a methyl group from betaine to L-Hcy, yielding L-methionine and dimethylglycine (DMG). BHMT-2 catalyzes the transfer of a methyl group from S-methylmethionine (SMM) to L-Hcy as well, yielding two molecules of L-methionine. Disorders in Hcy metabolism could lead to the so called hyper- homocysteinemia and homocystinuria, which can be connected with several pathological conditions. BHMT is already relatively well characterized enzyme. Its crystal structure and reaction mechanism have been described and a series of BHMT inhibitors have been prepared. The specific inhibitors enabled further in vivo studies and, recently, Bhmt-/- mice model has been successfully developed. In contrast, the research of BHMT-2 is still at the beginning and physiological functions of the enzyme are unknown so far. The reason is that BHMT-2 is a highly unstable enzyme and also there is a lack of selective BHMT-2 inhibitors. BHMT and BHMT-2 are very similar enzymes which have 73% amino acid identity. This thesis provides new...
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Molecular mechanisms in homocystinuria: spatial arrangement of human cystathionine β-synthase
Hnízda, Aleš ; Kožich, Viktor (advisor) ; Holada, Karel (referee) ; Jiráček, Jiří (referee)
Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...
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