The Compartmentalisation of Phosphorylated Free Oligosaccharides in Cells from a CDG Ig Patient Reveals a Novel ER-to-Cytosol Translocation Process

BACKGROUND: Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5)GlcNAc(2)-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3)Man(9)GlcNAc(2)-PP-dolichol). After transfer of Glc(3)Man(9)GlcNAc(2) onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation. METHODS AND PRINCIPAL FINDINGS: In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7)GlcNAc(2)-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7)GlcNAc(2)-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7)GlcNAc(2)-P appears in the cytosol without detectable generation of ER luminal Man(7)GlcNAc(2)-P. CONCLUSIONS AND SIGNIFICANCE: The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO

Functional study of the microRNA pathway in tumoral cells biology

Les micro-ARNs (miRNAs) sont des ARNs de 20-22 nucléotides, transcrits à partir du génome, dont la fonction est de réguler l’expression génique en s’appariant à des ARNm cibles, inhibant ainsi leur traduction et/ou entrainant leur dégradation. Dans les cancers, l’expression des miRNAs est fortement dérégulée. Une majorité de miRNAs est diminuée dans les tissus tumoraux par rapport aux tissus normaux, et un lien causal a été décrit entre inhibition globale des miRNAs et tumorigenèse. Par ailleurs, des miRNAs agissant comme des suppresseurs de tumeurs et d’autres comme des oncogènes ont été décrits. Dans ce contexte impliquant de plus en plus les miRNAs dans les pathologies néoplasiques, l’objectif de ce travail était d’étudier le rôle de la voie miRNA dans la biologie des cellules tumorales. Afin d’identifier des cellules tumorales dépendant de miRNAs oncogènes endogènes pour survivre ou proliférer, nous avons développé une stratégie d’inhibition globale de la biogenèse des miRNAs en ciblant Drosha ou DGCR8, les deux composants du microprocesseur, complexe nucléaire de maturation des miRNAs. Cette stratégie nous a permis d’identifier des lignées cellulaires tumorales dans lesquelles l’inhibition du microprocesseur conduit à un phénotype d’arrêt de prolifération durable. Nous avons mis à profit cette dépendance à la voie miRNA pour réaliser un crible positif de complémentation du défaut de prolifération observé grâce à l’expression de miRNAs individuels. Nous avons ainsi pu mettre en évidence des miRNAs capables de soutenir individuellement la prolifération de ces cellules tumorales. Cette stratégie nous a également permis de montrer des différences fonctionnelles entre miRNAs homologues ou de la même famille. La recherche des cibles régulées par ces miRNAs nous a permis d’élaborer des hypothèses concernant les cibles potentiellement impliquées dans le phénotype observé. Nous avons ainsi démontré la participation du suppresseur de tumeur PTEN à l’arrêt de prolifération induit par l’inhibition du microprocesseur. La stratégie d’inhibition globale de la voie miRNA suivie d’une complémentation phénotypique par des miRNAs individuels permet de s’affranchir de la grande redondance de séquence et de fonction des miRNAs et devrait pouvoir s’appliquer d’une manière plus générale à l’étude d’autres processus régulés par les miRNAs.MicroRNAs (miRNAs) are 20-22 nucleotides RNAs, transcribed from the genome, which regulate gene expression by base-pairing to target mRNAs, thus inhibiting their translation and/or leading to their degradation. In cancers, miRNAs expression is strongly deregulated. A majority of miRNAs is diminished in tumoral tissues compared to normal tissues, and a causal link has been established between global inhibition of the miRNA pathway and tumorigenesis. In addition, miRNAs acting like tumor suppressors or oncogenes have been described. In this context of growing evidences implicating miRNAs in neoplasic diseases, this work aimed to investigate the role played by miRNA pathway in the biology of tumoral cells. In order to identify tumoral cells depending on endogenous oncogenic miRNAs to proliferate or survive, we developed a strategy of global inhibition of miRNAs biogenesis by targeting Drosha or DGCR8, the two components of the “microprocessor”, the nuclear miRNA maturation complex. This strategy allowed us to identify tumoral cell lines in which microprocessor inhibition led to a sustained growth arrest. We took advantage of this miRNA pathway dependency to screen for individual miRNAs able to complement the observed growth defect. This complementation screen allowed us to identify individual miRNAs able to sustain growth in those tumoral cells. This strategy also highlighted functional differences between homologous miRNAs or between miRNAs from the same family. The search for targets regulated by those miRNAs allowed us to develop hypothesis concerning the potential targets involved in the observed phenotype. By using this approach, we demonstrated that the tumor suppressor PTEN was involved in the growth arrest induced by microprocessor inhibition. The strategy of global miRNA pathway inhibition followed by phenotypic complementation by individual miRNAs allows overcoming the high sequence and function redundancy of miRNAs. We thus think it could be applied more generally to the study of other cellular processes regulated by miRNAs

Etude fonctionnelle de la voie micro-ARN dans la biologie des cellules tumorales

MicroRNAs (miRNAs) are 20-22 nucleotides RNAs, transcribed from the genome, which regulate gene expression by base-pairing to target mRNAs, thus inhibiting their translation and/or leading to their degradation. In cancers, miRNAs expression is strongly deregulated. A majority of miRNAs is diminished in tumoral tissues compared to normal tissues, and a causal link has been established between global inhibition of the miRNA pathway and tumorigenesis. In addition, miRNAs acting like tumor suppressors or oncogenes have been described. In this context of growing evidences implicating miRNAs in neoplasic diseases, this work aimed to investigate the role played by miRNA pathway in the biology of tumoral cells. In order to identify tumoral cells depending on endogenous oncogenic miRNAs to proliferate or survive, we developed a strategy of global inhibition of miRNAs biogenesis by targeting Drosha or DGCR8, the two components of the “microprocessor”, the nuclear miRNA maturation complex. This strategy allowed us to identify tumoral cell lines in which microprocessor inhibition led to a sustained growth arrest. We took advantage of this miRNA pathway dependency to screen for individual miRNAs able to complement the observed growth defect. This complementation screen allowed us to identify individual miRNAs able to sustain growth in those tumoral cells. This strategy also highlighted functional differences between homologous miRNAs or between miRNAs from the same family. The search for targets regulated by those miRNAs allowed us to develop hypothesis concerning the potential targets involved in the observed phenotype. By using this approach, we demonstrated that the tumor suppressor PTEN was involved in the growth arrest induced by microprocessor inhibition. The strategy of global miRNA pathway inhibition followed by phenotypic complementation by individual miRNAs allows overcoming the high sequence and function redundancy of miRNAs. We thus think it could be applied more generally to the study of other cellular processes regulated by miRNAs.Les micro-ARNs (miRNAs) sont des ARNs de 20-22 nucléotides, transcrits à partir du génome, dont la fonction est de réguler l’expression génique en s’appariant à des ARNm cibles, inhibant ainsi leur traduction et/ou entrainant leur dégradation. Dans les cancers, l’expression des miRNAs est fortement dérégulée. Une majorité de miRNAs est diminuée dans les tissus tumoraux par rapport aux tissus normaux, et un lien causal a été décrit entre inhibition globale des miRNAs et tumorigenèse. Par ailleurs, des miRNAs agissant comme des suppresseurs de tumeurs et d’autres comme des oncogènes ont été décrits. Dans ce contexte impliquant de plus en plus les miRNAs dans les pathologies néoplasiques, l’objectif de ce travail était d’étudier le rôle de la voie miRNA dans la biologie des cellules tumorales. Afin d’identifier des cellules tumorales dépendant de miRNAs oncogènes endogènes pour survivre ou proliférer, nous avons développé une stratégie d’inhibition globale de la biogenèse des miRNAs en ciblant Drosha ou DGCR8, les deux composants du microprocesseur, complexe nucléaire de maturation des miRNAs. Cette stratégie nous a permis d’identifier des lignées cellulaires tumorales dans lesquelles l’inhibition du microprocesseur conduit à un phénotype d’arrêt de prolifération durable. Nous avons mis à profit cette dépendance à la voie miRNA pour réaliser un crible positif de complémentation du défaut de prolifération observé grâce à l’expression de miRNAs individuels. Nous avons ainsi pu mettre en évidence des miRNAs capables de soutenir individuellement la prolifération de ces cellules tumorales. Cette stratégie nous a également permis de montrer des différences fonctionnelles entre miRNAs homologues ou de la même famille. La recherche des cibles régulées par ces miRNAs nous a permis d’élaborer des hypothèses concernant les cibles potentiellement impliquées dans le phénotype observé. Nous avons ainsi démontré la participation du suppresseur de tumeur PTEN à l’arrêt de prolifération induit par l’inhibition du microprocesseur. La stratégie d’inhibition globale de la voie miRNA suivie d’une complémentation phénotypique par des miRNAs individuels permet de s’affranchir de la grande redondance de séquence et de fonction des miRNAs et devrait pouvoir s’appliquer d’une manière plus générale à l’étude d’autres processus régulés par les miRNAs

Etude fonctionnelle de la voie micro-ARN dans la biologie des cellules tumorales

Les micro-ARNs (miRNAs) sont des ARNs de 20-22 nucléotides, transcrits à partir du génome, dont la fonction est de réguler l expression génique en s appariant à des ARNm cibles, inhibant ainsi leur traduction et/ou entrainant leur dégradation. Dans les cancers, l expression des miRNAs est fortement dérégulée. Une majorité de miRNAs est diminuée dans les tissus tumoraux par rapport aux tissus normaux, et un lien causal a été décrit entre inhibition globale des miRNAs et tumorigenèse. Par ailleurs, des miRNAs agissant comme des suppresseurs de tumeurs et d autres comme des oncogènes ont été décrits. Dans ce contexte impliquant de plus en plus les miRNAs dans les pathologies néoplasiques, l objectif de ce travail était d étudier le rôle de la voie miRNA dans la biologie des cellules tumorales. Afin d identifier des cellules tumorales dépendant de miRNAs oncogènes endogènes pour survivre ou proliférer, nous avons développé une stratégie d inhibition globale de la biogenèse des miRNAs en ciblant Drosha ou DGCR8, les deux composants du microprocesseur, complexe nucléaire de maturation des miRNAs. Cette stratégie nous a permis d identifier des lignées cellulaires tumorales dans lesquelles l inhibition du microprocesseur conduit à un phénotype d arrêt de prolifération durable. Nous avons mis à profit cette dépendance à la voie miRNA pour réaliser un crible positif de complémentation du défaut de prolifération observé grâce à l expression de miRNAs individuels. Nous avons ainsi pu mettre en évidence des miRNAs capables de soutenir individuellement la prolifération de ces cellules tumorales. Cette stratégie nous a également permis de montrer des différences fonctionnelles entre miRNAs homologues ou de la même famille. La recherche des cibles régulées par ces miRNAs nous a permis d élaborer des hypothèses concernant les cibles potentiellement impliquées dans le phénotype observé. Nous avons ainsi démontré la participation du suppresseur de tumeur PTEN à l arrêt de prolifération induit par l inhibition du microprocesseur. La stratégie d inhibition globale de la voie miRNA suivie d une complémentation phénotypique par des miRNAs individuels permet de s affranchir de la grande redondance de séquence et de fonction des miRNAs et devrait pouvoir s appliquer d une manière plus générale à l étude d autres processus régulés par les miRNAs.MicroRNAs (miRNAs) are 20-22 nucleotides RNAs, transcribed from the genome, which regulate gene expression by base-pairing to target mRNAs, thus inhibiting their translation and/or leading to their degradation. In cancers, miRNAs expression is strongly deregulated. A majority of miRNAs is diminished in tumoral tissues compared to normal tissues, and a causal link has been established between global inhibition of the miRNA pathway and tumorigenesis. In addition, miRNAs acting like tumor suppressors or oncogenes have been described. In this context of growing evidences implicating miRNAs in neoplasic diseases, this work aimed to investigate the role played by miRNA pathway in the biology of tumoral cells. In order to identify tumoral cells depending on endogenous oncogenic miRNAs to proliferate or survive, we developed a strategy of global inhibition of miRNAs biogenesis by targeting Drosha or DGCR8, the two components of the microprocessor , the nuclear miRNA maturation complex. This strategy allowed us to identify tumoral cell lines in which microprocessor inhibition led to a sustained growth arrest. We took advantage of this miRNA pathway dependency to screen for individual miRNAs able to complement the observed growth defect. This complementation screen allowed us to identify individual miRNAs able to sustain growth in those tumoral cells. This strategy also highlighted functional differences between homologous miRNAs or between miRNAs from the same family. The search for targets regulated by those miRNAs allowed us to develop hypothesis concerning the potential targets involved in the observed phenotype. By using this approach, we demonstrated that the tumor suppressor PTEN was involved in the growth arrest induced by microprocessor inhibition. The strategy of global miRNA pathway inhibition followed by phenotypic complementation by individual miRNAs allows overcoming the high sequence and function redundancy of miRNAs. We thus think it could be applied more generally to the study of other cellular processes regulated by miRNAs.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Impairing the microRNA biogenesis pathway induces proteome modifications characterized by size bias and enrichment in antioxidant proteins

International audienc

Cytostatic effect of repeated exposure to simvastatin, a mechanism for chronic myotoxicity revealed by the use of mesodermal progenitors derived from human pluripotent stem cells.

International audienceStatin 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 two 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

Characterisation of negatively charged oligosaccharide-like material derived from different cell lines.

<p>A. Equal amounts of radioactivity associated with material that was eluted from AG-1 columns with 3M FA as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#pone-0011675-g002" target="_blank">Fig. 2A</a> were subjected to QAE-Sephadex chromatography as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#s2" target="_blank">Material and Methods</a>. The column was eluted with increasing concentrations of NaCl (indicated on the right hand y axis). Fractions were collected and assayed for radioactivity by scintillation counting. B. Aliquots of the negatively charged oligosaccharide-like material derived from EBV CDG Ig cells were analysed by thin layer chromatography (TLC) before and after treatment with either alkaline phosphatase (<i>P'ase</i>) or endo-β-N-acetylglucosaminidase H (<i>EndoH</i>). Abbreviations: lines to the left of the TLC fluorograms indicate the migration position of the oligosaccharide (Man₇GlcNAc₂; M₇GN₂) that was derived by mild acid hydrolysis of Man₇GlcNAc₂-PP-dolichol isolated from CDG Ig cells. This oligosaccharide was also treated with endoH to yield Man₇GlcNAc (M₇GN). The structure of the oligosaccharide moiety known to occur in the Man₇GlcNAc₂-PP-dolichol that accumulates in cells from CDG Ig is shown to the right of the TLC (mannose; green circles, N-acetylglucosamine; blue squares). The di-N-acetylchitobiose moiety of this oligosaccharide is sensitive to endoH. C. Aliquots of the negatively charged oligosaccharide-like material derived from DPM synthase-deficient Thy^-1 mouse lymphoma cells were analysed by thin layer chromatography (TLC) before and after treatment with either alkaline phosphatase (<i>P'ase</i>) or 20 mM HCl. The structure of the oligosaccharide moiety known to occur in the Man₅GlcNAc₂-PP-dolichol that accumulates in these cells is shown to the right of the TLC (mannose; green circles, N-acetylglucosamine; blue squares). The di-N-acetylchitobiose moiety of this oligosaccharide is not sensitive to endoH. The line to the left of the fluorograph indicates the migration position of Man₅GlcNAc₂ (M₅GN₂) that was released by mild acid acid treatment of Man₅GlcNAc₂-PP-dolichol derived from Thy^-1 cells. D. [¹⁴C]glucose-1-phosphate (Glc1P) and [¹⁴C]glucose-6-phosphate (Glc6P) were subjected to ion-exchange chromatography on AG-1(acetate) before and after either alkaline phosphatase or mild acid treatment as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#s2" target="_blank">Materials and Methods</a>. Neutralised material was assayed by scintillation counting and expressed as a percentage of input radioactivity.</p

fOSGN2-P are generated in streptolysin O-permeabilised cells.

<p>A. EBV CDG Ig cells were pulse radiolabeled for 30 min with [2-³H]mannose and then permeabilised with streptolysin O (SLO) at 4°C in permeabilisation buffer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#s2" target="_blank">Materials and Methods</a>. After centrifugation fOSGN2-P and neutral fOS were recovered from both the supernatant containing cytosolic components (<i>Cyt</i>) and the permeabilised cell pellet containing intact membrane bound compartments (<i>MBC</i>). After dephosphorylation with mild acid treatment fOSGN2-P and fOS were examined by TLC. The migration position of Man₇GlcNAc₂ (M₇GN₂), derived by mild acid hydrolysis of Man₇GlcNAc₂-PP-dolichol isolated from CDG Ig cells, is indicated to the left of each pair of chromatograms. B. EBV CDG Ig cells were pulse radiolabeled for 30 min with [2-³H]mannose and then permeabilised with SLO in incubation buffer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011675#s2" target="_blank">Materials and Methods</a>. After incubation of permeabilised cells in the presence of 20 µM each of UDP-Glc, GDP-Man, and UDP-GlcNAc for various times at 37°C, <i>Cyt</i> and <i>MBC</i> fractions were generated as described above. Neutral fOS (C) and fOSGN2-P (D) were recovered from the <i>Cyt</i> and <i>MBC</i> fractions and assayed by scintillation counting. E. DLO (L) and fOSGN2-P (P) recovered from the incubations described in C and D were hydrolysed using mild acid treatment and the resulting oligosaccharides were analysed by TLC. Pyrophosphate 10 mM was added to the indicated reaction mixture. The migration positions of standard oligosaccharides are indicated by the solid lines to the left of the chromatograms. The oligosaccharide migrating slightly slower than Man₉GlcNAc₂ (indicated with the dotted line) was not characterised but migrates as Glc₁Man₉GlcNAc₂ or Glc₃Man₇GlcNAc₂. The TLC plate on which DLO-derived oligosaccharides were resolved was exposed to film for 7 days whereas that on which fOSGN2-P-derived oligosaccharides were resolved was exposed for 14 days.</p

The dolichol cycle and protein N-glycosylation.

<p>The dolichol cycle consists of a series of reactions (heavy dashed blue lines) involved in the construction of the oligosaccharide precursor (Glc₃Man₉GlcNAc₂) on a dolichol carrier. The cycle is completed by reactions (heavy dashed pink lines) involved in the recycling of dolichol-phosphate (dol-P). Mature lipid linked oligosaccharide (DLO) is generated by the transfer of two residues of N-acetylglucosamine (blue squares), 9 residues of mannose (green circles) and 3 residues of glucose (blue circles) onto the lipid carrier dolichol-P (zig zag line). The monosaccharides are added sequentially, in the order indicated by their numbers, by glycosyltransferases whose gene names are shown in yellow ovals and whose order of action is also indicated. The first seven sugars are added by cytoplasmically orientated UDP-GlcNAc- and GDP-Man-requiring glycosyltransferases. The growing DLO is then flipped into the lumen of the ER by a process thought to involve the RFT1 gene product. Subsequently dolichol-P-Man (DPM)- and dolichol-P-Glc (DPG)-requiring glycosyltransferases complete DLO biosynthesis. The addition of the last glucose residue to the growing DLO allows efficient oligosaccharyltransferase (OST)-mediated transfer of the oligosaccharide from lipid onto nascent polypeptides (-N-X-T/S-) in the ER. As indicated by the heavy dashed pink lines, a series of reactions carried out by gene products indicated in the yellow ovals, ensure that the lumenally orientated dol-P and dol-PP molecules that are generated during the construction of mature DLO are reorientated towards the cytosolic face of the ER. The different type I congenital disorders of glycosylation subtypes (CDG Ia-p, indicated by letters in red circles on the gene name) are caused by mutations in genes encoding enzymes involved in either the construction of mature DLO or dolichol recycling.</p