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

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

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

Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths.

Biological membranes contain a large variety of lipids species compartmentalized in different domains heterogeneous in size, composition and dynamics. Cholesterol induces membrane ordered domains thanks to its affinity for saturated lipids. Membrane domains had been studied with fluorescent probes either linked to phospholipids and proteins or as individual fluorophore. However, no efficient formulation of a cholesterol probe has been available so far. Herein, we described a cholesterol-pyrene probe behaviour in heterogeneous membranes. We characterised the pyrene fluorescence spectra in liquid-ordered (Lo) and liquid-disordered (Ld) membranes. Using statistical multivariate analysis, we found out the most appropriate wavelengths for membrane domains studies. 373 nm and 379 nm were the most discriminant wavelengths to follow the liquid-ordered and the liquid-disordered environments. Cholesterol clustering behaviour was quantified by the modulation of the cholesterol-pyrene excimers peak (474 nm). In liquid-ordered membranes at low temperature, cholesterol-pyrene was found as multimers and as monomers. At high temperature, the liquid-ordered status of the membrane decreases and cholesterol-pyrene tends to cluster. In liquid-disordered membranes, cholesterol-pyrene was present mostly as monomers and the small quantity of excimers increased with temperature. Cholesterol-pyrene was used to test the ceramide effect on membranes, and presented a behaviour in agreement with the cholesterol behaviour reported in the literature. Overall, the presented data show that cholesterol-pyrene is an efficient sensor to study liquid ordered and liquid disordered organisation in membranes

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

The Ca 2+ -and phospholipid-binding protein Annexin A2 is able to increase and decrease plasma membrane order

International audienceAnnexin A2 (AnxA2) is a calcium-and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, HII). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes