Basic cell penetrating peptides induce plasma membrane positive curvature, lipid domain separation and protein redistribution

International audienceBasic cell penetrating peptides are tools for molecular cellular internalization of non membrane permeable molecules. Their uptake mechanisms involve energy-dependent and energy-independent pathways such as endocytosis, direct translocation or physical endocytosis. These mechanisms are ruled by both, the peptides physicochemical properties and structure and by the membrane lipids characteristics and organisation. Herein we used plasma membrane spheres and membrane models to study the membrane perturbations induced by three arginine-rich cell penetrating peptides. Nona-arginine (R9) and the amphipathic peptide RWRRWWRRW (RW9) induced positive membrane curvature in the form of buds and membrane tubes. Membranous tubes underwent rolling resulting in formation of multilamellar membrane particles at the surface of the plasma membrane spheres. The amphipathic peptides RW9 and RRWRRWWRRWWRRWRR (RW16) provoked lipid and membrane associated protein domain separation as well as changes in membrane fluidity and cholesterol redistribution. These data suggest that membrane domains separation and the formation of multilamellar membranous particles would be involved in arginine-rich cell penetrating peptides internalization

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

Structure-Function Relations in Oxaloacetate Decarboxylase Complex. Fluorescence and Infrared Approaches to Monitor Oxomalonate and Na+ Binding Effect

ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the carboxyltransferase catalytic site. characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements

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

Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?

Membrane proteins (MPs) are essential for cellular functions. Understanding the functions of MPs is crucial as they constitute an important class of drug targets. However, MPs are a challenging class of biomolecules to analyze because they cannot be studied outside their native environment. Their structure, function and activity are highly dependent on the local lipid environment, and these properties are compromised when the protein does not reside in the cell membrane. Mammalian cell membranes are complex and composed of different lipid species. Model membranes have been developed to provide an adequate environment to envisage MP reconstitution. Among them, tethered-Bilayer Lipid Membranes (tBLMs) appear as the best model because they allow the lipid bilayer to be decoupled from the support. Thus, they provide a sufficient aqueous space to envisage the proper accommodation of large extra-membranous domains of MPs, extending outside. Additionally, as the bilayer remains attached to tethers covalently fixed to the solid support, they can be investigated by a wide variety of surface-sensitive analytical techniques. This review provides an overview of the different approaches developed over the last two decades to achieve sophisticated tBLMs, with a more and more complex lipid composition and adapted for functional MP reconstitution

La créatine kinase mitochondriale - organisatrice de la membrane mitochondriale ? (ségrégation de la cardiolipine sur des monocouches phospholipidiques)

En utilisant comme modèle membranaire les monocouches de Langmuir à l interface air-tampon et des techniques de caractérisation spécifiques, nous avons obtenu des nouvelles informations sur l interaction entre la créatine kinase mitochondriale et la membrane interne de la mitochondrie. Par microscopie à l angle de Brewster nous avons pu visualiser la formation de complexes spécifiques cardiolipine-CKmt, avec ségrégation de la cardiolipine dans la membrane biomimétique. La fixation de la CKmt a lieu avec insertion de domaines protéiques parmi les lipides et abouti à une stabilisation globale du film interfacial, comme indiqué par des mesures de capacité différentielle d une électrode de mercure. Une réorientation des domaines protéiques, principalement des hélices a, a également pu être mise en évidence par spectroscopie infrarouge de réflexion-absorption par modulation de la polarisation (PM-IRRAS). L effet de la CKmt sur la morphologie de la monocouche dépend du degré d insaturation des chaînes acyles. Ceci est en relation avec la forte proportion d acide linoléique (C18:2), caractéristique des molécules de cardiolipine dans les cellules musculaires. Le phénomène de ségrégation induit par la CKmt, pourrait contribuer à la modulation de la distribution de cardiolipine et avoir des conséquences sur la fixation d autres molécules comme le cytochrome c ou les protéines de la famille Bcl-2. La membrane mitochondriale étant siège des nombreux phénomènes de peroxydation, de part sa structure et sa localisation, la CKmt est une cible des molécules oxydantes générées. Parmi ces molécules, le 4-hydroxynonénal, inactive l enzyme, modifie sa structure et induit l accumulation de complexes protéiques agrégés au niveau de la membrane. L interaction de la CKmt avec la membrane biomimétique est fortement perturbée, aussi bien en monocouche que sur les liposomesaThe interaction between the mitochondrial isoform of creatine kinase (mtCK) and a mitochondrial inner membrane biomimetic model was analysed using phospholipid monolayers at the air-buffer interface. New insight on the structural and morphological consequences both on protein and membrane was obtained. As visualised by Brewster angle microscopy, mtCK induces formation of specific protein cardiolipin complexes, leading to the segregation of cardiolipin molecules. MtCK binding occurs with partial insertion between lipids and results in an overall stabilisation of the membrane, as indicated by differential capacity measurements. A reorientation of protein -helical structures was also observed by polarisation modulation infrared reflexion-absorption spectroscopy (PM-IRRAS). Formation of mtCK cardiolipin complexes was found to be highly dependable of acyl chain unsaturation. This is of physiological relevance as one of the main characteristics of cardiolipin in muscle mitochondria is the high percentage of linoleic acid (C18:2). MtCK-induced segregation may contribute to membrane organisation by modulating cardiolipin distribution, and consequently membrane binding of other molecules such as cytochrome c or Bcl-2 family proteins. Mitochondrial membranes are the site of numerous peroxydation processes. MtCK structure and localisation in the vicinity of the membrane make it a target for oxidative molecules produced during such processes. Among these molecules, 4-hydroxynonenal, induces enzyme inactivation, modifies its structure and induces accumulation of aggregates at membranes. MtCK binding to model membranes, liposomes or monolayers, is thus disturbed. All these complex perturbation were analysed and debated under a physiological point of view along this thesis workLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Basic cell penetrating peptides induce plasma membrane positive curvature, lipid domain separation and protein redistribution

International audienceBasic cell penetrating peptides are tools for molecular cellular internalization of non membrane permeable molecules. Their uptake mechanisms involve energy-dependent and energy-independent pathways such as endocytosis, direct translocation or physical endocytosis. These mechanisms are ruled by both, the peptides physicochemical properties and structure and by the membrane lipids characteristics and organisation. Herein we used plasma membrane spheres and membrane models to study the membrane perturbations induced by three arginine-rich cell penetrating peptides. Nona-arginine (R9) and the amphipathic peptide RWRRWWRRW (RW9) induced positive membrane curvature in the form of buds and membrane tubes. Membranous tubes underwent rolling resulting in formation of multilamellar membrane particles at the surface of the plasma membrane spheres. The amphipathic peptides RW9 and RRWRRWWRRWWRRWRR (RW16) provoked lipid and membrane associated protein domain separation as well as changes in membrane fluidity and cholesterol redistribution. These data suggest that membrane domains separation and the formation of multilamellar membranous particles would be involved in arginine-rich cell penetrating peptides internalization

Basic cell penetrating peptides induce plasma membrane positive curvature, lipid domain separation and protein redistribution.

International audience: Basic cell penetrating peptides are tools for molecular cellular internalization of non membrane permeable molecules. Their uptake mechanisms involve energy-dependent and energy-independent pathways such as endocytosis, direct translocation or physical endocytosis. These mechanisms are ruled by both, the peptides physicochemical properties and structure and by the membrane lipids characteristics and organisation. Herein we used plasma membrane spheres and membrane models to study the membrane perturbations induced by three arginine-rich cell penetrating peptides. Nona-arginine (R9) and the amphipathic peptide RWRRWWRRW (RW9) induced positive membrane curvature in the form of buds and membrane tubes. Membranous tubes underwent rolling resulting in formation of multilamellar membrane particles at the surface of the plasma membrane spheres. The amphipathic peptides RW9 and RRWRRWWRRWWRRWRR (RW16) provoked lipid and membrane associated protein domain separation as well as changes in membrane fluidity and cholesterol redistribution. These data suggest that membrane domains separation and the formation of multilamellar membranous particles would be involved in arginine-rich cell penetrating peptides internalization

Mitochondrial creatine kinase interaction with heterogeneous monolayers : effect on lipid lateral organization

International audienceOur study highlights the tight relationship between protein binding to monolayers and the phase-state of the phospholipids. Interaction of mitochondrial creatine kinase with phospholipidic membranes was analysed using a two-phase monolayer system containing anionic phospholipids under chain mismatch conditions. Monolayers were made up of mixtures of DMPC/DPPG or DPPC/DMPG containing 40% negatively charged phospholipids which is approximately the negative charge content of the mitochondrial inner membrane. Langmuir isotherms of these monolayers showed that they underwent a phase transition from a liquid expanded state to a liquid-condensed phase at about 2 mN/m and 5 mN/m respectively. Interface morphology modifications caused by injection of mtCK under these monolayers at low or high surface pressure were monitored by Brewster angle microscopy. This work provides evidence that the presence at the air/water interface of discrete domains with increased charge density, may lead to difference in partition of soluble proteins such as mtCK, interacting with the lipid monolayer. Conversely these proteins may help to organize charged phospholipid domains in a membrane

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