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Copper is a critical element for eukaryotic life involved in numerous cellular functions, including redox balance, but is toxic in excess. Therefore, tight regulation of copper acquisition and homeostasis is essential for cell physiology and survival. Here, we identify a different regulatory mechanism for cellular copper homeostasis that requires the presence of an endogenous retroviral envelope glycoprotein called Refrex1. We show that cells respond to elevated extracellular copper by increasing the expression of Refrex1, which regulates copper acquisition through interaction with the main copper transporter CTR1. Downmodulation of Refrex1 results in intracellular copper accumulation leading to reactive oxygen species (ROS) production and subsequent apoptosis, which is prevented by copper chelator treatment. Our results show that Refrex1 has been co-opted for its ability to regulate copper entry through CTR1 in order to limit copper excess, redox imbalance, and ensuing cell death, strongly suggesting that other endogenous retroviruses may have similar metabolic functions among vertebrates.

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. StemCells2015;33:2936–2948

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.

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.

Les rétro-éléments constituent un moteur de la diversité du monde vivant du fait de leurs propriétés de transposition. Parmi eux se distinguent les rétrovirus, dont la «transposition» se propage en une véritable infection de la cellule initiale aux cellules et organismes voisins. Une étape clé de la conversion d’un rétrotransposon en agent infectieux est l’acquisition d’une glycoprotéine d’enveloppe, désignée Env. Nous présentons ici différents exemples où a pu être retracée cette «capture d’enveloppe», qui a conduit à la constitution de rétrovirus infectieux de mammifères, et montrons qu’un tel événement de capture s’est probablement produit dans le cas de la protéine Env du rétrovirus humain HTLV. Ces captures expliqueraient l’étroite parenté de la protéine Env de différents rétrovirus pourtant phylogéniquement distants. L’élucidation des diverses « étymologies » rétrovirales devrait aider à mieux comprendre la phylogénie des rétrovirus et la physiopathologie des infections rétrovirales, voire mener à la découverte de nouveaux rétro-éléments.

The human T-cell leukemia virus type 1 (HTLV) is the first isolated human retrovirus, but its receptor has yet to be identified, in part due to its ubiquitous expression. Here we report that quiescent CD4 and CD8 T lymphocytes do not express this receptor, as monitored with a soluble receptor-binding domain derived from the HTLV envelope. However, HTLV receptor is an early activation marker in neonatal and adult T lymphocytes, detected as early as 4 hours following T-cell–receptor (TCR) stimulation. This induced surface expression of the HTLV receptor requires de novo protein synthesis and results in a wide distribution on the surface of activated lymphocytes. Moreover, the distribution of the HTLV receptor is independent of TCR/CD3-capped membrane structures, as observed by confocal immunofluorescence microscopy. To determine whether HTLV receptor up-regulation specifically requires TCR-mediated signals or, alternatively, is dependent on more generalized cell cycle entry/proliferation signals, its expression was monitored in interleukin 7 (IL-7)–stimulated neonatal and adult T cells. Neonatal, but not adult, lymphocytes proliferate in response to IL-7 and HTLV receptor expression is restricted to the former population. Thus, HTLV receptor expression appears to be an early marker of cell cycle entry. Up-regulation of the HTLV receptor, via signals transmitted through the IL-7 cytokine receptor as well as the TCR, is likely to contribute to the mother-to-infant transmission and spreading of HTLV-1.

The human T-cell leukemia virus type 1 (HTLV) is the first isolated human retrovirus, but its receptor has yet to be identified, in part due to its ubiquitous expression. Here we report that quiescent CD4 and CD8 T lymphocytes do not express this receptor, as monitored with a soluble receptor-binding domain derived from the HTLV envelope. However, HTLV receptor is an early activation marker in neonatal and adult T lymphocytes, detected as early as 4 hours following T-cell–receptor (TCR) stimulation. This induced surface expression of the HTLV receptor requires de novo protein synthesis and results in a wide distribution on the surface of activated lymphocytes. Moreover, the distribution of the HTLV receptor is independent of TCR/CD3-capped membrane structures, as observed by confocal immunofluorescence microscopy. To determine whether HTLV receptor up-regulation specifically requires TCR-mediated signals or, alternatively, is dependent on more generalized cell cycle entry/proliferation signals, its expression was monitored in interleukin 7 (IL-7)–stimulated neonatal and adult T cells. Neonatal, but not adult, lymphocytes proliferate in response to IL-7 and HTLV receptor expression is restricted to the former population. Thus, HTLV receptor expression appears to be an early marker of cell cycle entry. Up-regulation of the HTLV receptor, via signals transmitted through the IL-7 cytokine receptor as well as the TCR, is likely to contribute to the mother-to-infant transmission and spreading of HTLV-1.

ABSTRACTTransduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.

ABSTRACTHydrophilic loops in the receptor binding domain of the amphotropic murine leukemia virus (MLV) envelope glycoprotein (SU) are predicted and may participate in SU-receptor interactions. We have replaced five segments of 6 to 15 amino acids located in each of these regions with an 11-amino-acid tag from the vesicular stomatitis virus glycoprotein (VSV-G). Substitution was compatible with envelope processing, transport, and incorporation into virions. However, three substitution mutants showed a temperature-dependent phenotype, suggesting structural unstability. Accessibility of the tagging epitope for a monoclonal anti-VSV-G antibody was greater in oligomeric than in monomeric SUs when insertion was done in VRA, a domain essential for receptor recognition. In contrast, accessibility was independent of structural constraints when insertion was done in VRB, a domain playing an accessory role in receptor binding. Interaction with the amphotropic receptor was investigated by interference assay and study of binding and infection of target cells with MLV particles coated with the substituted envelopes. Envelope-receptor interaction was abolished when substitution was performed in a potential loop-forming segment located at the N-terminal half of VRA. Although interaction was affected to variable extents, depending on the substituted segment, other mutants conserved the ability to interact with the amphotropic receptor. These experiments indicate the 14-amino-acid segment between positions 50 and 64 of SU as an essential determinant of amphotropic-receptor recognition. They also show that a foreign linear epitope can be tolerated in several locations of the amphotropic SU receptor binding site, and this result has implications for the design of targeted retroviral vectors.

The 243 N-terminal residues of Friend Murine Leukemia Virus envelope glycoprotein (SU) fold into a structurally and functionally autonomous domain which contains the determinants for binding to the ecotropic virus receptor. The two N-linked glycosylation sites present in this N-terminal portion of the viral SU were removed by site-directed mutagenesis without disturbing its biosynthesis and incorporation into infectious virions. A truncated version of the mutant protein which included only the N-terminal domain was poorly transported but still able to interact with the receptor. Interference assays Indicated that the interaction between the mutated protein and the virus receptor was weaker. We conclude that the elimination of N-linked oligosaccharide chains in the envelope N-terminal domain do not prevent receptor interaction but results in subtle conformational changes that may alter recognition and binding. Copyright 1994, 1999 Academic Press

Glucose provides a key supply of energy and carbon and its transport is achieved via multimembrane-spanning glucose transporters (GLUTs). The human erythrocyte is the cell type expressing the highest level of the GLUT1 glucose transporter, harboring greater than 200,000 molecules per cell. We now demonstrate that GLUT1 transcripts increase by 3-logs during erythropoiesis and high GLUT1 surface expression is observed following passage through the basophilic erythroblast stage. Paradoxically though, glucose transport significantly decreases. As GLUT1 also transports L-dehydroascorbic acid (DHA), the oxidized form of ascorbic acid (AA), transport of this molecule was assessed and indeed, it increases dramatically during erythropoiesis. The switch from glucose to DHA transport is coupled to the physical association of GLUT1 with stomatin, an integral erythrocyte membrane protein. We find that stomatin inversely regulates the relative transports of glucose and DHA by GLUT1. Moreover, in a patient with overhydrated hereditary stomatocytosis, a rare genetic disorder of red cell membrane permeability wherein stomatin is absent, glucose uptake is significantly higher but there is a concomitant 50% reduction in DHA transport. Intriguingly, erythrocyte-specific DHA transport is not conserved amongst all mammalian species; we did not detect GLUT1 on mature murine erythrocytes where DHA uptake is minimal. Notably though, humans differ from the vast majority of the greater than 5,000 mammalian species in that they are unable to synthesize ascorbic acid from glucose. This trait is shared only with other higher primates, guinea pigs and and fruit bats. We have determined that erythrocyte GLUT1 expression and associated DHA transport are specific features of these diverse ascorbic acid-deficient mammals. Within the primate order, defective ascorbic acid synthesis is due to an inactivation of the L-gulonolactone oxidase (GLO) enzyme at the Haplorrhini-Strespsirrhini split. In accord with the data presented above, we find that erythrocyte GLUT1 expression and associated DHA transport are characteristic of erythrocytes from primates within the Haplorrhini suborder but not Strepsirrhini lemurs. Thus, the erythrocyte-specific co-expression of GLUT1 and stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize the essential ascorbic acid metabolite.

Glucose provides a key supply of energy and carbon and its transport is achieved via multimembrane-spanning glucose transporters (GLUTs). The human erythrocyte is the cell type expressing the highest level of the GLUT1 glucose transporter, harboring greater than 200,000 molecules per cell. We now demonstrate that GLUT1 transcripts increase by 3-logs during erythropoiesis and high GLUT1 surface expression is observed following passage through the basophilic erythroblast stage. Paradoxically though, glucose transport significantly decreases. As GLUT1 also transports L-dehydroascorbic acid (DHA), the oxidized form of ascorbic acid (AA), transport of this molecule was assessed and indeed, it increases dramatically during erythropoiesis. The switch from glucose to DHA transport is coupled to the physical association of GLUT1 with stomatin, an integral erythrocyte membrane protein. We find that stomatin inversely regulates the relative transports of glucose and DHA by GLUT1. Moreover, in a patient with overhydrated hereditary stomatocytosis, a rare genetic disorder of red cell membrane permeability wherein stomatin is absent, glucose uptake is significantly higher but there is a concomitant 50% reduction in DHA transport. Intriguingly, erythrocyte-specific DHA transport is not conserved amongst all mammalian species; we did not detect GLUT1 on mature murine erythrocytes where DHA uptake is minimal. Notably though, humans differ from the vast majority of the greater than 5,000 mammalian species in that they are unable to synthesize ascorbic acid from glucose. This trait is shared only with other higher primates, guinea pigs and and fruit bats. We have determined that erythrocyte GLUT1 expression and associated DHA transport are specific features of these diverse ascorbic acid-deficient mammals. Within the primate order, defective ascorbic acid synthesis is due to an inactivation of the L-gulonolactone oxidase (GLO) enzyme at the Haplorrhini-Strespsirrhini split. In accord with the data presented above, we find that erythrocyte GLUT1 expression and associated DHA transport are characteristic of erythrocytes from primates within the Haplorrhini suborder but not Strepsirrhini lemurs. Thus, the erythrocyte-specific co-expression of GLUT1 and stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize the essential ascorbic acid metabolite.