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...

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...

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...