Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) protein that can be secreted and protects dopamine neurons and cardiomyocytes from ER stress and apoptosis. The mechanism of action of extracellular MANF has long been elusive. From a genetic screen for mutants with abnormal ER stress response, we identified the gene Y54G2A.23 as the evolutionarily conserved C. elegans MANF orthologue. We find that MANF binds to the lipid sulfatide, also known as 3-O-sulfogalactosylceramide present in serum and outer-cell membrane leaflets, directly in isolated forms and in reconstituted lipid micelles. Sulfatide binding promotes cellular MANF uptake and cytoprotection from hypoxia-induced cell death. Heightened ER stress responses of MANF-null C. elegans mutants and mammalian cells are alleviated by human MANF in a sulfatide-dependent manner. Our results demonstrate conserved roles of MANF in sulfatide binding and ER stress response, supporting sulfatide as a long-sought lipid mediator of MANF's cytoprotection.
- MeSH
- Caenorhabditis elegans MeSH
- cytoprotekce * MeSH
- HEK293 buňky MeSH
- lidé MeSH
- metabolismus lipidů MeSH
- neurotrofní faktory metabolismus MeSH
- proteiny Caenorhabditis elegans metabolismus MeSH
- stres endoplazmatického retikula * MeSH
- sulfoglykosfingolipidy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Two enzymes in the transsulfuration pathway of homocysteine -cystathionine beta-synthase (CBS) and gamma-cystathionase (CTH)-use cysteine and/or homocysteine to produce the important signaling molecule hydrogen sulfide (H2S) and simultaneously the thioethers lanthionine, cystathionine or homolanthionine. In this study we explored whether impaired flux of substrates for H2S synthesis and/or deficient enzyme activities alter production of hydrogen sulfide in patients with homocystinurias. As an indirect measure of H2S synthesis we determined by LC-MS/MS concentrations of thioethers in plasma samples from 33 patients with different types of homocystinurias, in 8 patient derived fibroblast cell lines, and as reaction products of seven purified mutant CBS enzymes. Since chaperoned recombinant mutant CBS enzymes retained capacity of H2S synthesis in vitro it can be stipulated that deficient CBS activity in vivo may impair H2S production. Indeed, in patients with classical homocystinuria we observed significantly decreased cystathionine and lanthionine concentrations in plasma (46% and 74% of median control levels, respectively) and significantly lower cystathionine in fibroblasts (8% of median control concentrations) indicating that H2S production from cysteine and homocysteine may be also impaired. In contrast, the grossly elevated plasma levels of homolanthionine in CBS deficient patients (32-times elevation compared to median of controls) clearly demonstrates a simultaneous overproduction of H2S from homocysteine by CTH. In the remethylation defects the accumulation of homocysteine and the increased flux of metabolites through the transsulfuration pathway resulted in elevation of cystathionine and homolanthionine (857% and 400% of median control values, respectively) indicating a possibility of an increased biosynthesis of H2S by both CBS and CTH. This study shows clearly disturbed thioether concentrations in homocystinurias, and modeling using these data indicates that H2S synthesis may be increased in these conditions. Further studies are needed to confirm our findings and to explore the possible implications for pathophysiology of these disorders.
- MeSH
- alanin analogy a deriváty metabolismus MeSH
- cystathionin-beta-synthasa metabolismus MeSH
- cystathionin metabolismus MeSH
- fibroblasty metabolismus patologie MeSH
- homocystinurie metabolismus patologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- sulfan metabolismus MeSH
- sulfidy metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
O-Acetylserine(thiol)lyases (OAS-TLs) play a pivotal role in a sulfur assimilation pathway incorporating sulfide into amino acids in microorganisms and plants, however, these enzymes have not been found in the animal kingdom. Interestingly, the genome of the roundworm Caenorhabditis elegans contains three expressed genes predicted to encode OAS-TL orthologs (cysl-1-cysl-3), and a related pseudogene (cysl-4); these genes play different roles in resistance to hypoxia, hydrogen sulfide and cyanide. To get an insight into the underlying molecular mechanisms we purified the three recombinant worm OAS-TL proteins, and we determined their enzymatic activities, substrate binding affinities, quaternary structures and the conformations of their active site shapes. We show that the nematode OAS-TL orthologs can bind O-acetylserine and catalyze the canonical reaction although this ligand may more likely serve as a competitive inhibitor to natural substrates instead of being a substrate for sulfur assimilation. In addition, we propose that S-sulfocysteine may be a novel endogenous substrate for these proteins. However, we observed that the three OAS-TL proteins are conformationally different and exhibit distinct substrate specificity. Based on the available evidences we propose the following model: CYSL-1 interacts with EGL-9 and activates HIF-1 that upregulates expression of genes detoxifying sulfide and cyanide, the CYSL-2 acts as a cyanoalanine synthase in the cyanide detoxification pathway and simultaneously produces hydrogen sulfide, while the role of CYSL-3 remains unclear although it exhibits sulfhydrylase activity in vitro. All these data indicate that C. elegans OAS-TL paralogs have distinct cellular functions and may play different roles in maintaining hydrogen sulfide homeostasis.
- MeSH
- buněčné jádro chemie enzymologie genetika MeSH
- Caenorhabditis elegans enzymologie genetika MeSH
- cysteinsynthasa chemie genetika metabolismus MeSH
- homeostáza fyziologie MeSH
- katalytická doména MeSH
- kyanidy metabolismus MeSH
- proteiny Caenorhabditis elegans chemie genetika metabolismus MeSH
- serin analogy a deriváty chemie genetika metabolismus MeSH
- substrátová specifita MeSH
- sulfan chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins.
- MeSH
- biokatalýza MeSH
- Caenorhabditis elegans enzymologie MeSH
- cystathionin-beta-synthasa chemie genetika metabolismus MeSH
- cytoplazma enzymologie MeSH
- homeostáza MeSH
- homocystein metabolismus MeSH
- konzervovaná sekvence MeSH
- kvarterní struktura proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- orgánová specificita MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- terciární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
