Inhibition of the post-translational processing of microvillar hydrolases is associated with a specific decreased expression of sucrase-isomaltase and an increased turnover of glucose in Caco-2 cells treated with monensin

AbstractThe biosynthesis and post-translational processing of sucrase-isomaltase and dipeptidylpeptidase IV were studied by L-[35S]methionine labeling, immunoisolation with monoclonal antibodies and SDS-PAGE in post-confluent Caco-2 cells treated with monensin (10 μM, 48 h). In addition to its classical effect on the post-translational processing of both hydrolases, i.e. an inhibition of the conversion of the high-mannose to the complex glycosylated form of the enzymes, monensin was found to have two other effects: a marked decrease of sucrase-isomaltase expression, but not of dipeptidylpeptidase IV; an increased turnover of glucose, as substantiated by increased rates of glucose consumption and lactic acid production and a decreased glycogen content. Whether these two effects are related to the particular differentiation and metabolic status of Caco-2 cells is discussed, as well as a possible role for the drug-induced modifications of glucose turnover on the decreased expression of sucrase-isomaltase

Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin

International audienceCellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization. 2 Keywords: Membrane invagination/ metabolic energy/ penetratin/ penetrating peptide/ physical endocytosis/ self-induced endocytosis

Monensin inhibits the expression of sucrase-isomaltase in Caco-2 cells at the mRNA level

AbstractUsing L-[35S]methionine labeling, SDS-PAGE and Northern blot analysis of sucrase-isomaltase mRNA, two different concentrations of monensin were used to delineate in Caco-2 cells the effect of the drug on the conversion of the high mannose to the complex form of sucrase-isomaltase from its dual effect on the biosynthesis of the enzyme and on the rate of glucose consumption. At 0.1 μM the drug has no effect on the rate of glucose consumption and, although it inhibits the conversion of the high mannose to the complex form of the enzyme, it has no effect on the level of sucrase-isomaltase mRNA and on the amount of neosynthesized enzyme. At 1 μM, in addition to its inhibiting effect on the maturation of the enzyme, monensin provokes concomitantly an increase in the rate of glucose consumption and a decrease in the level of sucrase-isomaltase mRNA and in the amount of neosynthesized enzyme. All these effects are reversible within 48 h after removal of the drug

Rotavirus-Like Particles: A Novel Nanocarrier for the Gut

The delivery of bioactive molecules directly to damaged tissues represents a technological challenge. We propose here a new system based on virus-like particles (VLP) from rotavirus, with a marked tropism for the gut to deliver bio-active molecules to intestinal cells. For this, nonreplicative VLP nanoparticles were constructed using a baculovirus expression system and used to deliver an exogenous biomolecule, the green fluorescent protein (GFP), into either MA104 cells or intestinal cells from healthy and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated mice. Our results show that expression of rotavirus capsid proteins in baculovirus led to the auto assembly of VLP that display similar properties to rotavirus. In vitro experiments showed that VLP were able to enter into MA104 cells and deliver the reporter protein. Intragastric administration of fluorescent VLP in healthy and TNBS-treated mice resulted in the detection of GFP and viral proteins in intestinal samples. Our results demonstrate an efficient entry of non-replicative rotavirus VLP into the epithelial cell line MA104 and provide the first in vivo evidence of the potential of these nanoparticles as a promising safe candidate for drug delivery to intestinal cells

The Homeodomain Derived Peptide Penetratin Induces Curvature of Fluid Membrane Domains

BACKGROUND:Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS:Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains) in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation) are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like) membrane domains. CONCLUSIONS/SIGNIFICANCE:The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we showed that the membrane targets for these molecules are preferentially the fluid membrane domains and that the mechanism involves the induction of membrane negative curvature. Consequences on cellular uptake are discussed

Non-Metabolic Membrane Tubulation and Permeability Induced by Bioactive Peptides

BACKGROUND: Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P. METHODOLOGY/PRINCIPAL FINDINGS: Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. CONCLUSIONS/SIGNIFICANCE: We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated “physical endocytosis,” which represents a new pathway for peptide cellular internalization

Distinct Behaviour of the Homeodomain Derived Cell Penetrating Peptide Penetratin in Interaction with Different Phospholipids

Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial.In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG) to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β–sheet contributions.These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import) and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution

Caractérisation biochimique de structures macromoléculaires induites durant l'infection de cellules par le rotavirus

L infection de cellules par le rotavirus implique la formation de nombreux assemblages macromoléculaires qui recrutent, en plus des protéines virales, de nombreuses protéines cellulaires nécessaires à la morphogenèse virale, à la signalisation et à la mise en place de défenses anti-virales. Cette stratégie est commune à de nombreux virus. La connaissance de la composition, de la structure et du fonctionnement de ces complexes, dont certains ont été baptisés usines virales , est d un intérêt majeur pour la biologie cellulaire fondamentale et pour la conception de stratégies thérapeutiques innovantes contre le rotavirus. Ce court travail de thèse a été consacré à l isolement et à la caractérisation de différents complexes viraux correspondant à des étapes précoces de la morphogenèse virale. Un protocole de purification permettant le fractionnement du cytoplasme de cellules infectées par le rotavirus a été mis au point afin (i) d isoler des complexes macromoléculaires contenant la protéine de la capside intermédiaire, VP6, (ii) d en analyser le contenu protéique par électrophorèse bidimensionnelle avec une première dimension en conditions non dénaturantes (Blue Native PAGE) suivi d une deuxième dimension en conditions dénaturantes, (iii) de réaliser une étude protéomique fine pour identifier les protéines impliquées dans ces complexes. Cette approche a permis d identifier quatre familles de protéines : (a) des chaperonnes cytoplasmiques, parmi lesquelles Hsc70, Hsp70, Hsp90, (b) des chaperonnes réticulaires, telles que la calréticuline, Grp78 et la protéine disulfide isomérase (PDI), (c) des protéines associées à l actine dont l Ezrine, l a actinine 4, la GAPDH et (d) un groupe plus hétérogène contenant des protéines ayant des fonctions métaboliques et des co-chaperonnes. Des études d immunofluorescence en microscopie optique et des expériences de co-immunoprécipitation ont permis de confirmer l interaction, au sein de ces complexes macromoléculaires, de la protéine VP6 du rotavirus avec Hsp70, Hsp90, PDI et la calréticuline. Ce travail démontre ainsi la faisabilité d une approche originale qui doit permettre d avancer dans la compréhension des mécanismes précoces de l assemblage et du trafic du rotavirus.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Interaction de la protéine de spicule VP4 de rotavirus avec le cystosquelette d'actine

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF