RESUMEN
Plasmodium falciparum invasion of the red blood cell is reliant upon the essential interaction of PfRh5 with the host receptor protein basigin. Basigin exists as part of one or more multiprotein complexes, most notably through interaction with the monocarboxylate transporter MCT1. However, the potential requirement for basigin association with MCT1 and the wider role of basigin host membrane context and lateral protein associations during merozoite invasion has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate the ability to uncouple basigin ectodomain presentation from its transmembrane domain-mediated interactions, including with MCT1. Merozoite invasion of reticulocytes is unaffected by disruption of basigin-MCT1 interaction and by removal or replacement of the basigin transmembrane helix. Therefore, presentation of the basigin ectodomain at the red blood cell surface, independent of its native association with MCT1 or other interactions mediated by the transmembrane domain, is sufficient to facilitate merozoite invasion.
Asunto(s)
Plasmodium falciparum , Simportadores , Plasmodium falciparum/metabolismo , Basigina/genética , Basigina/metabolismo , Eritrocitos/metabolismo , Dominios Proteicos , Simportadores/metabolismoRESUMEN
Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.
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Arginasa , Gripe Humana , Animales , Humanos , Ratones , Arginasa/genética , Arginasa/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Glutamina , Cinética , Pulmón/metabolismo , MamíferosRESUMEN
BACKGROUND AND OBJECTIVE: GLUT1 deficiency syndrome (Glut1DS) is a treatable neurometabolic disease that causes a wide range of neurologic symptoms in children and adults. However, its diagnosis relies on an invasive test, that is, a lumbar puncture (LP) to measure glycorrhachia, and sometimes complex molecular analyses of the SLC2A1 gene. This procedure limits the number of patients able to receive the standard of care. We wished to validate the diagnostic performance of METAglut1, a simple blood test that quantifies GLUT1 on the erythrocyte surface. METHODS: We performed a multicenter validation study in France, involving 33 centers. We studied 2 patient cohorts: a prospective cohort consisting of patients with a clinical suspicion of Glut1DS explored through the reference strategy, that is, LP and analyses of the SLC2A1 gene, and a retrospective cohort that included patients previously diagnosed with Glut1DS. All patients were blind-tested with METAglut1. RESULTS: We analyzed 428 patients in the prospective cohort, including 15 patients newly diagnosed with Glut1DS, and 67 patients in the retrospective cohort. METAglut1 was 80% sensitive and >99% specific for the diagnosis of Glut1DS. Concordance analyses showed a substantial agreement between METAglut1 and glycorrhachia. In the prospective cohort, the positive predictive value of METAglut1 was slightly higher than that of glycorrhachia. METAglut1 succeeded to identify patients with Glut1DS with SCL2A1 mosaicism and variants of unknown significance. DISCUSSION: METAglut1 is an easily performed, robust, and noninvasive diagnostic test for the diagnosis of Glut1DS, which allows wide screening of children and adults, including those with atypical forms of this treatable condition. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that a positive METAglut1 test accurately distinguishes patients with suspected GLUT1 deficiency syndrome from other neurologic syndromes as compared with invasive and genetic testing.
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Errores Innatos del Metabolismo de los Carbohidratos , Adulto , Niño , Humanos , Estudios Retrospectivos , Estudios Prospectivos , Errores Innatos del Metabolismo de los Carbohidratos/diagnóstico , Errores Innatos del Metabolismo de los Carbohidratos/genética , Proteínas de Transporte de Monosacáridos/genéticaRESUMEN
Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.
Asunto(s)
Proteómica , Espermidina , Humanos , Espermidina/metabolismo , Factores de Iniciación de Péptidos/genética , Diferenciación Celular , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1-/- mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis.
Asunto(s)
Adenosina Monofosfato/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Eritropoyesis , Células Madre Hematopoyéticas/metabolismo , Homeostasis , Animales , Tranportador Equilibrativo 1 de Nucleósido/genética , Humanos , Ratones , Ratones NoqueadosRESUMEN
The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.
Asunto(s)
Ácido Ascórbico/metabolismo , Eritropoyesis/genética , Isocitrato Deshidrogenasa/metabolismo , Mitocondrias/metabolismo , Diferenciación Celular , HumanosRESUMEN
Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.
Asunto(s)
Homeostasis , Fosfatos de Inositol/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virales/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Línea Celular , Humanos , Fosfatos de Inositol/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Primary familial brain calcification (PFBC) is a rare neurological disease characterized by deposits of calcium phosphate in the basal ganglia and other regions of the brain. Pathogenic variants in the XPR1/SLC53A1 gene, which encodes the only known inorganic phosphate exporter, cause an autosomal dominant form of PFBC. These variants are typically located in the SPX N-terminal domain of the protein. Here, we characterize three XPR1 variants outside of SPX in three PFBC patients with an apparently sporadic presentation: c.1375C > T p.(R459C), c.1855A > G p.(N619D) and c.1886T > G p.(I629S), with the latter identified as the first XPR1/SLC53A1 de novo mutation to occur in a PFBC proband. When tested in an in vitro physiological complementation assay, the three XPR1 variants were impaired in phosphate export function, although they were normally expressed at the cell surface and could serve as functional receptors for retrovirus entry. Moreover, peripheral blood cells from the p.N619D patient could be assayed ex vivo and displayed significantly impaired phosphate export. Our results establish for the first time the clinical and molecular characteristics of XPR1 variants located outside the SPX domain and assert a direct link between these variants, deficient phosphate export, and PFBC. Moreover, we unveiled new structural features in XPR1 C-terminal domain that play a role in phosphate export and disease.
Asunto(s)
Encefalopatías/patología , Calcinosis/patología , Mutación , Hormonas Peptídicas/genética , Fosfatos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Encefalopatías/genética , Calcinosis/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Linaje , Dominios Proteicos , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
HIV persists in long-lived infected cells that are not affected by antiretroviral treatment. These HIV reservoirs are mainly located in CD4+ T cells, but their distribution is variable in the different subsets. Susceptibility to HIV-1 increases with CD4+ T cell differentiation. We evaluated whether the metabolic programming that supports the differentiation and function of CD4+ T cells affected their susceptibility to HIV-1. We found that differences in HIV-1 susceptibility between naive and more differentiated subsets were associated with the metabolic activity of the cells. Indeed, HIV-1 selectively infected CD4+ T cells with high oxidative phosphorylation and glycolysis, independent of their activation phenotype. Moreover, partial inhibition of glycolysis (1) impaired HIV-1 infection in vitro in all CD4+ T cell subsets, (2) decreased the viability of preinfected cells, and (3) precluded HIV-1 amplification in cells from HIV-infected individuals. Our results elucidate the link between cell metabolism and HIV-1 infection and identify a vulnerability in tackling HIV reservoirs.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/metabolismo , Subgrupos de Linfocitos T/metabolismo , Linfocitos T CD4-Positivos/patología , Diferenciación Celular , Células Cultivadas , Glucólisis/inmunología , Infecciones por VIH/patología , VIH-1 , Humanos , Activación de Linfocitos , Fosforilación Oxidativa , Subgrupos de Linfocitos T/patologíaRESUMEN
The susceptibility of CD4 T cells to human immunodeficiency virus 1 (HIV-1) infection is regulated by glucose and glutamine metabolism, but the relative contributions of these nutrients to infection are not known. Here we show that glutaminolysis is the major pathway fuelling the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) in T-cell receptor-stimulated naïve, as well as memory CD4, subsets and is required for optimal HIV-1 infection. Under conditions of attenuated glutaminolysis, the α-ketoglutarate (α-KG) TCA rescues early steps in infection; exogenous α-KG promotes HIV-1 reverse transcription, rendering both naïve and memory cells more sensitive to infection. Blocking the glycolytic flux of pyruvate to lactate results in altered glucose carbon allocation to TCA and pentose phosphate pathway intermediates, an increase in OXPHOS and augmented HIV-1 reverse transcription. Moreover, HIV-1 infection is significantly higher in CD4 T cells selected on the basis of high mitochondrial biomass and OXPHOS activity. Therefore, the OXPHOS/aerobic glycolysis balance is a major regulator of HIV-1 infection in CD4 T lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Ciclo del Ácido Cítrico , Glucosa/metabolismo , Glutamina/metabolismo , VIH-1/patogenicidad , Linfocitos T CD4-Positivos/inmunología , Humanos , Activación de LinfocitosRESUMEN
HIV-2 groups have emerged from sooty mangabey SIV and entered the human population in Africa on several separate occasions. Compared to world pandemic HIV-1 that arose from the chimpanzee SIVcpz virus, the SIVsm-derived HIV-2, largely confined to West Africa, is less replicative, less transmissible and less pathogenic. Here, we evaluated the interactions between host cellular factors, which control HIV-1 infection and target the capsid, and HIV-2 capsids obtained from primary isolates from patients with different disease progression status. We showed that, like HIV-1, all HIV-2 CA we tested exhibited a dependence on cyclophilin A. However, we observed no correlation between HIV-2 viremia and susceptibility to hu-TRIM5alpha or dependence to CypA. Finally, we found that all CA from HIV-2 primary isolates exploit Nup358 and Nup153 for nucleus transposition. Altogether, these findings indicate that the ability to use the two latter nucleoporins is essential to infection of human cells for both HIV-1 and HIV-2. This dependence provides another molecular target that could be used for antiviral strategies against both HIV-1 and 2, based on both nucleoporins.
Asunto(s)
Proteínas Portadoras/metabolismo , Ciclofilina A/metabolismo , Infecciones por VIH/virología , VIH-2/patogenicidad , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , África Occidental , Animales , Factores de Restricción Antivirales , Cápside/metabolismo , Cápside/fisiología , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células HEK293 , Infecciones por VIH/metabolismo , VIH-1/metabolismo , VIH-1/patogenicidad , VIH-2/metabolismo , Humanos , Ratones , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Replicación ViralRESUMEN
The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/metabolismo , Homeostasis , Proteínas Mitocondriales/metabolismo , Piel/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Membrana Basal/metabolismo , Adhesión Celular , Células Cultivadas , Microambiente Celular , Proteínas de Unión al ADN/deficiencia , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/genética , Células Epidérmicas , Epidermis/metabolismo , Regulación de la Expresión Génica , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones Noqueados , Proteínas Mitocondriales/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Piruvatos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras , Células Madre/metabolismo , Factores de Transcripción/deficiencia , Ubiquitina-Proteína LigasasRESUMEN
Mutations in XPR1, a gene encoding an inorganic phosphate exporter, have recently been identified in patients with primary familial brain calcification (PFBC). Using Sanger sequencing, we screened XPR1 in 18 unrelated patients with PFBC and no SLC20A2, PDGFB, or PDGFRB mutation. XPR1 variants were tested in an in vitro physiological complementation assay and patient blood cells were assessed ex vivo for phosphate export. We identified a novel c.260T > C, p.(Leu87Pro) XPR1 variant in a 41-year-old man complaining of micrographia and dysarthria and demonstrating mild parkinsonism, cerebellar ataxia and executive dysfunction. Brain (123)I-Ioflupane scintigraphy showed marked dopaminergic neuron loss. Peripheral blood cells from the patient exhibited decreased phosphate export. XPR1 in which we introduced the mutation was not detectable at the cell surface and did not lead to phosphate export. These results confirm that loss of XPR1-mediated phosphate export function causes PFBC, occurring in less than 8 % of cases negative for the other genes, and may be responsible for parkinsonism.
Asunto(s)
Encefalopatías/genética , Calcinosis/genética , Salud de la Familia , Mutación/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Adulto , Encefalopatías/diagnóstico por imagen , Calcinosis/diagnóstico por imagen , Femenino , Estudios de Asociación Genética , Humanos , Imagen por Resonancia Magnética , Masculino , Nortropanos/farmacocinética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Cintigrafía , Transfección , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Mammalian retroviruses cause a variety of diseases in their hosts, including hematological and immunodeficiency disorders. Both human T-cell leukemia (HTLV) and human immunodeficiency (HIV) viruses originated from several independent zoonotic transmissions, indicating that cross-species transmissions from animal to humans may still occur. Thus, as the risk for retroviral transmissions from animals to humans increase, we investigated whether mammalian retroviruses are involved in selected pediatric idiopathic diseases whose symptoms evoke retroviral infections. Blood samples, sera, and synovial fluids, or bone marrow cells were collected from pediatric patients under 18 years of age with different autoimmune idiopathic diseases. Overall, we screened clinical samples from 110 children using sensitive nested and semi-nested PCR strategies targeting env genes, and a C-type retrovirus reverse transcriptase (RT) activity kit. All clinical samples were free of retroviral signatures, indicating the unlikelihood of an etiological role of the retroviruses we assessed in the pediatric diseases we tested.
Asunto(s)
Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/patología , Infecciones por Retroviridae/patología , Infecciones por Retroviridae/virología , Retroviridae/aislamiento & purificación , Adolescente , Niño , Preescolar , Productos del Gen env/genética , Humanos , Lactante , Reacción en Cadena de la Polimerasa , ADN Polimerasa Dirigida por ARN/análisis , Retroviridae/genéticaRESUMEN
Statin treatment of hypercholesterolemia can lead to chronic myotoxicity which is, in most cases, alleviated by drug withdrawal. Cellular and molecular mechanisms of this adverse effect have been elusive, in particular because of the lack of in vitro models suitable for long-term exposures. We have taken advantage of the properties of human pluripotent stem cell-derived mesodermal precursors, that can be maintained unaltered in vitro for a long period of time, to develop a model of repeated exposures to simvastatin during more than 2 weeks. This approach unveiled major differences, both in functional and molecular terms, in response to single versus repeated-dose exposures to simvastatin. The main functional effect of the in vitro simvastatin-induced long-term toxicity was a loss of proliferative capacity in the absence of concomitant cell death, revealing that cytostatic effect could be a major contributor to statin-induced myotoxicity. Comparative analysis of molecular modifications induced by simvastatin short-term versus prolonged exposures demonstrated powerful adaptive cell responses, as illustrated by the dramatic decrease in the number of differentially expressed genes, distinct biological pathway enrichments, and distinct patterns of nutrient transporters expressed at the cell surface. This study underlines the potential of derivatives of human pluripotent stem cells for developing new approaches in toxicology, in particular for chronic toxicity testing.
Asunto(s)
Hipercolesterolemia/tratamiento farmacológico , Mesodermo/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Simvastatina/efectos adversos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/patología , Mesodermo/citología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Células Madre Pluripotentes/citología , Simvastatina/administración & dosificación , Transcriptoma/efectos de los fármacosRESUMEN
Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Calcinosis/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Escala de Lod , Masculino , Persona de Mediana Edad , Mutación Missense , Enfermedades Neurodegenerativas/genética , Linaje , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/metabolismo , Linaje de la Célula , Regulación de la Expresión Génica , Glucosa/metabolismo , Glutamina/metabolismo , Células Madre Hematopoyéticas/citología , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Transporte Biológico , Diferenciación Celular , Cromatografía Liquida , Eritrocitos/citología , Glucólisis , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: Many species of non-human primates in Africa are naturally infected by simian immunodeficiency viruses (SIV) and humans stand at the forefront of exposure to these viruses in Sub-Saharan Africa. Cross-species transmission and adaptation of SIV to humans have given rise to human immunodeficiency viruses (HIV-1 and HIV-2) on twelve accountable, independent occasions. However, the determinants contributing to a simian-to-human lasting transmission are not fully understood. Following entry, viral cores are released into the cytoplasm and become the principal target of host cellular factors. Here, we evaluated cellular factors likely to be involved in potential new SIV cross-species transmissions. We investigated the interactions of capsids from naturally circulating SIV isolates with both HIV-1 restricting (i.e. TRIM5 proteins) and facilitating (i.e. cyclophilin A and nucleopore-associated Nup358/RanBP2 and Nup153) factors in single-round infectivity assays that reproduce early stages of the viral life-cycle. RESULTS: We show that human TRIM5α is unlikely to prevent cross-species transmission of any SIV we tested and observed that the SIV CA-CypA interaction is a widespread but not a universal feature. Moreover, entry in the nucleus of different SIV appeared to follow pathways that do not necessarily recruit Nup358/RanBP2 or Nup153, and this regardless of their interaction with CypA. Nevertheless, we found that, like HIV-1, human-adapted HIV-2 infection was dependent on Nup358/RanBP2 and Nup153 interactions for optimal infection. Furthermore, we found that, unlike HIV CA, SIV CA did not require a direct interaction with the Cyp-like domain of Nup358/RanBP2 to carry out successful infection. CONCLUSIONS: Circulating SIV present a variety of phenotypes with regard to CA-interacting restricting or facilitating factors. Altogether, we unveiled unidentified pathways for SIV CA, which could also be exploited by HIV in different cellular contexts, to drive entry into the nucleus. Our findings warrant a closer evaluation of other potential defenses against circulating SIV.
Asunto(s)
Proteínas de la Cápside/metabolismo , Interacciones Huésped-Patógeno , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Integración Viral , Replicación Viral , Animales , Línea Celular , VIH-1/fisiología , VIH-2/fisiología , Humanos , Pan troglodytesRESUMEN
Inorganic phosphate uptake is a universal function accomplished by transporters that are present across the living world. In contrast, no phosphate exporter has ever been identified in metazoans. Here, we show that depletion of XPR1, a multipass membrane molecule initially identified as the cell-surface receptor for xenotropic and polytropic murine leukemia retroviruses (X- and P-MLV), induced a decrease in phosphate export and that reintroduction of various XPR1 proteins, from fruit fly to human, rescued this defect. Inhibition of phosphate export was also obtained with a soluble ligand generated from the envelope-receptor-binding domain of X-MLV in all human cell lines tested, as well as in diverse stem cells and epithelial cells derived from renal proximal tubules, the main site of phosphate homeostasis regulation. These results provide new insights on phosphate export in metazoans and the role of Xpr1 in this function.
Asunto(s)
Fosfatos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células CACO-2 , Cricetulus , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Células 3T3 NIH , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Especificidad de la Especie , Transfección , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.