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

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Analyse des éléments structuraux de l' homéoprotéine Engrailed 2 impliqués dans les fonctions de reconnaissance par résonance magnétique nucléaire

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

Implication des cystéines dans l'internalisation et la toxicité des peptides pénétrants

Les CPPs peuvent transporter à l intérieur des cellules différents types de molécules bioactives. Nous avons étudié, à l aide d une méthode basée sur l analyse par spectrométrie de masse MALDI-TOF, l effet de la présence de cystéines lors de l internalisation de deux séquences : pAntp et (R/W)9. L importance des propriétés redox des cystéines lors de la translocation a été montrée ; cette interactivité conduirait à la séquestration du peptide dans la membrane ou à une nouvelle voie d internalisation. Deux nouveaux protocoles permettant de différencier chimiquement le peptide membranaire ont permis de montrer que la cyclisation ou la conjugaison améliore l internalisation. De plus, ces deux séquences non cytotoxiques présentent de fortes activités antibactériennes, modulées par l état structural du peptide. Nous avons enfin montré que, lors d analyses par microscopie confocale, les informations obtenues sur cellules vivantes ou après reconnaissance biotine/avidine sont complémentaires.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Nouveaux vecteurs pseudo-peptidiques de structure linéaire, branchée ou dendrimérique (étude du transport d'une cargaison peptidique dans les cellules de mammifères)

L OBJECTIF DE CE TRAVAIL A ÉTÉ LE DÉVELOPPEMENT DE NOUVEAUX VECTEURS PSEUDO-PEPTIDIQUES, DE STRUCTURE VARIABLE, STABLES EN MILIEU BIOLOGIQUE, CAPABLES DE TRANSPORTER PLUS EFFICACEMENT UNE CARGAISON PEPTIDIQUE DANS DES CELLULES CHO. Afin de pouvoir accéder à des transporteurs de structure variée, la bis-ornithine, un nouvel acide aminé C , -disubstitué a été synthétisé. Ce composé est la pièce maîtresse d unités monomériques, dont l agencement modulable permet d accéder à des structures hétérofonctionnalisées : linéaires, branchées ou dendrimériques. Neuf nouveaux transporteurs ont ainsi été synthétisés sur support solide. Leur aptitude à transporter une espèce peptidique (le PKCi : inhibiteur des protéines kinases C) dans des cellules CHO a été évaluée, grâce à une nouvelle méthode de quantification, utilisant la spectrométrie de masse MALDI-TOF. Certains de ces composés sont jusqu'à 10 fois plus efficaces que les peptides vecteurs les plus utilisés. Une étude, par microscopie confocale, de la localisation intracellulaire de la cargaison, a permis de souligner une distribution, fonction de la nature du transporteur utilisé.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Synthèse de thiazolidines fonctionalisées, incorporation dans des PNA et étude des propriétés d'appariement

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

When cationic cell-penetrating peptides meet hydrocarbons to enhance in-cell cargo delivery

International audienceCell-penetrating peptides (CPPs) are short sequences often rich in cationic residues with the remarkable ability to cross cell membranes. In the past 20 years, CPPs have gained wide interest and have found numerous applications in the delivery of bioactive cargoes to the cytosol and even the nucleus of living cells. The covalent or non-covalent addition of hydrocarbon moieties to cationic CPPs alters the hydrophobicity/hydrophilicity balance in their sequence. Such perturbation dramatically influences their interaction with the cell membrane, might induce self-assembling properties and modifies their intracellular trafficking. In particular, the introduction of lipophilic moieties changes the subcellular distribution of CPPs and might result in a dramatically increase of the internalization yield of the co-transported cargoes. Herein, we offer an overview of different aspects of the recent findings concerning the properties of CPPs covalently or non-covalently associated to hydrocarbons. We will focus on the impact of the hydrocarbon moieties on the delivery of various cargoes, either covalently or non-covalently bound to the modified CPPs. We will also provide some key elements to rationalize the influence of the hydrocarbons moieties on the cellular uptake. Furthermore, the recent in vitro and in vivo successful applications of acylated CPPs will be summarized to provide a broad view of the versatility of these modified CPPs as small-molecules and oligonucleotides vectors

Peptides de transduction : analyse des relations "structure-fonction" à l'aide de membranes modèles

Dans ce travail, nous nous intéressons à une étape clef de l'internalisation des peptides dans les cellules : l'interaction peptide-membrane. Nous avons étudié les effets de trois peptides : La substance P, la pénétratine et un peptide amphipatique (RL)16 sur deux types de membranes modèles, les vésicules géantes unilamellaires (GUVs) et les vésicules larges unilamellaires (LUVs). Nous avons observé que la pénétratine provoque la formation de tubules à l'intérieur des vésicules géantes sans perte de perméabilité. Le peptide amphipatique (RL)16, quant à lui, affecte profondément la perméabilité des membranes ce qui se traduit notamment par l'éclatement des vésicules géantes. Enfin, la substance P ne semble pas avoir d'effet sur des deux types de membranes modèles

Tubular structures in heterogeneous membranes induced by the cell penetrating peptide penetratin

The delivery of active molecules into cells requires the efficient translocation of the plasma membrane barrier. Penetratin is a promising cell penetrating peptide is which crosses the cell membrane by a receptor and metabolic energy-independent mechanism. In previous work, we have shown that basic peptides induce membrane invaginations (i.e., tubes formation by induction of negative curvature of membranes) suggesting a new mechanism for cellular uptake of cell penetrating peptides: “physical endocytosis”. These effects on membrane curvature are favored in pure liquid disordered but not in pure liquid ordered (raft-like) membrane domains. Herein, we present experiments in heterogeneous membranes composed of mixed domains. The results show that Penetratin is able to induce invaginations in membranes in which liquid ordered and liquid disordered membranes coexist. We suggest that Penetratin is able to recruit specific lipids locally forming fluid membrane patches dispersed inside a liquid ordered membrane zone resulting in the invagination of tubes composed of heterogeneous membrane domains