Energy-independent translocation of cell-penetrating peptides occurs without formation of pores. A biophysical study with pep-1

Pep-1 is a cell-penetrating peptide (CPP) with the ability to translocate across biological membranes and introduce active proteins inside cells. The uptake mechanism used by this CPP is, as yet, unknown in detail. Previous results show that such a mechanism is endocytosis-independent and suggests that physical-chemical interactions between the peptide and lipid bilayers govern the translocation mechanism. Formation of a transmembrane pore has been proposed but this issue has always remained controversial. In this work the secondary structure of pep-1 in the absence/presence of lipidic bilayers was determined by CD and ATR-FTIR spectroscopies and the occurrence of pore formation was evaluated through electrophysiological measurements with planar lipid membranes and by confocal microscopy using giant unilamellar vesicles. Despite pep-1 hydrophobic domain tendency for amphipathic alpha-helix conformation in the presence of lipidic bilayers, there was no evidence for membrane pores in the presence of pep-1. Furthermore, alterations in membrane permeability only occurred for high peptide/lipid ratios, which induced the complete membrane disintegration. Such observations indicate that electrostatic interactions are of first importance in the pep-1-membrane interactions and show that pores are not formed. A peptide-lipid structure is probably formed during peptide partition, which favours peptide translocation.     

Consequences of nonlytic membrane perturbation to the translocation of the cell penetrating peptide pep-1 in lipidic vesicles

The action of the cell penetrating pep-1 at the molecular level is not clearly understood. The ability of the peptide to induce (1) vesicle aggregation, (2) lipidic fusion, (3) anionic lipid segregation, (4) pore or other lytic structure formation, (5) asymmetric lipidic flip-flop, and (6) peptide translocation across the bilayers in large unilamellar vesicles was studied using photophysical methodologies mainly related to fluorescence spectroscopy. Neflometry and turbidimetry techniques show that clustering of vesicles occurs in the presence of the peptide in a concentration- and anionic lipid content-dependent manner. Results from Forst?r resonance energy transfer-based methodologies prove lipidic fusion and anionic lipid segregation, but no evidence for pores or other lytic structures was found. Asymmetric lipid flip-flop was not detected either. A specific method related to the quenching of the rhodamine-labeled lipids by pep-1 was developed to study the eventual translocation of the peptide. Translocation does not occur in symmetrical neutral and negatively charged vesicles, except when a valinomycin-induced transmembrane potential exists. Our work strongly suggests that the main driving force for peptide translocation is charge asymmetry between the outer and inner leaflet of biological membranes and reveals that pep-1 is able to perturb membranes without being cytotoxic. This nonlytic perturbation is probably mandatory for translocation to occur.     

Translocation or membrane disintegration? Implication of peptide-membrane interactions in pep-1 activity.

The Cell membrane is impermeable for most peptides, proteins, and oligonucleotides. Moreover, some cationic peptides, the so-called cell-penetrating peptides (CPPs), are able to translocate across the membrane. This observation has attracted much attention because these peptides can be covalently coupled to different macromolecules, which are efficiently delivered inside the cell. The mechanism used by these peptides to pass across the membrane is a controversial matter of debate. It has been suggested that endocytosis is the main mechanism of internalization and this was confirmed by several studies for different peptides. Pep-1 is an exception worthy of attention for its ability to translocate cargo macromolecules without the need to be covalently attached to them. A preferential internalization by an endocytosis-independent mechanism was demonstrated both in vitro and in vivo. Pep-1 has a high affinity to lipidic membranes, it is able to insert and induce local destabilization in the lipidic bilayer, although without pore formation. No cytotoxic effects were found for pep-1 concentrations where translocation is fully operative. At much higher concentrations, membrane disintegration takes place by a detergent-like mechanism that resembles anti-microbial peptide activity. In this review, the ability of pep-1 to transverse the membrane by an endocytosis-independent mechanism, not mediated by pores as well as an ability to induce membrane disintegration at high peptide concentration, is demonstrated.     

Pore formation by a Bax-derived peptide: effect on the line tension of the membrane probed by AFM

Bax is a critical regulator of physiological cell death that increases the permeability of the outer mitochondrial membrane and facilitates the release of the so-called apoptotic factors during apoptosis. The molecular mechanism of action is unknown, but it probably involves the formation of partially lipidic pores induced by Bax. To investigate the interaction of Bax with lipid membranes and the physical changes underlying the formation of Bax pores, we used an active peptide derived from helix 5 of this protein (Bax-alpha5) that is able to induce Bax-like pores in lipid bilayers. We report the decrease of line tension due to peptide binding both at the domain interface in phase-separated lipid bilayers and at the pore edge in atomic force microscopy film-rupture experiments. Such a decrease in line tension may be a general strategy of pore-forming peptides and proteins, as it affects the energetics of the pore and stabilizes the open state.     

Re-evaluating the role of strongly charged sequences in amphipathic cell-penetrating peptides: a fluorescence study using Pep-1

Cell-penetrating peptides (CPPs) are able to translocate across biological membranes and deliver bioactive proteins. Cellular uptake and intracellular distribution of CPPs is commonly evaluated with fluorescent labels, which can alter peptide properties. The effect of carboxyfluorescein label in the Lys-rich domain of the amphipathic CPP pep-1, was evaluated and compared with non-labelled pep-1 in vitro and in vivo. A reduced membrane affinity and an endosomal-dependent translocation mechanism, at variance with non-labelled pep-1, were detected. Therefore, the charged domain is not a mere enabler of peptide adsorption but has a crucial role in the translocation pathway of non-labelled pep-1.     

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.     

Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential

The cell-penetrating peptide (CPP) pep-1 is capable of introducing large proteins into different cell lines, maintaining their biological activity. Two possible mechanisms have been proposed to explain the entrance of other CPPs in cells, endosomal-dependent and independent types. In this work, we evaluated the molecular mechanisms of pep-1-mediated cellular uptake of beta-galactosidase (beta-Gal) from Escherichia coli in large unilamellar vesicles (LUV) and HeLa cells. Fluorescence spectroscopy was used to evaluate the translocation process in model systems (LUV). Immunofluorescence microscopy was used to study the translocation in HeLa cells. Enzymatic activity detection enabled us to monitor the internalization of beta-Gal into LUV and the functionality of the protein in the interior of HeLa cells. Beta-Gal translocated into LUV in a transmembrane potential-dependent manner. Likewise, the extent of beta-Gal incorporation was extensively decreased in depolarized cells. Furthermore, beta-Gal uptake efficiency and kinetics were temperature-independent, and beta-Gal did not colocalize with endosomes, lysosomes, or caveosomes. Therefore, beta-Gal translocation was not associated with the endosomal pathway. Although an excess of pep-1 was mandatory for beta-Gal translocation in vivo, transmembrane pores were not formed as concluded from the trypan blue exclusion method. These results altogether indicated that protein uptake both in vitro with LUV and in vivo with HeLa cells was mainly, if not solely, dependent on negative transmembrane potential across the bilayer, which suggests a physical mechanism governed by electrostatic interactions between pep-1 (positively charged) and membranes (negatively charged).     

A lipocentric view of peptide-induced pores

Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, nonmediated leakage is not completely improbable. A high activation energy normally keeps unassisted bilayer permeation at a very low frequency, but lipids are able to self-organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions such as phase coexistence, external stress or perturbation associated to binding of nonlipidic molecules. Here, we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation of peptides, lipidic pores induced by different types of stress, and spontaneous transient bilayer defects driven by thermal fluctuations. Within such a lipocentric framework, amphipathic peptides are best described as pore-inducing rather than pore-forming elements. Active peptides bound to membranes can be understood as a source of internal surface tension which facilitates pore formation by diminishing the high activation energy barrier. This first or immediate action of the peptide has some resemblance to catalysis. However, the presence of membrane-active peptides has the additional effect of displacing the equilibrium towards the pore-open state, which is then maintained over long times, and reducing the size of initial individual pores. Thus, pore-inducing peptides, regardless of their sequence and oligomeric organization, can be assigned a double role of increasing the probability of pore formation in membranes to high levels as well as stabilizing these pores after they appear.     

Molecular dynamics simulations of hydrophilic pores in lipid bilayers

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.     

An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes.

The peptide fragment of the carboxy-terminal region of the human immunodeficiency virus (HIV) transmembrane protein (gp41) has been implicated in T-cell death. This positively charged, amphipathic helix (amino acids 828 to 848) of the envelope protein is located within virions or cytoplasm. We studied the interaction of the isolated, synthetic amphipathic helix of gp41 with planar phospholipid bilayer membranes and with Sf9 cells using voltage clamp, potentiodynamic, and single-cell recording techniques. We found that the peptide binds strongly to planar membranes, especially to the negatively charged phosphatidylserine bilayer. In the presence of micromolar concentrations of peptide sufficient to make its surface densities comparable with those of envelope glycoprotein molecules in HIV virions, an increase in bilayer conductance and a decrease in bilayer stability were observed, showing pore formation in the planar lipid bilayers. These pores were permeable to both monovalent and divalent cations, as well as to chloride. The exposure of the inner leaflet of cell membranes to even 25 nM peptide increased membrane conductance. We suggest that the carboxy-terminal fragment of the HIV type 1 envelope protein may interact with the cell membrane of infected T cells to create lipidic pores which increase membrane permeability, leading to sodium and calcium flux into cells, osmotic swelling, and T-cell necrosis or apoptosis.     

Membrane translocation mechanism of the antimicrobial peptide buforin 2

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.     

Polar angle as a determinant of amphipathic alpha-helix-lipid interactions: a model peptide study

Various physicochemical properties play important roles in the membrane activities of amphipathic antimicrobial peptides. To examine the effects of the polar angle, two model peptides, thetap100 and thetap180, with polar angles of 100 degrees and 180 degrees, respectively, were designed, and their interactions with membranes were investigated in detail. These peptides have almost identical physicochemical properties except for polar angle. Like naturally occurring peptides, these peptides selectively bind to acidic membranes, assuming amphipathic alpha-helices, and formed peptide-lipid supramolecular complex pores accompanied by lipid flip-flop and peptide translocation. Despite its somewhat lower membrane affinity, thetap100 exhibited higher membrane permeabilization activity, a greater flip-flop rate, as well as more antimicrobial activity due to a higher pore formation rate compared with thetap180. Consistent with these results, the peptide translocation rate of thetap100 was higher. Furthermore, the number of peptides constituting thetap100 pores was less than that of thetap180, and thetap100 pores involved more lipid molecules, as reflected by its cation selectivity. The polar angle was found to be an important parameter determining peptide-lipid interactions.     

Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin in planar phospholipid bilayer membranes

alpha-Hemolysin (HlyA) is an extracellular protein toxin (117 kDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed these data to quantify how HlyA diminished the line tension of the membrane (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. We discuss these results in terms of models for proteolipidic pores.     

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

Insights into buforin II membrane translocation from molecular dynamics simulations

Buforin II is a histone-derived antimicrobial peptide that readily translocates across lipid membranes without causing significant membrane permeabilization. Previous studies showed that mutating the sole proline of buforin II dramatically decreases its translocation. As well, researchers have proposed that the peptide crosses membranes in a cooperative manner by forming transient toroidal pores. This paper reports molecular dynamics simulations designed to investigate the structure of buforin II upon membrane entry and evaluate whether the peptide is able to form toroidal pore structures. These simulations showed a relationship between protein–lipid interactions and increased structural deformations of the buforin N-terminal region promoted by proline. Moreover, simulations with multiple peptides show how buforin II can embed deeply into membranes and potentially form toroidal pores. Together, these simulations provide structural insight into the translocation process for buforin II in addition to providing more general insight into the role proline can play in antimicrobial peptides.     

Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad-spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle-encapsulated fluorescent markers in a size-dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid-containing pores. Such a membrane-permeabilizing function appears to be an evolutionarily conserved host-defense mechanism of antimicrobial peptides.     

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.