Metabolismus a signalizace sirovodíku: úloha proteinů příbuzných k CBS u Caenorhabditis elegans

Sirovodík (H2S) je toxický plyn, který ve vysokých koncentracích způsobuje respirační selhání a smrt organismu, zatímco v nízkých koncentracích hraje roli jako vasodilatátor, neuromodulátor, a chrání buňky a tkáně před reperfuzním poškozením, hypoxií, hyperglykémií či dysfunkcí endotelu. Ke studiu fyziologie a signalizace H2S je využíváno hned několik modelových organismů. Hlístice Caenorhabditis elegans je pozoruhodným modelem ke studiu fyziologie, vývojové biologie, a v neposlední řadě i signalizace H2S, nicméně metabolismus sirovodíku v tomto organismu není známý. Cystathionin-beta-synthasa (CBS) je jedním ze tří enzymů produkujících H2S u živočichů. Zajímavé je, že u C. elegans je postulováno hned šest genů kódující homologní proteiny k CBS (cbs-1, cbs-2, cysl-1, cysl -2, cysl -3 a cysl -4). Cílem této práce bylo zjistit funkci těchto genů v metabolismu a signalizaci H2S u C. elegans. Nejprve jsme identifikovali cbs-1 jako gen kódující CBS; rekombinantní purifikovaný protein CBS-1 vykázal CBS aktivitu a RNA interference cbs-1 vedla ke snížené CBS aktivitě a zvýšené hladině homocysteinu v červích homogenátech, což rekapituluje deficit CBS u savců. Je zajímavé, že hlístí a savčí CBS mají odlišnou doménovou architekturu a tedy i posttranslační regulaci. Dále jsme zjistili, že ostatní proteiny...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...Institute of Inherited Metabolic Disorders First Faculty of Medicine Charles University in PragueÚstav dědičných metabolických poruch 1.LF a VFN v PrazeFirst Faculty of Medicine1. lékařská fakult

Conserved roles of C. elegans and human MANFs in sulfatide binding and cytoprotection.

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

Novel structural arrangement of nematode cystathionine β-synthases: characterization of Caenorhabditis elegans CBS-1

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

The metabolism and signaling of hydrogen sulfide: the role of CBS-related proteins in Caenorhabditis elegans

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..

The receptor tyrosine kinase HIR-1 coordinates HIF-independent responses to hypoxia and extracellular matrix injury

Inadequate tissue oxygen, or hypoxia, is a central concept in the pathophysiology of ischemic disorders and cancer. Hypoxia promotes extracellular matrix (ECM) remodeling, cellular metabolic adaptation, and cancer cell metastasis. To discover new pathways through which cells respond to hypoxia, we performed a large-scale forward genetic screen in Caenorhabditis elegans and identified a previously uncharacterized receptor tyrosine kinase named HIR-1. Loss of function in hir-1 phenocopied the impaired ECM integrity associated with hypoxia or deficiency in the oxygen-dependent dual oxidase, heme peroxidases, or cuticular collagens involved in ECM homeostasis. Genetic suppressor screens identified NHR-49 and MDT-15 as transcriptional regulators downstream of HIR-1. Furthermore, hir-1 mutants showed defects in adapting to and recovering from prolonged severe hypoxia. We propose that C. elegans HIR-1 coordinates hypoxia-inducible factor-independent responses to hypoxia and hypoxia-associated ECM remodeling through mechanisms that are likely conserved in other organisms

Fluorescent reporter of Caenorhabditis elegans Parkin: Regulators of its abundance and role in autophagy-lysosomal dynamics [version 2; peer review: 2 approved]

Background: Parkin, which when mutated leads to early-onset Parkinson’s disease, acts as an E3 ubiquitin ligase. How Parkin is regulated for selective protein and organelle targeting is not well understood. Here, we used protein interactor and genetic screens in Caenorhabditis elegans (C. elegans) to identify new regulators of Parkin abundance and showed their impact on autophagy-lysosomal dynamics and alpha-Synuclein processing. Methods: We generated a transgene encoding mCherry-tagged C. elegans Parkin – Parkinson’s Disease Related 1 (PDR-1). We performed protein interactor screen using Co-immunoprecipitation followed by mass spectrometry analysis to identify putative interacting partners of PDR-1. Ribonucleic acid interference (RNAi) screen and an unbiased mutagenesis screen were used to identify genes regulating PDR-1 abundance. Confocal microscopy was used for the identification of the subcellular localization of PDR-1 and alpha-Synuclein processing. Results: We show that the mCherry::pdr-1 transgene rescues the mitochondrial phenotype of pdr-1 mutants and that the expressed PDR-1 reporter is localized in the cytosol with enriched compartmentalization in the autophagy-lysosomal system. We determined that the transgenic overexpression of the PDR-1 reporter, due to inactivated small interfering RNA (siRNA) generation pathway, disrupts autophagy-lysosomal dynamics. From the RNAi screen of putative PDR-1 interactors we found that the inactivated Adenine Nucleotide Translocator ant-1.1/hANT, or hybrid ubiquitin genes ubq-2/hUBA52 and ubl-1/hRPS27A encoding a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively, induced PDR-1 abundance and affected lysosomal dynamics. In addition, we demonstrate that the abundant PDR-1 plays a role in alpha-Synuclein processing. Conclusions: These data show that the abundant reporter of C. elegans Parkin affects the autophagy-lysosomal system together with alpha-Synuclein processing which can help in understanding the pathology in Parkin-related diseases

Conserved roles of C. elegans and human MANFs in sulfatide binding and cytoprotection.

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