Unassisted translocation of large polypeptide domains across phospholipid bilayers

Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can posttranslationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533-2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology.     

Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape

Cellular entry of peptide, protein, and nucleic acid biopharmaceuticals is severely impeded by the cell membrane. Linkage or assembly of such agents and cell-penetrating peptides (CPP) with the ability to cross cellular membranes has opened a new horizon in biomedical research. Nevertheless, the uptake mechanisms of most CPP have been controversially discussed and are poorly understood. We present data on two recently developed oligocationic CPP, the sweet arrow peptide SAP, a gamma-zein-related sequence, and a branched human calcitonin derived peptide, hCT(9-32)-br, carrying a simian virus derived nuclear localization sequence in the side chain. Uptake in HeLa cells and intracellular trafficking of N-terminally carboxyfluorescein labeled peptides was studied by confocal laser scanning microscopy and flow cytometry using biochemical markers in combination with quenching and colocalization approaches. Both peptides were readily internalized by HeLa cells through interaction with the extracellular matrix followed by lipid raft-mediated endocytosis as confirmed by reduced uptake at lower temperature, in the presence of endocytosis inhibitors and through cholesterol depletion by methyl-beta-cyclodextrin, supported by colocalization with markers for clathrin-independent pathways. In contrast to the oligocationic SAP and hCT(9-32)-br, interaction with the extracellular matrix, however, was no prerequisite for the observed lipid raft-mediated uptake of the weakly cationic, unbranched hCT(9-32). Transient involvement of endosomes in intracellular trafficking of SAP and hCT(9-32)-br prior to endosomal escape of both peptides was revealed by colocalization and pulse-chase studies of the peptides with the early endosome antigen 1. The results bear potential for CPP as tools for intracellular drug delivery.     

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.     

Membrane binding and translocation of cell-penetrating peptides

Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.     

Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient

Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian ratchet mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter.     

Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E. coli

The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C. The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG. In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.     

Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.     

Permeation of a beta-heptapeptide derivative across phospholipid bilayers

Based on a number of experiments it is concluded that the fluorescein labeled beta-heptapeptide fluoresceinyl-NH-CS-(S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the beta-heptapeptide (S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb(3+)-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled beta-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular beta-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.     

Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles

Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp ('penetratin'), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% alpha-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% beta-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of alpha-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.     

Lateral pressure dependence of the phospholipid transmembrane diffusion rate in planar-supported lipid bilayers

The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10⁻⁴ s⁻¹ to 1.03 × 10⁻⁴ s⁻¹) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 Å²/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed.     

Modeling the step of endosomal escape during cell infection by a nonenveloped virus

Widely disparate viruses enter the host cell through an endocytic pathway and travel the cytoplasm inside an endosome. For the viral genetic material to be delivered into the cytoplasm, these viruses have to escape the endosomal compartment, an event triggered by the conformational changes of viral endosomolytic proteins. We focus here on small nonenveloped viruses such as adeno-associated viruses, which contain few penetration proteins. The first time a penetration protein changes conformation defines the slowest timescale responsible for the escape. To evaluate this time, we construct what to our knowledge is a novel biophysical model based on a stochastic approach that accounts for the small number of proteins, the endosomal maturation, and the protease activation dynamics. We show that the escape time increases with the endosomal size, whereas decreasing with the number of viral particles inside the endosome. We predict that the optimal escape probability is achieved when the number of proteases in the endosome is in the range of 250-350, achieved for three viral particles.     

Catalysis of electron transfer across phospholipid bilayers by iron-porphyrin complexes

Phospholipid vesicles containing K₃Fe(CN)₆ were prepared from egg yolk phosphatidylcholine. Hemin dimethyl ester was incorporated into these vesicles during preparation in ratios of phospholipid to hemin dimethyl ester that varied from 200 : 1 to 45000 : 1. Electron transfer across the bilayer was measured anaerobically after injecting the vesicles into a solution containing reduced indigotetrasulfonic acid. Vesicles containing hemin dimethyl ester exhibited high rates of electron transfer (240 electrons/molecule hemin dimethyl ester per min). Conditions could be selected where the rate-limiting step for catalysis was either the bimolecular reaction between ferric hemin dimethyl ester and reduced indigotetrasulfonic acid or the movement of hemin dimethyl ester from interface to interface. The hemin dimethyl ester-catalyzed electron transfer went to completion within a few seconds, completely oxidizing the reduced indigotetrasulfonic acid. Valinomycin (in the presence of potassium) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were without effect on catalyzed electron transport. Thus, the electron transport is not electrogenic but is a coupled, neutral system. By specific assay, neither phosphate nor cyanide was significantly transported during electron transfer but evidence is provided to suggest that a coordinated hydroxide accompanies movement of Fe(III) hemin dimethyl ester from the inside surface to the outside surface of the bilayer. It was also demonstrated in a bulk phase transport system that hemin dimethyl ester readily catalyzes transfer of S¹⁴CN^? through a chloroform layer separating two aqueous phases. Another more hydrophobic iron-porphyrin complex, Fe(III) tetraphenylporphyrin, was found to be twice as effective as hemin dimethyl ester. Other porphyrin complexes were also tested as control systems. No significant catalysis was found for metal-free protoporphyrin IX dimethyl ester or Ni(II) tetraphenylporphyrin. The results are discussed in comparison with in vivo electron transport and the future usefulness of this model system.     

Real-time transmembrane translocation of penetratin driven by light-generated proton pumping

Cell penetrating peptides (CPPs) are small peptides that are able to penetrate the plasma membrane of mammalian cells. Because these peptides can also carry large hydrophilic cargos such as proteins, they could potentially be used to transport biologically active drugs across cell membranes to modulate in vivo biology. One characteristic feature of the CPPs is that they typically have a net positive charge. Therefore, a key issue associated with the transport mechanism is the role of the transmembrane electrochemical potential in driving the peptides across the membrane. In this study, we have reconstituted bacteriorhodopsin (bR) in large unilamellar vesicles (LUVs) with fluorescein-labeled CPP penetratin enclosed within the LUVs under conditions when the fluorescence is quenched. Illumination of the bacteriorhodopsin-containing LUVs resulted in creation of a transmembrane proton electrochemical gradient (positive on the inside). Upon generation of this gradient, an increase in fluorescence was observed, which shows that the proton gradient drives the translocation of penetratin. The mechanism most likely can be generalized to other CPPs.     

The translocation of Ca2+ across phospholipid bilayers induced by a synthetic neutral Ca2+ -ionophore.

The effect of a neutral synthetic Ca2+ -ligand, which induces selective Ca2+ transport in electrodialysis experiments in bulk membranes, on the Ca2+ permeability of phospholipid bilayers has been investigated. The ligand is able to promote the transport of Ca2+ across synthetic phospholipid bilayers and can therefore be classified as a Ca2+ -ionophore. Its activity is enhanced by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The efficiency of the neutral carrier-mediated Ca2+ transport is rather low as compared with that of the charged Ca2+ -ionophore X537A. The Ca2+ selectivity of the nuetral ionophore is decreased by its incorporation in the low dielectric ambient of the phospholipid bilayer.     

Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.     

Comparison of the interaction of methionine and norleucine-containing peptides with phospholipid bilayers

Norleucine is a structural analog of methionine with a methylene group replacing the thio ether. Despite the close structural similarity of these two amino acids, norleucine-containing peptides have markedly different behaviour with phospholipids compared with methionine-containing peptides. For example, HCO-L-Ahx-L-Leu-L-Phe-OMe behaves as a hydrophobic peptide when mixed with dimyristoylphosphatidylcholine. This peptide lowers the enthalpy of the lipid phase transition. The effect is independent of the rate of heating. With the homologous peptide, HCO-L-Met-L-Leu-L-Phe-OMe, the results are markedly dependent on scan rate with a higher enthalpy observed at faster scan rates. Only at a scan rate of 0.2 K min-1 do the two peptides approach similar behaviour. The higher enthalpy observed for samples with the methionine peptide at higher scan rates can be explained assuming that the peptide aggregates at low temperature. As the phase transition temperature is approached, the more hydrophilic methionine peptide partitions more slowly into the membrane than the norleucine peptide. Partitioning of the peptides between aqueous and lipid phases was measured at 37 degrees by centrifuging down the lipid-bound fraction. At a peptide concentration of 15 microM and a lipid concentration of 1.4 mM, 89% of the HCO-L-Ahx-L-Leu-L-PheOMe and 97% of the HCO-L-Met-L-Leu-L-PheOMe remained in the supernate; indicating a greater tendency of the norleucine-containing peptide to partition into the lipid phase. The peptides Ac-L-Phe-L-Met-L-Arg-L-Phe-NH2 and Ac-L-Phe-L-Ahx-L-Arg-L-Phe-NH2 are readily soluble in water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Domain formation in phosphatidylcholine bilayers containing transmembrane peptides: specific effects of flanking residues

Lateral segregation in biological membranes leads to the formation of domains. We have studied the lateral segregation in gel-state model membranes consisting of supported dipalmitoylphosphatidylcholine (DPPC) bilayers with various model peptides, using atomic force microscopy (AFM). The model peptides are derivatives of the Ac-GWWL(AL)(n)WWA-Etn peptides (the so-called WALP peptides) and have instead of tryptophans, other flanking residues. In a previous study, we found that WALP peptides induce the formation of extremely ordered, striated domains in supported DPPC bilayers. In this study, we show that WALP analogues with other uncharged residues (tyrosine, phenylalanine, or histidine at pH 9) can also induce the formation of striated domains, albeit in some cases with a slightly different pattern. The WALP analogues with positively charged residues (lysine or histidine at low pH) cannot induce striated domains and give rise to a completely different morphology: they induce irregularly shaped depressions in DPPC bilayers. The latter morphology is explained by the fact that the positively charged peptides repel each other and hence are not able to form striated domains in which they would have to be in close vicinity. They would reside in disordered, fluidlike lipid areas, appearing below the level of the ordered gel-state lipid domains, which would account for the irregularly shaped depressions.