Membrane destabilizing properties of cell-penetrating peptides

Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48-60) and oligoarginine, denoted TatP59W, TatLysP59W and R(7)W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thoren et al., Biochem. Biophys. Res. Commun. 307 (2003) 100-107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R(7)W. In this paper, the interactions of penetratin, R(7)W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R(7)W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties.     

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.     

Arginine-rich cell-penetrating peptides

Arginine-rich cell-penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell-penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell-penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example.     

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.     

Uptake of cell-penetrating peptides in yeasts

The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)(3)K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.     

Membrane perturbation through novel cell-penetrating peptides influences intracellular accumulation of imatinib mesylate in CML cells

Chronic myeloid leukemia is a stem cell disease with the presence of Philadelphia chromosome generated through reciprocal translocation of chromosome 9 and 22. The use of first- and second-generation tyrosine kinase inhibitors has been successful to an extent. However, resistance against such drugs is an emerging problem. Apart from several drug-resistant mechanisms, drug influx/efflux ratio appears to be one of the key determinants of therapeutic outcomes. In addition, intracellular accumulation of drug critically depends on cell membrane fluidity and lipid raft dynamics. Previously, we reported two novel cell-penetrating peptides (CPPs), namely, cationic IR15 and anionic SR11 present in tryptic digest of Abrus agglutinin. Here, the potential of IR15 and SR11 to influence intracellular concentration of imatinib has been evaluated. Fluorescent correlation spectroscopy and lifetime imaging were employed to map membrane fluidity and lipid raft distribution following peptide-drug co-administration. Results show that IR15 and SR11 are the two CPPs which can modulate membrane fluidity and lipid raft distribution in K562 cells. Both IR15 and SR11 significantly reduce the viability of CML cells in the presence of imatinib by increasing the intracellular accumulation of the drug.     

Studying the uptake of cell-penetrating peptides

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.     

Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers

Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.     

Membrane adsorption, folding, insertion and translocation of synthetic trans-membrane peptides

Spontaneous membrane adsorption, folding and insertion of the synthetic WALP16 and KALP16 peptides was studied by computer simulations starting from completely extended conformations. The peptides were simulated using an unmodified all-atom force field in combination with an efficient Monte Carlo sampling algorithm. The membrane is represented implicitly as a hydrophobic zone inside a continuum solvent modelled using the generalized Born theory of solvation. The method was previously parameterized to match insertion energies of hydrophobic side chain analogs into cyclohexane and no parameters were optimized for the present simulations. Both peptides rapidly precipitate out of bulk solution and adsorb to the membrane surface. Interfacial folding into a helical conformation is followed by membrane insertion. Both the peptide conformations and their location in the membrane are strongly temperature dependent. The temperature dependent behaviour can be summarized by fitting to a four-state model, separating the system into folded and unfolded conformers, which are either inserted into the membrane or located at the interfaces. As the temperature is lowered the dominant peptide conformation of the system changes from unfolded surface bound configurations to folded surface bound states. Folded trans-membrane conformers represent the dominant configuration at low temperatures. The analysis allows direct estimates of the free energies of peptide folding and membrane insertion. In the case of WALP the quality of the fit is excellent and the thermodynamic behaviour is in good agreement with expected theoretical consideration. For KALP the fit is more problematic due to the large solvation energies of the charged lysine residues.     

Membrane adsorption,folding, insertion and translocation of synthetic trans-membrane peptides

Spontaneous membrane adsorption, folding and insertion of the synthetic WALP16 and KALP16 peptides was studied by computer simulations starting from completely extended conformations. The peptides were simulated using an unmodified all-atom force field in combination with an efficient Monte Carlo sampling algorithm. The membrane is represented implicitly as a hydrophobic zone inside a continuum solvent modelled using the generalized Born theory of solvation. The method was previously parameterized to match insertion energies of hydrophobic side chain analogs into cyclohexane and no parameters were optimized for the present simulations. Both peptides rapidly precipitate out of bulk solution and adsorb to the membrane surface. Interfacial folding into a helical conformation is followed by membrane insertion. Both the peptide conformations and their location in the membrane are strongly temperature dependent. The temperature dependent behaviour can be summarized by fitting to a four-state model, separating the system into folded and unfolded conformers, which are either inserted into the membrane or located at the interfaces. As the temperature is lowered the dominant peptide conformation of the system changes from unfolded surface bound configurations to folded surface bound states. Folded trans-membrane conformers represent the dominant configuration at low temperatures. The analysis allows direct estimates of the free energies of peptide folding and membrane insertion. In the case of WALP the quality of the fit is excellent and the thermodynamic behaviour is in good agreement with expected theoretical consideration. For KALP the fit is more problematic due to the large solvation energies of the charged lysine residues.     

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.     

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 α-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.     

A brief introduction to cell-penetrating peptides

Cell membranes act as protective walls to exclude most molecules that are not actively imported by living cells. This is an efficient way for a cell to prevent uncontrolled influx or efflux of solutes, which otherwise would be harmful to it. Only compounds within a narrow range of molecular size, polarity and net charge are able to diffuse effectively through cell membranes. In order to overcome this barrier for effective delivery of membrane-impermeable molecules, several chemical and physical methods have been developed. These methods, e.g. electroporation, and more recent methods as cationic lipids/liposomes, have been shown to be effective for delivering hydrophobic macromolecules. The drawbacks of these harsh methods are, primarily, the unwanted cellular effects exerted by them, and, secondly, their limitation to in vitro applications. The last decade's discovery of cell-penetrating peptides translocating themselves across cell membranes of various cell lines, along with a cargo 100-fold their own size, via a seemingly energy-independent process, opens up the possibility for efficient delivery of DNA, antisense peptide nucleic acids, oligonucleotides, proteins and small molecules into cells both in vitro and in vivo.     

Screening of cell-penetrating peptides using mRNA display

Cell-penetrating peptides (CPPs) are attractive vectors for in vivo and in vitro cellular uptake. Their use is, however, limited by insufficient understanding of their preference for a target cell. Here, a new CPP screening method is presented that uses mRNA display. After incubating the target cell lines, such as human embryonic kidney 293 (HEK 293) and HeLa cells, with an mRNA display library for 3 h at 37°C, the CPP-mRNA nucleotide conjugates were harvested. These were amplified with PCR and subsequently sequenced. The screened CPPs for each cell line were identified after four rounds of selection. Among them, two peptides, MAMPGEPRRANVMAHKLEPASLQLR NSCA (CPPK) and MAPQRDTVGGRTTPPSWGPAKAQLRNSCA (CPPL) were selected, and the FITC-labeled peptides were evaluated for their ability to penetrate cells. The screened CPPs were superior to polyarginine (R(11) ), which is widely used as a standard peptide and shows good cell penetration efficiency. Our method can be applied to other target cells for which CPPs have not yet been elucidated.     

Internalisation of cell-penetrating peptides into tobacco protoplasts

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.     

Internalisation of cell-penetrating peptides into tobacco protoplasts

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.     

Membrane binding properties of blood coagulation Factor V and derived peptides

The interactions of factor V and factor Va light chain with phospholipid vesicles were compared. The results showed that the factor Va light chain bound with the same parameters as factor V when the proteins were present at similar densities on the membrane. The protein-vesicle collisional efficiency was 30-50% for both factor V and factor Va light chain. The factor Va light chain bound at a higher density, and the additional binding interactions had lower affinity. The dissociation process showed negative cooperativity, possibly due to competition for acidic phospholipids in the membrane. The higher molar packing density produced more rapid protein-membrane dissociation rate constants. However, when factor V and Va light chains were present at similar molar densities on the vesicle, the dissociation rates, estimated by two methods, were similar. Analysis of dissociation rates also showed that factor Va interacted with factor Xa on the membrane surface while factor Va light chain did not. Factor Va generated by thrombin digestion of factor V did not result in a major loss of membrane-bound protein mass unless ethylenenediaminetetraacetic acid was present; in the latter case the mass changes indicated that all peptides were removed from the membrane except factor Va light chain. Equilibrium and dynamic measurements showed that ionic strength had a major effect on the dissociation rate but not on the association process. The salt effect indicated interaction between oppositely charged species with the product of the number of charges equal to at least -5.5. Factor Va light chain appeared to interact with phospholipids via a general charge interaction rather than via a specific charge stoichiometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: correlations of structure to cellular uptake

This work reports on the binding and conformation of a series of CPPs in the bilayer membranes of large unilamellar vesicles and the effect of the presence of cholesterol. We show a negative correlation between alpha-helical structure and uptake efficiency for penetratin peptides where the two central arginine residues of penetratin are thought to be important for breaking the secondary structure. Penetratin alpha-helicity is also reduced upon incorporation of cholesterol into the membrane. Flow linear dichroism in the far-UV region shows that the penetratin peptides adopt a preferential orientation of the alpha-helix parallel to the bilayer, and the linear dichroism (LD) spectrum in the aromatic region indicates that the tryptophan residues are preferentially oriented parallel to the membrane. The Tat analogue TatP59W and the oligoarginine R7W, which are more efficient CPPs than penetratin, bind to membranes as random coils and do not show any orientation in LD, again indicating that alpha-helicity reduces uptake efficiency. Further, we observe large variations in tryptophan quantum yields for the five CPPs in this study and discuss this in terms of the ability to cause lipid rearrangement. Binding isotherms show that cholesterol increases the affinity of the peptide for the membrane, but tryptophan fluorescence lifetimes are essentially unaltered by incorporation of as much as 40 mol % cholesterol into the membrane, suggesting the absence of specific peptide-cholesterol interactions. Fluorescence emission maxima are insensitive to cholesterol and indicate that the peptide is positioned in the headgroup region. The results on peptide-membrane interactions are discussed in terms of possible uptake mechanisms.     

Synthesis of cell-penetrating conjugates of calpain activator peptides

Calpains, the intracellular proteolytic enzymes, play important roles in various processes in cells. The lack of calpain or its overexpression is thought to be an underlying factor in some diseases. In this study, we report the synthesis of a new group of cell-penetrating calpastatin-peptide conjugates with the activating capacity of m-calpain intracellularly. In these constructs, peptides related to the calpastatin A or C subunit with the capabiliy of activation of isolated m-calpain was covalently conjugated to the C-terminal of penetratin via amide, thioether, or disulfide bond. These conjugates were prepared by solid-phase synthesis and/or by chemical ligation and properly characterized (MS, HPLC). Our results using isolated m-calpain suggest that conjugation does not interfere with the enzyme-activating effect of the calpastatin peptides; in fact, the efficiency of the conjugates was markedly higher. The conjugates with different bonds showed essentially the same level of activation. Internalization experiments with fluorophore (4-[7-hydroxycoumaryl] acetic acid (Hca) at the N-terminal of penetratin and/or 5(6)-carboxyfluorescein (cf)) labeled conjugates show that these constructs are taken up by COS-7 cells. Using cell lysates produced after incubation with the 1:1 (mol/mol) mixture of calpastatin A and C peptide conjugates, we found a significant calpain activating effect. We also noticed that the conjugate even with a disulfide bond between the components seems to be stable and activate m-calpain after intracellular translocation under the conditions studied. To the best of our knowledge, this is the first report to describe conjugates with an m-calpain activating effect on isolated enzymes and more importantly within living cells after transmembrane delivery. Thus, these conjugates seem to be appropriate as molecular tools to activate intracellular m-calpain and to study calpain functions in living cells.