RICIN-BINDING PROTEINS ALONG THE ENDOCYTIC PATHWAY: THE MAJOR ENDOSOMAL RICIN-BINDING PROTEIN IS ENDOSOME-SPECIFIC

International audienceRicin is internalized after binding at the cell surface via lectin activity of the B-chain recognizing terminal galactose residues. Ricin-A chain is then translocated to the cytosol from various endocytic structures. Cell death is the result of catalytic inactivation of protein synthesis. Using125I-ricin overlays, we examined the distribution of ricin binding-proteins within highly purified preparations of plasma membrane vesicles, endosomes and lysosomes from lymphocytes. All compartments of the endocytic pathway had distinct profiles; some ricin-binding proteins were present throughout the pathway; others were restricted to the plasma membrane and endosomes. The major endosomal protein recognized by125I-ricin, a 166 kDa glycoprotein, was endosome-specific. When endosomal proteins were solubilized before chromatography onto ricin-agarose this protein was also by far the major specifically-bound glycoprotein. This 166 kDa glycoprotein might be involved in ricin translocation from this compartment

Translocation of Full-length Pseudomonas Exotoxin from Endosomes Is Driven by ATP Hydrolysis but Requires Prior Exposure to Acidic pH

International audienceWe attached human transferrin to Pseudomonas exotoxin A (PE) to specifically localize this toxin to the endosomal compartment and study its translocation from purified endosomes using a cell-free assay. Transferrin was linked to PE via a disulfide bond. Chemical derivatization inactivated the PE cell-binding domain, and transferrin-PE was found to be endocytosed via the transferrin receptor only. Transferrin was also conjugated to a truncated PE with no receptor-binding domain (PE46). After labeling mouse lymphocytes with radiolabeled transferrin-PE or transferrin-PE46 and endosome isolation, selective translocation of the full-sized toxin portion of the conjugate was observed in a cell-free system. This translocation was strictly dependent upon ATP hydrolysis and was not affected when the acidity of the endosome lumen was neutralized using weak bases, protonophores, or bafilomycin A1. Nevertheless, when present during cell labeling, inhibitors of endosome acidification prevented PE from acquiring translocation competence. Similar inhibition was observed when endocytosis was performed in the presence of brefeldin A, a drug known to interfere with the delivery of endocytic tracers to acidic endosomes. Our data indicate that full-length PE can be transferred to the cytosol directly from endosomes during intoxication by PE conjugates and that, although exposure to acidic pH is a prerequisite for translocation, ATP hydrolysis directly provides the energy required for PE translocation

Endosomes et translocation de toxines

Dans cette revue nous avons résumé les résultats que nous avons obtenus sur l’entrée de toxines, ricine, exotoxine A de Pseudomonas aeruginosa (PE) et toxine diphtérique (DT) principalement, dans les cellules animales. Nous avons surtout étudié le processus de translocation permettant à ces toxines de traverser la membrane endosomale et de passer dans le cytosol où se trouve leur cible : le système de synthèse des protéines qu’elles vont inactiver, tuant ainsi la cellule. Cette étape de translocation est limitante cinétiquement pour la toxicité de ces toxines. Nous avons montré que ces trois toxines traversent la membrane en utilisant des mécanismes différents. La translocation de la ricine ne dépend que de l’hydrolyse de l’ATP cytosolique, alors que PE requiert tout d'abord une exposition au pH acide endosomal (pH~6), probablement pour s’insérer dans la membrane, avant d’être transportée vers le cytosol via un mécanisme dépendant de l’hydrolyse de l’ATP cytosolique. La DT est transportée vers le cytosol en réponse au gradient de pH endosome-cytosol. En utilisant des conjugués avec la dihydrofolate réductase, nous avons pu montrer indirectement que la ricine et PE devaient être dépliées pour traverser la membrane endosomale. Par une approche de délétion, nous avons produit un mutant de PE qui traverse les membranes plus rapidement et présente donc une cytotoxicité améliorée

A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

International audiencePseudomonas exotoxin A (PE) is a cytotoxin composed of three structural domains. Domain I is responsible for cell binding, domain II for membrane translocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death. To investigate the role of the six alpha-helices (A-F) that form the translocation domain, we deleted them successively one at a time. All mutants showed native cell-binding and catalytic activities, indicating that deletions specifically affected translocation activity. This step of the intoxication procedure was examined directly using a cell-free translocation assay, and indirectly by monitoring cytotoxicity. Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limiting for PE intoxication. Deletion of B, C and D helices resulted in non-toxic and non-translocating molecules, whereas mutants lacking the A or E helix displayed significant cytotoxicity albeit 500-fold lower than native PE. We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparatively dispensable. The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% higher than PE, along with a three- to sixfold increase in cytotoxicity in all tested cell lines. This toxin is the most in vitro active PE mutant obtained until now. Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active molecule

Ricin A Chain Can Transport Unfolded Dihydrofolate Reductase into the Cytosol

International audienceRicin is a heterodimeric protein toxin. The ricin A chain is able to cross the membrane of intracellular compartments to reach the cytosol where it catalytically inactivates protein synthesis. It is linked via a disulfide bond to the B chain, a galactose-specific lectin, which allows ricin binding at the cell surface and endocytosis. To examine the potential of ricin A to carry proteins into the cytosol and the requirement for unfolding of the passenger protein, we connected mouse dihydrofolate reductase (DHFR) to ricin A by gene fusion via a spacer peptide. DHFR-ricin A expressed in Escherichia coli displayed the biological activities of the parent proteins and associated quantitatively with ricin B to form DHFR-ricin. The resulting toxin was highly cytotoxic to cells (4-8-fold less than recombinant ricin). DHFR-ricin cytotoxicity was inhibited by methotrexate, a DHFR inhibitor stabilizing DHFR-ricin A in a folded conformation. The DHFR moiety of DHFR ricin bound to the plasma membrane. Although methotrexate prevented this binding, it did not significantly affect DHFR-ricin endocytosis, which proceeded via ricin B chain. Intoxication kinetics data and a cell-free translocation assay demonstrated that protection of cells from DHFR-ricin cytotoxicity resulted from a selective inhibition by methotrexate of DHFR-ricin A translocation. We conclude that ricin A is a potential carrier of proteins to the cytosol, provided that the passenger protein is able to unfold for transmembrane transport

Expression of Functional Ricin B Chain using the Baculovirus System

International audienceThe ricin B chain (RTB) was expressed using a baculovirus expression system. The RTB coding sequence downstream of the preproricin signal sequence was inserted in the baculovirus transfer vector pM34T. After cotransfection of Spodoptera frugiperda Sf9 cells with linearized baculovirus DNA, recombinant viruses were selected. cloned and amplified. Upon infection of Sf9 cells with these recombinant baculoviruses, RTB production was revealed by immunoblotting. RTB expression using this system was optimum 72 h after infection of the cells at a multiplicity of infection of 3. RTB produced was glycosylated and had an apparent molecular mass of 34 kDa. Most of the signal sequence was removed, but the resulting recombinant RTB had a 13‐residue N‐terminus extension. Immunofluorescence analysis showed that this protein was located in the endoplasmic reticulum/Golgi region of the cell. RTB was not present at the plasma membrane. Secretion was enhanced by the addition of lactose to the cell‐culture medium up to 50 mM. Purification was achieved from both cells and media using immobilized lactose and the lectin activity of RTB. Results obtained with the purified recombinant protein (more than 2 mg/l culture) were identical to those obtained with native RTB in all assays for biological activity; binding, internaliza‐tion and reassociation with the ricin A chain to produce toxic ricin. Moreover, the RTB translocation capacity was not altered by the N‐terminal peptide, showing that recombinant RTB could be used to deliver antigenic peptides to the cytosol for the induction of cell‐mediated immunity

Influence of Deletions within Domain II of Exotoxin A on Its Extracellular Secretion from Pseudomonas aeruginosa

International audiencePseudomonas aeruginosa is a gram-negative bacterium that secretes many proteins into the extracellular medium via the Xcp machinery. This pathway, conserved in gram-negative bacteria, is called the type II pathway. The exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. This information may be present within a conformational motif. The nature of this signal has been examined for P. aeruginosa exotoxin A (PE). Previous studies failed to identify a common minimal motif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one found additional information at the C terminus. In this study, we assess the role of the central PE domain II composed of six α-helices (A to F). The secretion behavior of PE derivatives, individually deleted for each helix, was analyzed. Helix E deletion has a drastic effect on secretion of PE, which accumulates within the periplasm. The conformational rearrangement induced in this variant is predicted from the three-dimensional PE structure, and the molecular modification is confirmed by gel filtration experiments. Helix E is in the core of the molecule and creates close contact with other domains (I and III). Deletion of the surface-exposed helix F has no effect on secretion, indicating that no secretion information is contained in this helix. Finally, we concluded that disruption of a structured domain II yields an extended form of the molecule and prevents formation of the conformational secretion motif

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal (125)I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect (125)I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when (125)I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired (125)I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure

Care management in a French cohort with down syndrome from the AnDDI-Rares/CNSA study

Down syndrome (DS) is a genetic neurodevelopmental disorder. In individuals with DS, a multidisciplinary approach to care is required to prevent multiple medical complications. The aim of this study was to describe the rehabilitation, medical care, and educational and social support provided to school-aged French DS patients with varying neuropsychological profiles. A mixed study was conducted. Quantitative data were obtained from a French multicentre study that included patients aged 4-20 years with diverse genetic syndromes. Qualitative data were collected by semi-structured face-to-face interviews and focus groups. Ninety-five DS subjects with a mean age of 10.9 years were included. Sixty-six per cent had a moderate intellectual disability (ID) and 18.9% had a severe ID. Medical supervision was generally multidisciplinary but access to medical specialists was often difficult. In terms of education, 94% of children under the age of six were in typical classes. After the age of 15, 75% were in medico-social institutions. Analysis of multidisciplinary rehabilitation conducted in the public and private sectors revealed failure to access physiotherapy, psychomotor therapy and occupational therapy, but not speech therapy. The main barrier encountered by patients was the difficulty accessing appropriate facilities due to a lack of space and long waiting lists. In conclusion, children and adolescents with DS generally received appropriate care. Though the management of children with DS has been improved considerably, access to health facilities remains inadequate