Structure-activity relationship study of the cell-penetrating peptide pVEC

The peptide pVEC is a recently described cell-penetrating peptide, derived from the murine vascular endothelial-cadherin protein. In order to define which part of this 18-amino acid long peptide is important for the cellular translocation, we performed a structure-activity relationship study of pVEC. Together with the l-alanine substituted peptides, the retro-pVEC, D-pVEC and the scramble pVEC are studied for comparison. The peptide analogues are labeled with carboxyfluorescein at the N-terminus for monitoring the cellular uptake into human Bowes melanoma cells with different efficacy. We show that all the Fl-pVEC analogues internalize in live Bowes melanoma cells. l-Alanine substitution of the five respective N-terminal hydrophobic amino acids significantly decreases the translocation property, while replacing of Arg⁶, Arg⁸ or Ser¹⁷ by alanine enhances the uptake. The uptake of pVEC is significantly reduced by treatment with an endocytosis inhibitor wortmannin. Treatment with heparinase III, nystatin and EIPA had no effect on the peptide uptake. The data presented here show that the N-terminal hydrophobic part of pVEC is crucial for efficient cellular translocation.     

VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier functions

Cell-penetrating peptides, CPPs, have been shown to translocate into living cells by a receptor-independent mechanism and to carry macromolecules over the plasma membrane. This article reports studies of the internalization of pVEC, an 18-amino acid-long peptide derived from the murine sequence of the cell adhesion molecule vascular endothelial cadherin, amino acids 615-632. Fluorophore-labeled pVEC entered four different cell lines tested: human aortic endothelial cells, brain capillary endothelial cells, Bowes melanoma cells, and murine brain endothelial cells. In order to evaluate the translocation efficiency of pVEC, we performed a side-by-side comparison with penetratin, a well-characterized CPP. The cellular uptake of pVEC was highest for murine brain endothelial cells. All cell lines tested contained equal or slightly higher concentrations of pVEC than penetratin. pVEC mainly accumulated in nuclear structures but was also found throughout the cells. Furthermore, pVEC functioned as a transporter of both a hexameric peptide nucleic acid molecule of 1.7 kDa and a 67-kDa protein, streptavidin-FITC, and cellular uptake of fluorophore-labeled pVEC took place at 4 degrees C, suggesting a nonendocytotic mechanism of translocation. In conclusion, our results indicate that pVEC is efficiently and rapidly taken up into cells and functions as a potent carrier peptide.     

Deletion analogues of transportan

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.     

Insights into membrane translocation of the cell-penetrating peptide pVEC from molecular dynamics calculations

Discovery of cargo carrying cell-penetrating peptides has opened a new gate in the development of peptide-based drugs that can effectively target intracellular enzymes. Success in application and development of cell-penetrating peptides in drug design depends on understanding their translocation mechanisms. In this study, our aim was to examine the bacterial translocation mechanism of the cell-penetrating pVEC peptide (LLIILRRRIRKQAHAHSK) using steered molecular dynamics (SMD) simulations. The significance of specific residues or regions for translocation was studied by performing SMD simulations on the alanine mutants and other variants of pVEC. Residue-based analysis showed that positively charged residues contribute to adsorption to the lipid bilayer and to electrostatic interactions with the lipid bilayer as peptides are translocated. Translocation takes place in three main stages; the insertion of the N-terminus into the bilayer, the inclusion of the whole peptide inside the membrane and the exit of the N-terminus from the bilayer. These three stages mirror the three regions on pVEC; namely, the hydrophobic N-terminus, the cationic midsection, and the hydrophilic C-terminus. The N-terminal truncated pVEC, I3A, L5A, R7A mutants and scramble-pVEC make weaker interactions with the lipids during translocation highlighting the contribution of the N-terminal residues and the sequence of the structural regions to the translocation mechanism. This study provides atomistic detail about the mechanism of pVEC peptide translocation and can guide future peptide-based drug design efforts.     

Cellular uptake of cell-penetrating peptides pVEC and transportan in plants.

Internalization of fluorescently labeled CPPs, pVEC, transportan and scrambled pVEC, in a range of plant cells was investigated. Cellular uptake of the peptides was found to be tissue dependent. pVEC and transportan were distinctly internalized in triticale mesophyll protoplasts, onion epidermal cells, leaf bases and root tips of seven-day old triticale seedlings but showed negligible florescence in coleoptile and leaf tips as observed under a fluorescence microscope. Further, pVEC and transportan uptake studies were focused on mesophyll protoplasts as a system of investigation. In fluorimetric studies transportan showed 2.3 times higher cellular internalization than pVEC in protoplasts, whereas scrambled pVEC failed to show any significant fluorescence. Effect of various factors on cellular internalization of pVEC and transportan in protoplasts was also investigated. The cellular uptake of both the peptides was concentration dependent and nonsaturable. The cellular uptake of pVEC and transportan was enhanced at low temperature (4 degrees C). The presence of endocytic/macropinocytosis inhibitors did not reduce the cellular uptake of the peptides, suggesting direct cell penetration, receptor-independent internalization of pVEC and transportan into the plant cells.     

pVEC hydrophobic N‐terminus is critical for antibacterial activity

Cell‐penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure‐activity relationship and sequence alignment studies have suggested that the hydrophobic N‐terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N‐terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.     

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.     

In vitro uptake and stability study of pVEC and its all-D analog

A key step in the development of new hydrophilic pharmaceuticals is to get them through biological barriers. Cell-penetrating peptides, CPPs, have been shown previously to enter cells both in vitro and in vivo by a non-endocytotic mechanism and to be able to carry large cargo molecules with them. Recently, we showed that a small peptide, pVEC, from murine vascular endothelial cadherin, has the characteristics to be classified as a protein derived CPP. Here we have further investigated pVEC together with its all-D analog for cellular uptake, intra- and extracellular stability, and their enzymatic degradation. The two peptides, pVEC and all-D pVEC, translocate into aortic endothelial cells and murine fibroblasts by a non-endocytotic mechanism. In phosphate buffer, pVEC remains intact while the C-terminal lysine is quickly removed in human serum and serum-containing media. Both pVEC and pVEC without the C-terminal Lys were detected by mass spectrometry inside the two cell types tested. The pVEC half-life is 10.5 min in phosphate buffer containing 10 units of trypsin and 44.6 min in phosphate buffer containing 4.2 units of carboxypeptidase A and 18 units of carboxypeptidase B. In contrast topVEC, the all-D analog remains intact in serum and resists enzymatic degradation.     

Bowes human melanoma cell line. An immunocytochemical study

Bowes human melanoma cell line was investigated immunocytochemically to localize S-100 protein, HMB-45 and intermediate filament proteins. The majority of the cells showed strong positive staining with antibodies directed against S-100 protein, HMB-45 and vimentin filaments. Antibodies directed against the other cytoskeletal proteins failed to show any reactivity. These findings suggest that this transformed cell line does not dedifferentiate in culture and continues to express the specific antigens of human melanoma cells. Thus, Bowes cell line provides a useful model for studying the cellular biology and pathology of malignant melanoma.     

Translocation properties of novel cell penetrating transportan and penetratin analogues

Novel analogues of the cell-penetrating peptides penetratin and transportan were synthesized. The distribution of the biotin-labeled peptides in Bowes melanoma cell line has been investigated by indirect fluorescence with fluorescein-streptavidin detection. The time course of uptake of (125)I-labeled transportan analogues has been characterized in the same cell line. Molecular modeling was used to analyze the penetration and the orientation of molecules in a simulated biological membrane. The results, both from molecular modeling and fluorescence studies, imply that penetratin and transportan do not enter the cells by related mechanisms and that they do not belong to the same family of translocating peptides.     

Cellular uptake of transportan 10 and its analogs in live cells: Selectivity and structure-activity relationship studies

Transportan 10 (TP10) is an amphipathic cell-penetrating peptide with high translocation ability. In order to obtain more details of structure-activity relationship of TP10, we evaluated the effects of structure and charge on its translocation ability. Our results demonstrated that disrupting the helical structure or Arg substitution could remarkably decrease the cellular uptake of TP10. However, increasing the number of positive charge was an effective strategy to enhance translocation ability of TP10. Furthermore, the molecular dynamics simulation supported the results derived from experiments, suggesting that higher membrane disturbance leads to higher cellular uptake of peptides. In addition, our study also demonstrated TP10 and its analogs preferentially entered cancer cells rather than normal cells. The uptake selectivity toward cancer cells makes TP10 and its analogs as potent CPPs for drug delivery.     

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.     

The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study

Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Biophysical and biological studies of end-group-modified derivatives of Pep-1

Pep-1 is a tryptophane-rich cell-penetrating peptide (CPP) that has been previously proposed to bind protein cargoes by hydrophobic assembly and translocate them across cellular membranes. To date, however, the molecular mechanisms responsible for cargo binding and translocation have not been clearly identified. This study was conducted to gain insight into the interaction between Pep-1 with its cargo and the biological membrane to identify the thereby involved structural elements crucial for translocation. We studied three peptides differing in their N- and C-termini: (i) Pep-1, carrying an acetylated N-terminus and a C-terminal cysteamine elongation, (ii) AcPepWAmide, with an acetylated N-terminus and an amidated C-terminus, and (iii) PepW, with two free termini. Thioredoxin (TRX) and beta-galactosidase were used as protein cargoes. To study CPP-membrane interactions, we performed biophysical as well as biological assays. To mimic biological membranes, we used phospholipid liposomes in a dye leakage assay and surfactant micelles for high-resolution NMR studies. In addition, membrane integrity, cell viability, and translocation efficiency were analyzed in HeLa cells. An alpha-helical structure was found for all peptides in the hydrophobic N-terminal region encompassing residues 4-13, whereas the hydrophilic region remained unstructured in the presence of micelles. Our results show that the investigated peptides interacted with the micelles as well as with the protein cargo via their tryptophan-rich domain. All peptides displayed an orientation parallel to the micelle surface. The C-terminal cysteamine group formed an additional membrane anchor, leading to more efficient translocation properties in cells. No membrane permeabilization was observed, and our data were largely compatible with an endocytic pathway for cellular uptake.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

Secondary structure of cell‐penetrating peptides during interaction with fungal cells

Cell‐penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep‐1, MPG, pVEC, TP‐10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α‐helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep‐1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular‐level interaction with the cell membrane, and these simulations suggested that pVEC, TP‐10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α‐helical conformation. In contrast, penetratin, Pep‐1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena.     

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.     

Conjugation of arginine-glycine-aspartic acid peptides to poly(ethylene oxide)-b-poly(epsilon-caprolactone) micelles for enhanced intracellular drug delivery to metastatic tumor cells

An arginine-glycine-aspartic acid (RGD) containing model peptide was conjugated to the surface of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles as a ligand that can recognize adhesion molecules overexpressed on the surface of metastatic cancer cells, that is, integrins, and that can enhance the micellar delivery of encapsulated hydrophobic drug into a tumor cell. Toward this goal, PEO-b-PCL copolymers bearing acetal groups on the PEO end were synthesized, characterized, and assembled to polymeric micelles. The acetal group on the surface of the PEO-b-PCL micelles was converted to reactive aldehyde under acidic condition at room temperature. An RGD-containing linear peptide, GRGDS, was conjugated on the surface of the aldehyde-decorated PEO-b-PCL micelles by incubation at room temperature. A hydrophobic fluorescent probe, that is, DiI, was physically loaded in prepared polymeric micelles to imitate hydrophobic drugs loaded in micellar carrier. The cellular uptake of DiI loaded GRGDS-modified micelles by melanoma B16-F10 cells was investigated at 4 and 37 degrees C by fluorescent spectroscopy and confocal microscopy techniques and was compared to the uptake of DiI loaded valine-PEO-b-PCL micelles (as the irrelevant ligand decorated micelles) and free DiI. GRGDS conjugation to polymeric micelles significantly facilitated the cellular uptake of encapsulated hydrophobic DiI most probably by intergrin-mediated cell attachment and endocytosis. The results indicate that acetal-terminated PEO-b-PCL micelles are amenable for introducing targeting moieties on the surface of polymeric micelles and that RGD-peptide conjugated PEO-b-PCL micelles are promising ligand-targeted carriers for enhanced drug delivery to metastatic tumor cells.     

Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides

In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides.Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane.In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.