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

Basic cell penetrating peptides are tools for molecular cellular internalization of nonmembrane 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 organization. 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.     

Cell biology meets biophysics to unveil the different mechanisms of penetratin internalization in cells

Although cell-penetrating peptides are widely used as molecular devices to cross membranes and transport molecules or nanoparticles inside cells, the underlying internalization mechanism for such behavior is still studied and discussed. One of the reasons for such a debate is the wide panel of chemically different cell-penetrating peptides or cargo that is used. Indeed the intrinsic physico-chemical properties of CPP and conjugates strongly affect the cell membrane recognition and therefore the internalization pathways. Altogether, the mechanisms described so far should be shared between two general pathways: endocytosis and direct translocation. As it is established now that one cell-penetrating peptide can internalize at the same time by these two different pathways, the balance between the two pathways relies on the binding of the cell-penetrating peptide or conjugate to specific cell membrane components (carbohydrates, lipids). Like endocytosis which includes clathrin- and caveolae-dependent processes and macropinocytosis, different translocation mechanisms could co-exist, an idea that emerges from recent studies. In this review, we will focus solely on penetratin membrane interactions and internalization mechanisms.     

Membrane interactions of two arginine-rich peptides with different cell internalization capacities

Cell penetrating peptides (CPPs) can cross cell membranes in a receptor independent manner and transport cargo molecules inside cells. These peptides can internalize through two independent routes: energy dependent endocytosis and energy independent translocation across the membrane, but the exact mechanisms are still unknown. The interaction of the CPP with different membrane components is certainly a preliminary key point that triggers internalization, such as the interaction with lipids to lead to the translocation process. In this study, we used two arginine-rich peptides, RW9 (RRWWRRWRR-NH(2)), which is a potent CPP, and RL9 (RRLLRRLRR-NH(2)) that, although binding tightly and accumulating on membranes, does not enter into cells. Using a set of experimental and theoretical techniques, we studied the binding, insertion and orientation of the peptides into different model membranes as well as the subsequent membrane reorganization. Herein we show that although the two peptides had rather similar behavior regarding lipid membrane interaction, subtle differences were found concerning the depth of peptide insertion, effect on the lipid chain ordering and kinetics of peptide insertion in the membrane, which altogether might explain their different cell internalization capacities. Molecular dynamics simulation studies show that some peptide molecules flipped their orientation over the course of the simulation such that the hydrophobic residues penetrated deeper in the lipid core region while Arg-residues maintained H-bonds with the lipid headgroups, serving as a molecular hinge in a conformation that appeared to correspond to the equilibrium one.     

Investigation of the sequence and length dependence for cell-penetrating prenylated peptides

Cell penetrating peptides are useful delivery tools for introducing molecules of interest into cells. A new class of cell penetrating molecules has been recently reported—cell penetrating, prenylated peptides. In this study a series of such peptides was synthesized to examine the relationship between peptide sequence and level of peptide internalization and to probe their mechanism of internalization. This study revealed that prenylated peptides internalize via a non-endocytotic pathway regardless of sequence. Sequence length and identity was found to play a role in peptide uptake but prenylated sequences as short as two amino acids were found to exhibit significant cell penetrating properties.     

Effect of the electrostatic potential on the internalization mechanism of cell penetrating peptides derived from TIRAP

In order to develop future therapeutic applications for cell penetrating peptides (CPPs), it is essential to characterize their internalization mechanisms, as they might affect the stability and the accessibility of the carried drug. Several internalization mechanisms have been described in literature, such as endocytosis and transduction. In this work we study the internalization mechanism in HeLa cells of two TIRAP derived peptides: pepTIRAP and pepTIRAP^ALA, where some of the cationic amino acids were replaced with alanines. Detailed analysis of internalization and the peptides electrostatic potential was carried out, to shed light on the internalization mechanism involved. Molecular modeling studies showed that the main difference identified between pepTIRAP and pepTIRAP^ALA is the distribution of their electrostatic potential field. The structure of pepTIRAP displays a predominantly positive potential when compared to pepTIRAP^ALA, which has a more balanced potential distribution. In addition, docking experiments show that interactions between pepTIRAP and negatively charged molecules on the cellular surface such as heparan sulfate are stronger than the ones exhibited by pepTIRAP^ALA. A mathematical model was proposed to quantify the amount of peptide internalized or non-specifically bound to the membrane. The model indicates a stronger interaction of pepTIRAP with the plasma membrane, compared to pepTIRAP^ALA. We propose these discrepancies are related to the differences in the electrostatic potential characteristics of each peptide. In the case of pepTIRAP, these interactions lead to the formation of nucleation zones, which are the first stage of the transduction internalization mechanism. These results should be considered for effective design of a cell penetrating peptide.     

Studies on the internalization mechanism of cationic cell-penetrating peptides

A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43-58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43-58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation.     

The CPP Tat enhances eGFP cell internalization and transepithelial transport by the larval midgut of Bombyx mori (Lepidoptera, Bombycidae)

Cell-Penetrating Peptides (CPPs) are short peptides that are able to translocate across the cell membrane a wide range of cargoes. In the past decade, different mammalian cell lines have been used to clarify the mechanism of CPPs penetration and to characterize the internalization process, which has been described either as an energy-independent direct penetration through the plasma membrane, or as endocytic uptake. Whatever the mechanism involved, the cell penetration properties of these peptides make their use very attractive as vector for promoting the cellular uptake of coupled bioactive macromolecules, such as peptides, proteins and oligonucleotides. Here we demonstrate, for the first time in insect, that cultured columnar cells from the larval midgut of Bombyx mori more readily internalize eGFP (enhanced Green Fluorescent Protein) when fused to CPP Tat. Tat-eGFP translocates across the plasma membrane of absorptive cells in an energy-independent and non-endocytic manner, since no inhibition of the fusion protein uptake is exerted by metabolic inhibitors and by drugs that interfere with the endocytic uptake. Moreover, the CPP Tat enhances the internalization of eGFP in the columnar cells of intact midgut tissue, mounted in a suitable perfusion apparatus, and the transepithelial flux of the protein. These results open new perspectives for effective delivery of insecticidal macromolecules targeting receptors located both within the insect gut epithelium and behind the gut barrier, in the hemocoel compartment.     

Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic pathways can be divided into clathrin-dependent and clathrin-independent pathways. While clathrin-dependent pathways are known to be involved in a variety of cellular processes in plants, clathrin-independent pathways have so far only been identified in animal and yeast cells. Here we show that internalization of fluorescent glucose into BY-2 cells leads to accumulation of the sugar in compartments of the endocytic pathway. This endocytic uptake of glucose was not blocked by ikarugamycin, an inhibitor of clathrin-dependent endocytosis, suggesting a role for clathrin-independent endocytosis in glucose uptake. Investigations of fusion and fission of single vesicles by membrane capacitance measurements revealed stimulation of endocytic activity by extracellular glucose. Glucose-stimulated fission of vesicles was not affected by addition of ikarugamycin or blocking of clathrin coat formation by transient over-expression of HUB1 (the C-terminal part of the clathrin heavy chain). These data demonstrate that clathrin-independent endocytosis does occur in plant cells. This pathway may represent a common mechanism for the uptake of external nutrients.     

Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis

Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasma-membrane-resident proteins including PIN auxin efflux carriers.     

Protein cargo delivery properties of cell-penetrating peptides. A comparative study

Application of cell-penetrating peptides for delivering various hydrophilic macromolecules with biological function into cells has gained much attention in recent years. We compared the protein transduction efficiency of four cell-penetrating peptides: penetratin, Tat peptide, transportan, and pVEC and studied the effects of various medium parameters on the uptake. Depletion of cellular energy and lowering of temperature strongly impaired the internalization of protein complexed with cell-penetrating peptides, confirming the endocytotic mechanism of peptide-mediated protein cellular transduction. Peptide-induced protein association with HeLa cells decreased 3-6-fold in energy-depleted cells. Inhibition of clathrin-dependent endocytosis by the hyperosmolar medium decreased the uptake of peptide-avidin complexes 1.5-3-fold and the removal of cholesterol from the plasma membrane 1.2-2-fold, suggesting that both clathrin-dependent and independent endocytosis were involved in peptide-induced cellular delivery of avidin. However, even under conditions of cellular energy depletion, ceasing of cellular traffic, and partial depolarization of plasma membrane, peptide-protein complexes associated with HeLa cells, as observed by FACS analysis and spectrofluorimetry. Among the studied peptides, pTat and transportan revealed higher protein transduction efficiency than penetratin or pVEC.     

A comprehensive model for the cellular uptake of cationic cell-penetrating peptides

The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so-called cell-penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis-independent cellular entry. In this study, we provide evidence that the Antennapedia-homeodomain-derived antennapedia (Antp) peptide, nona-arginine and the HIV-1 Tat-protein-derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis-independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant-negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.     

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.     

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The penetrating properties of the tumor homing peptide LyP‐1 in model lipid membranes

Cell‐penetrating peptides (CPPs) have the property to cross the plasma membrane and enhance its permeability. CPPs were successfully used to deliver numerous cargoes such as drugs, proteins, nucleic acids, imaging and radiotherapeutic agents, gold and magnetic nanoparticles, or liposomes inside cells. Although CPPs were intensively investigated over the past 20 years, the exact molecular mechanisms of translocation across membranes are still controversial and vary from passive to active mechanisms. LyP‐1 is a cyclic 9‐amino‐acids homing peptide that specifically binds to p32 receptors overexpressed in tumor cells. tLyP‐1 peptide is the linear truncated form of LyP‐1 and recognizes neuropilin (NRP) receptors expressed in glioma tumor tissue. Here, we investigate the interaction of the cyclic LyP‐1 peptide and linear truncated tLyP‐1 peptide with model plasma membrane in order to understand their passive, energy‐independent mechanism of uptake. The experiments reveal that internalization of tLyP‐1 peptides depends on membrane lipid composition. Inclusion of negatively charged phosphatidylserine (PS) or cone‐shaped phosphatidylethanolamine (PE) lipids in the composition of giant unilamellar vesicles facilitates the membrane adsorption and direct penetration but without inducing pore formation in membranes. In contrast, cyclic LyP‐1 peptide mostly accumulates on the membrane, with very low internalization, regardless of the lipid composition. Thus, the linear tLyP‐1 peptide has enhanced penetrating properties compared with the cyclic LyP‐1 peptide. Development of a mutant peptide containing higher number of arginine amino acids and preserving the homing properties of tLyP‐1 may be a solution for new permeable peptides that facilitate the internalization in cells and further the endosomal escape as well.     

Enhanced cellular uptake and metabolic stability of lipo-oligoarginine peptides

Developing efficient cellular delivery vectors is crucial for designing novel therapeutic agents to enhance their plasma membrane permeability and metabolic stability in cells. Previously, we engineered cell penetrating peptide vectors named as "lipo-oligoarginine peptides" (LOAPs) by conjugating a proper combination of fatty acid and oligoarginine that translocated into cell easily without adverse effect on cell viability. In the present study, we report a systemic evaluation of cellular uptake and metabolic stability of LOAPs in Jurkat cells by introducing different combination of D-Arg residues in the peptide backbone. The cellular uptake and intracellular fate, cell viability, and metabolic stability and proteolytic degradation patterns of various LOAPs consisted of different combination of L- and D-Arg sequences were confirmed by flow cytometry, cytotoxicity assay, and analytical RP-HPLC with MALDI-TOF mass. All investigated LOAPs penetrated the cell efficiently with low cellular toxicity. The LOAPs having D-Arg residues at their N-termini seemed to have better metabolic stability than the LOAPs having C-terminal D-Arg residues. The metabolic degradation patterns were similar among all investigated LOAPs. The major hydrolytic site was between lauroyl group and β-Ala residue. Without the lipid chain, the oligoarginine peptide was pumped out ofcells easily. The results presented in this study suggest that structurally modified LOAPs could be used as a novel CPP design toward improved therapeutic application.     

Translocation and Endocytosis for Cell-penetrating Peptide Internalization

Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells.     

Glycosphingolipid-facilitated membrane insertion and internalization of cobra cardiotoxin. The sulfatide.cardiotoxin complex structure in a membrane-like environment suggests a lipid-dependent cell-penetrating mechanism for membrane binding polypeptides

Cobra cardiotoxins, a family of basic polypeptides having lipid- and heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 angstroms resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.     

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.     

Stochastic entry of enveloped viruses: fusion versus endocytosis

Infection by membrane-enveloped viruses requires the binding of receptors on the target cell membrane to glycoproteins, or "spikes," on the viral membrane. The initial entry mechanism is usually classified as fusogenic or endocytotic. However, binding of viral spikes to cell surface receptors not only initiates the viral adhesion and the wrapping process necessary for internalization, but can simultaneously initiate direct fusion with the cell membrane. Both fusion and internalization have been observed to be viable pathways for many viruses. We develop a stochastic model for viral entry that incorporates a competition between receptor-mediated fusion and endocytosis. The relative probabilities of fusion and endocytosis of a virus particle initially nonspecifically adsorbed on the host cell membrane are computed as functions of receptor concentration, binding strength, and number of spikes. We find different parameter regimes where the entry pathway probabilities can be analytically expressed. Experimental tests of our mechanistic hypotheses are proposed and discussed.