Assessment of the Cytotoxicity, Mutagenicity, and Genotoxicity of Two Traditional Chinese Herbs: Aristolochia baetica and Magnolia officinalis

peer reviewedHerbal remedies used in traditional medicine often contain several compounds combined in order to potentiate their own intrinsic properties. However, herbs can sometimes cause serious health troubles. In Belgium, patients who developed severe aristolochic acid nephropathy ingested slimming pills containing root extracts of an Aristolochia species, as well as the bark of Magnolia officinalis. The goal of the study was to evaluate, on a human renal cell line, Aristolochia and Magnolia extracts for their cytotoxicity by a resazurin cell viability assay, and their genotoxicity by immunodetection and quantification of the phosphorylated histone γ-H2AX. The present study also sought to assess the mutagenicity of these extracts, employing an OECD recognized test, the Ames test, using four Salmonella typhimurium strains with and without a microsomial fraction. Based on our results, it has been demonstrated that the Aristolochia–Magnolia combination (aqueous extracts) was more genotoxic to human kidney cells, and that this combination (aqueous and methanolic extracts) was more cytotoxic to human kidney cells after 24 and 48 h. Interestingly, it has also been shown that the Aristolochia–Magnolia combination (aqueous extracts) was mutagenic with a TA98 Salmonella typhimurium strain in the presence of a microsomial liver S9 fraction. This mutagenic effect appears to be dose-dependent

Transport nucléo-cytoplasmique des protéines de la famille TIS11 et formation des granules de stress: deux nouveaux rôles potentiels des Transportines

The nucleo-cytoplasmic compartmentalization enables eukaryotic cells to develop sophisticated post-transcriptional regulations of gene expression. However, managing the exchanges of macromolecules between the two compartments also represents a formidable challenge for the cells. Nucleo-cytoplasmic exchanges rely on specialized soluble carriers and take place at nuclear pore complexes that span the nuclear envelope. Active nucleo-cytoplasmic transport of proteins, in particular, is performed mainly by a family of carriers called karyopherins, which includes about twenty members in mammals. Some of them, called importins, recognize nuclear localization signals (NLSs) in their substrates and convey them into the nucleus. Others, called exportins, recognize nuclear export signals (NESs) in their substrates and bring them back to the cytoplasm. <p>Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, where they can often fulfill different functions. RBPs also frequently localize into specialized microdomains that are not delimited by a membrane but in which specific factors are concentrated. Those include processing bodies and stress granules, which are cytoplasmic foci associated with mRNA decay, storage and translational repression. Post-transcriptional regulations mediated by RBPs can therefore be modulated rapidly and efficiently through changes in the localization of RBPs.<p>The first part of this work focuses on the subcellular localization and nucleo-cytoplasmic transport of the Drosophila RBP dTIS11. Like its mammalian and yeast homologues, dTIS11 binds AU-rich elements in the 3’UTR of its target mRNAs, and stimulates their rapid deadenylation and decay. Here, we have observed that although dTIS11 appears to be located mostly in the cytoplasm, it is constantly shuttling in and out of the nucleus. We show that the export of dTIS11 from the nucleus depends on the CRM1 exportin and is mediated by a hydrophobic NES that encompasses residues 101 to 113 in dTIS11 sequence. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This PY-NLS partially overlaps the second zinc finger (ZnF2) of dTIS11. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zn2+ ion unmask dTIS11 and TTP PY-NLS and promote nuclear import. Taken together, our results indicate that the nuclear export of Drosophila and mammalian TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism might rely on a conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.<p>In the second part, we present preliminary results which implicate the nucleo-cytoplasmic transport machinery in the assembly of stress granules (SGs) in mammalian cells. SGs contain silenced mRNPs which resemble stalled initiation complexes, and they form transiently in response to acute stress, concomitantly with a global arrest of translation. While their exact role remains undefined, it seems clear that SGs are able to exchange mRNPs with polysomes and with PBs, and that they are connected to post-transcriptional and translational regulations of gene expression during stress. Here, we show that inhibition of Transportin-1 expression or function does not affect the translational status of cells but impairs the assembly of stress granules. Finally, we show that Transportin-1 and -2B, but not -2A, localize into stress granules in response to several stresses. <p>In conclusion, we suggest two potential new roles for Transportins, in the nucleo-cytoplasmic traffic of TIS11 proteins on the one hand and in the assembly of stress granules on the other hand.<p>/<p>Le compartimentage nucléo-cytoplasmique permet aux cellules eucaryotes de réguler l’expression génétique par des mécanismes post-transcriptionnels élaborés. Les ARN messagers subissent plusieurs étapes de maturation dans le noyau avant d’être exportés vers le cytoplasme où ils sont traduits et dégradés. Ces processus sont effectués via des protéines de liaison à l’ARN, ou RBPs. Beaucoup de RBPs exercent des fonctions différentes dans le noyau et dans le cytoplasme, et leur activité peut dès lors être rapidement modulée par une modification de leur localisation.<p>Le transport nucléo-cytoplasmique actif des protéines s’effectue à travers les pores nucléaires et fait majoritairement appel à des transporteurs solubles de la famille des karyophérines. Ceux-ci reconnaissent au sein des protéines à transporter une séquence-passeport appelée NLS (nuclear localization signal) ou NES (nuclear export signal) selon la direction nécessitée. <p>Le présent travail comporte deux parties. La première porte sur la localisation subcellulaire et le transport nucléo-cytoplasmique des protéines de la famille TIS11, et plus particulièrement de dTIS11 qui est le seul représentant de cette famille chez la Drosophile. Comme ses homologues dans d’autres espèces, dTIS11 est une RBP qui favorise la déadénylation et la dégradation de ses ARN messagers cibles. Nos résultats démontrent que dTIS11 fait la navette entre le noyau et le cytoplasme. L’export de dTIS11 hors du noyau est réalisé par la karyophérine CRM1 et fait appel à un NES différent de celui présent chez les protéines TIS11 mammaliennes. Nous identifions également un NLS cryptique au sein du domaine à deux doigts de zinc avec lequel dTIS11 lie l’ARN. Ce NLS correspond partiellement au signal consensus reconnu par la Transportine. Il est démasqué par la mutation du second doigt de zinc ;dans ces conditions, il permet l’import de dTIS11 par la Transportine. Enfin, nous montrons qu’il est conservé dans d’autres protéines de la famille TIS11. <p>Dans la seconde partie, nous nous intéressons aux granules de stress, qui sont des microdomaines cytoplasmiques dans lesquels se concentrent des RBPs et des ARN messagers non traduits en réponse à un stress cellulaire. Nous montrons que les karyophérines appartenant à la sous-famille des Transportines sont présentes dans ces granules et que l’inhibition de l’expression ou de la fonction des Transportines réduit la formation de ces granules en réponse à divers stress cellulaires. Nous écartons la possibilité que ce résultat soit un effet indirect d’un ralentissement du métabolisme traductionnel. Nos résultats suggèrent donc une implication des Transportines dans la formation des granules de stress. <p>Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Transport nucléo-cytoplasmique des protéines de la famille TIS11 et formation des granules de stress: deux nouveaux rôles potentiels des Transportines

The nucleo-cytoplasmic compartmentalization enables eukaryotic cells to develop sophisticated post-transcriptional regulations of gene expression. However, managing the exchanges of macromolecules between the two compartments also represents a formidable challenge for the cells. Nucleo-cytoplasmic exchanges rely on specialized soluble carriers and take place at nuclear pore complexes that span the nuclear envelope. Active nucleo-cytoplasmic transport of proteins, in particular, is performed mainly by a family of carriers called karyopherins, which includes about twenty members in mammals. Some of them, called importins, recognize nuclear localization signals (NLSs) in their substrates and convey them into the nucleus. Others, called exportins, recognize nuclear export signals (NESs) in their substrates and bring them back to the cytoplasm. Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, where they can often fulfill different functions. RBPs also frequently localize into specialized microdomains that are not delimited by a membrane but in which specific factors are concentrated. Those include processing bodies and stress granules, which are cytoplasmic foci associated with mRNA decay, storage and translational repression. Post-transcriptional regulations mediated by RBPs can therefore be modulated rapidly and efficiently through changes in the localization of RBPs.The first part of this work focuses on the subcellular localization and nucleo-cytoplasmic transport of the Drosophila RBP dTIS11. Like its mammalian and yeast homologues, dTIS11 binds AU-rich elements in the 3’UTR of its target mRNAs, and stimulates their rapid deadenylation and decay. Here, we have observed that although dTIS11 appears to be located mostly in the cytoplasm, it is constantly shuttling in and out of the nucleus. We show that the export of dTIS11 from the nucleus depends on the CRM1 exportin and is mediated by a hydrophobic NES that encompasses residues 101 to 113 in dTIS11 sequence. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This PY-NLS partially overlaps the second zinc finger (ZnF2) of dTIS11. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zn2+ ion unmask dTIS11 and TTP PY-NLS and promote nuclear import. Taken together, our results indicate that the nuclear export of Drosophila and mammalian TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism might rely on a conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.In the second part, we present preliminary results which implicate the nucleo-cytoplasmic transport machinery in the assembly of stress granules (SGs) in mammalian cells. SGs contain silenced mRNPs which resemble stalled initiation complexes, and they form transiently in response to acute stress, concomitantly with a global arrest of translation. While their exact role remains undefined, it seems clear that SGs are able to exchange mRNPs with polysomes and with PBs, and that they are connected to post-transcriptional and translational regulations of gene expression during stress. Here, we show that inhibition of Transportin-1 expression or function does not affect the translational status of cells but impairs the assembly of stress granules. Finally, we show that Transportin-1 and -2B, but not -2A, localize into stress granules in response to several stresses. In conclusion, we suggest two potential new roles for Transportins, in the nucleo-cytoplasmic traffic of TIS11 proteins on the one hand and in the assembly of stress granules on the other hand./Le compartimentage nucléo-cytoplasmique permet aux cellules eucaryotes de réguler l’expression génétique par des mécanismes post-transcriptionnels élaborés. Les ARN messagers subissent plusieurs étapes de maturation dans le noyau avant d’être exportés vers le cytoplasme où ils sont traduits et dégradés. Ces processus sont effectués via des protéines de liaison à l’ARN, ou RBPs. Beaucoup de RBPs exercent des fonctions différentes dans le noyau et dans le cytoplasme, et leur activité peut dès lors être rapidement modulée par une modification de leur localisation.Le transport nucléo-cytoplasmique actif des protéines s’effectue à travers les pores nucléaires et fait majoritairement appel à des transporteurs solubles de la famille des karyophérines. Ceux-ci reconnaissent au sein des protéines à transporter une séquence-passeport appelée NLS (nuclear localization signal) ou NES (nuclear export signal) selon la direction nécessitée. Le présent travail comporte deux parties. La première porte sur la localisation subcellulaire et le transport nucléo-cytoplasmique des protéines de la famille TIS11, et plus particulièrement de dTIS11 qui est le seul représentant de cette famille chez la Drosophile. Comme ses homologues dans d’autres espèces, dTIS11 est une RBP qui favorise la déadénylation et la dégradation de ses ARN messagers cibles. Nos résultats démontrent que dTIS11 fait la navette entre le noyau et le cytoplasme. L’export de dTIS11 hors du noyau est réalisé par la karyophérine CRM1 et fait appel à un NES différent de celui présent chez les protéines TIS11 mammaliennes. Nous identifions également un NLS cryptique au sein du domaine à deux doigts de zinc avec lequel dTIS11 lie l’ARN. Ce NLS correspond partiellement au signal consensus reconnu par la Transportine. Il est démasqué par la mutation du second doigt de zinc ;dans ces conditions, il permet l’import de dTIS11 par la Transportine. Enfin, nous montrons qu’il est conservé dans d’autres protéines de la famille TIS11. Dans la seconde partie, nous nous intéressons aux granules de stress, qui sont des microdomaines cytoplasmiques dans lesquels se concentrent des RBPs et des ARN messagers non traduits en réponse à un stress cellulaire. Nous montrons que les karyophérines appartenant à la sous-famille des Transportines sont présentes dans ces granules et que l’inhibition de l’expression ou de la fonction des Transportines réduit la formation de ces granules en réponse à divers stress cellulaires. Nous écartons la possibilité que ce résultat soit un effet indirect d’un ralentissement du métabolisme traductionnel. Nos résultats suggèrent donc une implication des Transportines dans la formation des granules de stress. Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Transportin-1 and Transportin-2: protein nuclear import and beyond.

Nearly 20 years after its identification as a new β-karyopherin mediating the nuclear import of the RNA-binding protein hnRNP A1, Transportin-1 is still commonly overlooked in comparison with its best known cousin, Importin-β. Transportin-1 is nonetheless a considerable player in nucleo-cytoplasmic transport. Over the past few years, significant progress has been made in the characterization of the nuclear localization signals (NLSs) that Transportin-1 recognizes, thereby providing the molecular basis of its diversified repertoire of cargoes. The recent discovery that mutations in the Transportin-dependent NLS of FUS cause mislocalization of this protein and result in amyotrophic lateral sclerosis illustrates the importance of Transportin-dependent import for human health. Besides, new functions of Transportin-1 are emerging in processes other than nuclear import. Here, we summarize what is known about Transportin-1 and the related β-karyopherin Transportin-2.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: re.jinfo:eu-repo/semantics/publishe

Conformation-regulated lateral segregation of the Can1 permease

Can1, the arginine permease of yeast, is dynamically localized in the Membrane Compartment occupied by Can1 (MCC), a plasma membrane microdomain associated with a cytoplasmic protein scaffold termed eisosome (1). Can1 undergoes ubiquitin (Ub)-dependent endocytosis in response to substrate transport (2). Yet the physiological significance of the enrichment of Can1 in the MCC with respect to its functionality and substrate-induced endocytosis remains controversial (3,4). We have now investigated the potential role of lipids and Ub in the lateral plasma membrane distribution of Can1. We found that the MCC enrichment of Can1 requires proper sphingolipid biosynthesis. In keeping with previous studies, addition of arginine promotes exit of Can1 from the MCCs. Our results show that this redistribution is dependent on Can1 activity but not on its ubiquitylation. Furthermore, an inactive Can1 mutant seems to adopt a conformation favoring its exit from the MCCs. Our data are consistent with a model where some conformational changes adopted by Can1 during substrate transport promote its segregation out of the MCCs, followed by its ubiquitylation and endocytosis.info:eu-repo/semantics/nonPublishe

A new candidate protein for the function of the apical thyrocyte iodide channel

The iodide uptake in the thyroid follicular lumen requires its first active transport by the Na+/I-symporter NIS, at the basal side followed by its efflux from the cell at the luminal side. The latter step is generally believed to be mediated by pendrin.We have identified a new candidate thyroid apical iodide channel (TAIC). We show that the TAIC is expressed in human and rat thyroid.To determine the properties of TAIC and pendrin we have expressed both pendrin and TAIC in HEK293T cells and compared their properties to those of the existing channel in PCCl3 and FRTL5 rat thyroid cell lines. The properties of TAIC but not those of pendrin nicely fit those of the channel functional in the cell lines. We therefore conclude that TAIC is a functional iodide channel in rat thyroid. It is not excluded that in different species several channels, including pendrin, may carry this function.info:eu-repo/semantics/nonPublishe

FTY720-induced endocytosis of yeast and human amino acid transporters is preceded by reduction of their inherent activity and TORC1 inhibition

FTY720 is a sphingoid base analog that acts as an anticancer agent in animal models. Its effect on tumor cells stems largely from its ability to trigger endocytosis of several nutrient transporters. The observation that FTY720 similarly stimulates downregulation of amino acid permeases in yeast suggests that the cellular mechanisms it targets, which are still poorly characterized, are evolutionarily conserved. We here report that adding FTY720 to yeast cells results in rapid inhibition of the intrinsic activity of multiple permeases. This effect is associated with inhibition of the TORC1 kinase complex, which in turn promotes ubiquitin-dependent permease endocytosis. Further analysis of the Gap1 permease showed that FTY720 elicits its ubiquitylation via the same factors that promote this modification when TORC1 is inhibited by rapamycin. We also show that FTY720 promotes endocytosis of the LAT1/SLC7A5 amino acid transporter in HeLa cells, this being preceded by loss of its transport activity and by mTORC1 inhibition. Our data suggest that in yeast, TORC1 deactivation resulting from FTY720-mediated inhibition of membrane transport elicits permease endocytosis. The same process seems to occur in human cells even though our data and previous reports suggest that FTY720 promotes transporter endocytosis via an additional mechanism insensitive to rapamycin.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Molecular insights into Adgra2/Gpr124 and Reck intracellular trafficking

Adgra2, formerly known as Gpr124, is a key regulator of cerebrovascular development in vertebrates. Together with the GPI-anchored glycoprotein Reck, this adhesion GPCR (aGPCR) stimulates Wnt7-dependent Wnt/β-catenin signaling to promote brain vascular invasion in an endothelial cell-autonomous manner. Adgra2 and Reck have been proposed to assemble a receptor complex at the plasma membrane, but the molecular modalities of their functional synergy remain to be investigated. In particular, as typically found in aGPCRs, the ectodomain of Adgra2 is rich in protein-protein interaction motifs whose contributions to receptor function are unknown. In opposition to the severe ADGRA2 genetic lesions found in previously generated zebrafish and mouse models, the zebrafish ouchless allele encodes an aberrantly-spliced and inactive receptor lacking a single leucine-rich repeat (LRR) unit within its N-terminus. By characterizing this allele we uncover that, in contrast to all other extracellular domains, the precise composition of the LRR domain determines proper receptor trafficking to the plasma membrane. Using CRISPR/Cas9 engineered cells, we further show that Adgra2 trafficking occurs in a Reck-independent manner and that, similarly, Reck reaches the plasma membrane irrespective of Adgra2 expression or localization, suggesting that the partners meet at the plasma membrane after independent intracellular trafficking events

Three-dimensional visualization of termite (Apicotermitinae) enteric valve using confocal laser scanning microscopy

Humivorous termites are dominant members of tropical rainforest soil communities. In the soil-feeding subfamily Apicotermitinae (Termitidae), the enteric valve connecting the first section of the hindgut to the paunch often displays a complex sclerotized armature everted towards the lumen of the paunch. This structure is central in termite taxonomy but its function remains hypothetical. Here, we evaluate the potential of confocal laser scanning microscopy to provide detailed imaging of the valve of Anoplotermes parvus, by comparison with bright-field microscopy and scanning electron microscopy. We detected a strong far-red emission of the enteric valve armature that sharply contrasted with the surrounding tissues, providing a convenient method to highlight minute structural elements of the valve and its three-dimensional structure. The method is easy to use and is applicable to standard archival material as demonstrated by images of enteric valves of four other Apicotermitinae species. It may represent a valuable asset for the study of termite enteric valves, for the purpose of taxonomy or functional morphology.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Competitive interplay between the cytoplasmic role of dTIS11 in ARE-mediated decay and its Transportin-dependent nuclear import

info:eu-repo/semantics/nonPublishe