RESUMEN
Parkinson's disease (PD) is associated with various deficits in sensing and responding to reductions in oxygen availability (hypoxia). Here we summarize the evidence pointing to a central role of hypoxia in PD, discuss the relation of hypoxia and oxygen dependence with pathological hallmarks of PD, including mitochondrial dysfunction, dopaminergic vulnerability, and alpha-synuclein-related pathology, and highlight the link with cellular and systemic oxygen sensing. We describe cases suggesting that hypoxia may trigger Parkinsonian symptoms but also emphasize that the endogenous systems that protect from hypoxia can be harnessed to protect from PD. Finally, we provide examples of preclinical and clinical research substantiating this potential.
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Enfermedad de Parkinson , Trastornos Parkinsonianos , Humanos , Enfermedad de Parkinson/patología , alfa-Sinucleína , Trastornos Parkinsonianos/patología , Neuronas Dopaminérgicas/patología , Hipoxia/patología , OxígenoRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Cisteína Sintasa/metabolismo , Homeostasis/fisiología , Sulfuro de Hidrógeno/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Dominio Catalítico , Núcleo Celular/química , Núcleo Celular/enzimología , Núcleo Celular/genética , Cianuros/metabolismo , Cisteína Sintasa/química , Cisteína Sintasa/genética , Sulfuro de Hidrógeno/química , Serina/análogos & derivados , Serina/química , Serina/genética , Serina/metabolismo , Especificidad por SustratoRESUMEN
CBSs (cystathionine ß-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.
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Caenorhabditis elegans/enzimología , Cistationina betasintasa/metabolismo , Secuencia de Aminoácidos , Animales , Biocatálisis , Secuencia Conservada , Cistationina betasintasa/química , Cistationina betasintasa/genética , Citoplasma/enzimología , Homeostasis , Homocisteína/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Especificidad de Órganos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Hydrogen sulfide (H2S), long viewed as a toxic gas and environmental hazard, is emerging as a biological mediator with remarkable physiological and pathophysiological relevance. H2S is now viewed as the third main gasotransmitter in the mammalian body. Its pharmacological characteristic possesses similarities to the other two gasotransmitters - nitric oxide (NO) and carbon monoxide (CO). Many of the biological effects of H2S follow a bell-shaped concentration-response; at low concentration or at lower release rates it has beneficial and cytoprotective effects, while at higher concentrations or fast release rates toxicity becomes apparent. Cellular bioenergetics is a prime example for this bell-shaped dose-response, where H2S, at lower concentrations/rates serves as an inorganic substrate and electron donor for mitochondrial ATP generation, while at high concentration it inhibits mitochondrial respiration by blocking the Complex IV in the mitochondrial electron transport chain. The current review is aimed to focus on the following aspects of H2S biology: 1) a general overview of the general pharmacological characteristics of H2S, 2) a summary of the key H2S-mediated signal transduction pathways, 3) an overview of role of H2S in regulation of cellular bioenergetics, 4) key aspects of H2S physiology in C. elegans (a model system) and, finally 5) the therapeutic potential of H2S donating molecules in various disease states.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Sulfuro de Hidrógeno/química , Transducción de Señal , Adenosina Trifosfato/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/fisiología , Cistationina betasintasa/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Transporte de Electrón , Electrones , Metabolismo Energético , Humanos , Ratones , Mitocondrias/metabolismo , Modelos Biológicos , Ratas , Sulfuros/químicaRESUMEN
Idiopathic Parkinson's disease (PD) is characterized by progressive loss of dopaminergic (DA) neurons during aging. The pathological hallmark of PD is the Lewy body detected in postmortem brain tissue, which is mainly composed of aggregated α-Synuclein (αSyn). However, it is estimated that 90% of PD cases have unknown pathogenetic triggers. Here, we generated a new transgenic Caenorhabditis elegans PD model eraIs1 expressing green fluorescent protein- (GFP-) based reporter of human αSyn in DA neurons, and exhibited a nice readout of the developed αSyn inclusions in DA neurons, leading to their degeneration during aging. Using these animals in a preliminary reverse genetic screening of >100-PD genome-wide association study- (GWAS-) based susceptibility genes, we identified 28 orthologs of C. elegans and their inactivation altered the phenotype of eraIs1; 10 knockdowns exhibited reduced penetrance of αSyn:Venus inclusions formed in the axons of cephalic (CEP) DA neurons, 18 knockdowns exhibited increased penetrance of disrupted CEP dendrite integrity among which nine knockdowns also exhibited disrupted neuronal morphology independent of the expressed αSyn reporter. Loss-of-function alleles of the five identified genes, such as sac-2, rig-6 or lfe-2, unc-43, and nsf-1, modulated the corresponding eraIs1 phenotype, respectively, and supported the RNA interference (RNAi) data. The Western blot analysis showed that the levels of insoluble αSyn:Venus were not correlated with the observed phenotypes in these mutants. However, RNAi of 12 identified modulators reduced the formation of pro-aggregating polyglutamine Q40:YFP foci in muscle cells, suggesting the possible role of these genes in cellular proteotoxicity. Therefore, modulators identified by their associated biological pathways, such as calcium signaling or vesicular trafficking, represent new potential therapeutic targets for neurodegenerative proteopathies and other diseases associated with aging.
RESUMEN
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/h UBA52 and ubl-1/h RPS27A 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.
RESUMEN
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.
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Proteínas de Caenorhabditis elegans/genética , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Hipoxia , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Homeostasis/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
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Proteínas de Caenorhabditis elegans/metabolismo , Citoprotección , Estrés del Retículo Endoplásmico , Factores de Crecimiento Nervioso/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Animales , Caenorhabditis elegans , Células HEK293 , Humanos , Metabolismo de los LípidosRESUMEN
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.
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Alanina/análogos & derivados , Cistationina/metabolismo , Fibroblastos/metabolismo , Homocistinuria/metabolismo , Sulfuro de Hidrógeno/metabolismo , Sulfuros/metabolismo , Alanina/metabolismo , Células Cultivadas , Cistationina betasintasa/metabolismo , Femenino , Fibroblastos/patología , Homocistinuria/patología , Humanos , MasculinoRESUMEN
The C. elegans HIF-1 proline hydroxylase EGL-9 functions as an O(2) sensor in an evolutionarily conserved pathway for adaptation to hypoxia. H(2)S accumulates during hypoxia and promotes HIF-1 activity, but how H(2)S signals are perceived and transmitted to modulate HIF-1 and animal behavior is unknown. We report that the experience of hypoxia modifies a C. elegans locomotive behavioral response to O(2) through the EGL-9 pathway. From genetic screens to identify novel regulators of EGL-9-mediated behavioral plasticity, we isolated mutations of the gene cysl-1, which encodes a C. elegans homolog of sulfhydrylases/cysteine synthases. Hypoxia-dependent behavioral modulation and H(2)S-induced HIF-1 activation require the direct physical interaction of CYSL-1 with the EGL-9 C terminus. Sequestration of EGL-9 by CYSL-1 and inhibition of EGL-9-mediated hydroxylation by hypoxia together promote neuronal HIF-1 activation to modulate behavior. These findings demonstrate that CYSL-1 acts to transduce signals from H(2)S to EGL-9 to regulate O(2)-dependent behavioral plasticity in C. elegans.