RESUMEN
Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. We find that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. We propose that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program.
Asunto(s)
Cistina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ayuno , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Factores de Transcripción/metabolismo , Acetilcoenzima A/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Animales , Autofagia , Ciclo del Ácido Cítrico , Cisteína/metabolismo , Cisteína/farmacología , Citosol/metabolismo , Dieta con Restricción de Proteínas , Drosophila melanogaster/crecimiento & desarrollo , Cuerpo Adiposo/fisiología , Modelos Animales , Transducción de SeñalAsunto(s)
Proteínas de Drosophila/genética , Síndrome Nefrótico/genética , Animales , Drosophila , Riñón , MutaciónRESUMEN
The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm5s2U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Centrosoma/metabolismo , Nucleotidiltransferasas/metabolismo , Sulfurtransferasas/metabolismo , Aconitato Hidratasa/metabolismo , Sistemas CRISPR-Cas , Liasas de Carbono-Azufre/análisis , Centrosoma/ultraestructura , Coenzimas/metabolismo , Células HEK293 , Células HeLa , Humanos , Isocitrato Deshidrogenasa/metabolismo , Metaloproteínas/metabolismo , Cofactores de Molibdeno , Nucleotidiltransferasas/análisis , Nucleotidiltransferasas/genética , Pteridinas/metabolismo , ARN de Transferencia/metabolismo , Sulfito-Oxidasa/metabolismo , Sulfurtransferasas/análisis , Sulfurtransferasas/genéticaRESUMEN
Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, we screened for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, our findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.
Asunto(s)
Proteínas de Drosophila/metabolismo , Neuroglía/metabolismo , Sulfito-Oxidasa/metabolismo , Animales , Astrocitos/metabolismo , Drosophila , Proteínas de Drosophila/genética , Glutamatos/metabolismo , Sulfito-Oxidasa/genética , Sulfitos/metabolismoRESUMEN
Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.
RESUMEN
Assembly of iron-sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by l-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondrial targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved "LYR" motif is essential for the role of ISD11 in forming a stable and active complex with NFS1.
Asunto(s)
Liasas de Carbono-Azufre/química , Proteínas de Unión a Hierro/química , Proteínas Reguladoras del Hierro/química , Proteínas Hierro-Azufre/química , Hierro/química , Mitocondrias/metabolismo , Azufre/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Hierro/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Proteínas Reguladoras del Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mitocondrias/genética , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Transducción de Señal , Azufre/metabolismo , FrataxinaRESUMEN
APOL1 harbors C-terminal sequence variants (G1 and G2), which account for much of the increased risk for kidney disease in sub-Saharan African ancestry populations. Expression of the risk variants has also been shown to cause injury to podocytes and other cell types, but the underlying mechanisms are not understood. We used Drosophila melanogaster and Saccharomyces cerevisiae to help clarify these mechanisms. Ubiquitous expression of the human APOL1 G1 and G2 disease risk alleles caused near-complete lethality in D. melanogaster, with no effect of the G0 nonrisk APOL1 allele, corresponding to the pattern of human disease risk. We also observed a congruent pattern of cellular damage with tissue-specific expression of APOL1. In particular, expression of APOL1 risk variants in D. melanogaster nephrocytes caused cell-autonomous accumulation of the endocytic tracer atrial natriuretic factor-red fluorescent protein at early stages and nephrocyte loss at later stages. We also observed differential toxicity of the APOL1 risk variants compared with the APOL1 nonrisk variants in S. cerevisiae, including impairment of vacuole acidification. Yeast strains defective in endosomal trafficking or organelle acidification but not those defective in autophagy displayed augmented APOL1 toxicity with all isoforms. This pattern of differential injury by the APOL1 risk alleles compared with the nonrisk alleles across evolutionarily divergent species is consistent with an impairment of conserved core intracellular endosomal trafficking processes. This finding should facilitate the identification of cell injury pathways and corresponding therapeutic targets of interest in these amenable experimental platforms.
Asunto(s)
Apolipoproteínas/metabolismo , Apolipoproteínas/fisiología , Muerte Celular/fisiología , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/fisiología , Alelos , Animales , Apolipoproteína L1 , Apolipoproteínas/genética , Drosophila melanogaster/citología , Humanos , Concentración de Iones de Hidrógeno , Lipoproteínas HDL/genética , Transporte de Proteínas , Saccharomyces cerevisiae/citologíaRESUMEN
In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po(lpo)-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po(lpo) allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.
Asunto(s)
Aldehído Oxidasa/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Regulación Enzimológica de la Expresión Génica , Proteínas de Insectos/genética , Aldehído Oxidasa/química , Aldehído Oxidasa/metabolismo , Alelos , Animales , Drosophila melanogaster/química , Evolución Molecular , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Förster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Coenzimas/biosíntesis , Cisteína/metabolismo , Citosol/metabolismo , Metaloproteínas/biosíntesis , Azufre/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Cofactores de Molibdeno , Proteínas Mutantes/metabolismo , Neurospora/enzimología , Nitrato-Reductasa/metabolismo , Nucleotidiltransferasas/metabolismo , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Pteridinas , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , Sulfurtransferasas/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Friedreich ataxia (FRDA) is an inherited neurodegenerative disease caused by frataxin (FXN) deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III), which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.
Asunto(s)
Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patología , Proteínas de Unión a Hierro/metabolismo , Adulto , Anciano de 80 o más Años , Secuencia de Aminoácidos , Línea Celular , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Femenino , Ataxia de Friedreich/genética , Regulación de la Expresión Génica , Humanos , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/genética , Proteínas Hierro-Azufre/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Miocardio/metabolismo , Miocardio/patología , Especificidad de Órganos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , FrataxinaRESUMEN
The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an L-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human L-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from L-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco.