Cellular uptake of Antennapedia Penetratin peptides is a two-step process in which phase transfer precedes a tryptophan-dependent translocation

Several homeodomains and homeodomain-containing proteins enter live cells through a receptor- and energy-independent mechanism. Translocation through biological membranes is conferred by the third α-helix of the homeodomain, also known as Penetratin. Biophysical studies demonstrate that entry of Penetratin into cells requires its binding to surface lipids but that binding and translocation are differentially affected by modifications of some physico-chemical properties of the peptide, like helical amphipathicity or net charge. This suggests that the plasma membrane lipid composition affects the internalization of Penetratin and that internalization requires both lipid binding and other specific properties. Using a phase transfer assay, it is shown that negatively charged lipids promote the transfer of Penetratin from a hydrophilic into a hydrophobic environment, probably through charge neutralization. Accordingly, transfer into a hydrophobic milieu can also be obtained in the absence of negatively charged lipids, by the addition of DNA oligonucleotides. Strikingly, phase transfer by charge neutralization was also observed with a variant peptide of same charge and hydrophobicity in which the tryptophan at position 6 was replaced by a phenylalanine. However, Penetratin, but not its mutant version, is internalized by live cells. This underscores that charge neutralization and phase transfer represent only a first step in the internalization process and that further crossing of a biological membrane necessitates the critical tryptophan residue at position 6.     

Cellular uptake of the Antennapedia homeodomain polypeptide by macropinocytosis

Antennapedia homeodomain has been shown to be able to translocate from extracellular space into the cytoplasm of cells in a receptor-independent manner. Its third α-helix domain, designated as “Penetratin”, was proposed to be the functional transduction domain that is responsible for the translocation, and it is widely used for intracellular delivery of various exogenous proteins. Although Penetratin has been regarded to be the only element conferring the capacity on its parent polypeptide to penetrate through the plasma membrane, we found that the complete Antennapedia homeodomain exhibits an appreciably higher level of translocation efficiency as compared to Penetratin. Pharmacological analysis demonstrated that macropinocytic endocytosis plays a significant role underlying the process of the homeodomain internalization, and this is consistent with the observation that internalized polypeptide co-localizes with a fluid phase dye. Our results identify macropinocytosis as a major mechanism by which Antennapedia homeodomain obtains the access to the interior of cells, providing a novel perspective in the field of protein translocation and transduction.     

Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin.

Peptides derived from the third alpha-helix of the homeodomain (residues 43-58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N- and C-terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C-terminal segment 52Arg-Arg-Met-Lys-Trp-Lys-Lys58 of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide's basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.     

Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry

We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)9 and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS. Results of cargo delivery were compared to the amounts of free CPP internalized inside cells. The third helix of Knotted gave the best results concerning free CPP cellular uptake. It was also found to be the most efficient carrier. This peptide thus emerges as a new CPP with very promising properties.     

Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry

We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)₉ and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS. Results of cargo delivery were compared to the amounts of free CPP internalized inside cells. The third helix of Knotted gave the best results concerning free CPP cellular uptake. It was also found to be the most efficient carrier. This peptide thus emerges as a new CPP with very promising properties.     

Relationships between membrane binding, affinity and cell internalization efficacy of a cell-penetrating peptide: penetratin as a case study

Background

Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.

Methodology

Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAG^neg) and sialic acid-deficient (SA^neg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.

Principal Findings

Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SA^neg>WT>GAG^neg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SA^neg and GAG^neg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAG^neg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.

Conclusion/Significance

These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering.     

Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin

Cellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization.     

Comparison of penetratin and other homeodomain-derived cell-penetrating peptides: interaction in a membrane-mimicking environment and cellular uptake efficiency

Antennapedia and other homeoproteins have the unique ability to efficiently translocate across biological membranes, a property that is mediated by the third helix of the homeodomain. To analyze the effects of sequence divergence in the homeodomain, we have compared the cellular uptake efficiencies and interaction properties in a membrane-mimicking environment of four peptides corresponding to the third helix sequence of Antennapedia, Engrailed-2, HoxA-13, and Knotted-1. NMR studies revealed that these peptides adopt helical conformations in SDS micelles. Their localization with respect to the micelle was investigated using Mn(2+) as a paramagnetic probe. Peptides are positioned parallel to the micelle surface, but subtle differences in the depth of immersion were observed. Using a recently developed method for quantification of CPP cellular uptake based on MALDI-TOF mass spectrometry, all of these peptides were found to translocate into cells but with large differences in their uptake efficiencies. The peptide with the highest uptake efficiency was found to be the least deeply inserted within the micelle, indicating that electrostatic surface interactions may be a major determinant for membrane translocation. A new cell-penetrating peptide derived from Knotted-1 homeodomain with improved uptake properties compared to penetratin is introduced here.     

An induction gene trap for identifying a homeoprotein-regulated locus

An important issue in developmental biology is the identification of homeoprotein target genes. We have developed a strategy based on the internalization and nuclear addressing of exogenous homeodomains, using an engrailed homeodomain (EnHD) to screen an embryonic stem (ES) cell gene trap library. Eight integrated gene trap loci responded to EnHD. One is within the bullous pemphigoid antigen 1 (BPAG1) locus, in a region that interrupts two neural isoforms. By combining in vivo electroporation with organotypic cultures, we show that an already identified BPAG1 enhancer/promoter is differentially regulated by homeoproteins Hoxc-8 and Engrailed in the embryonic spinal cord and mesencephalon. This strategy can therefore be used for identifying and mutating homeoprotein targets. Because homeodomain third helices can internalize proteins, peptides, phosphopeptides, and antisense oligonucleotides, this strategy should be applicable to other intracellular targets for characterizing genetic networks involved in a large number of physiopathological states.     

Studies on the internalization mechanism of cationic cell-penetrating peptides

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

The antimicrobial peptide histatin-5 causes a spatially restricted disruption on the Candida albicans surface, allowing rapid entry of the peptide into the cytoplasm

Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.     

Membrane interaction and cellular internalization of penetratin peptides.

Penetratin is a 16-residue peptide [RQIKIWFQNRRMKWKK(43-58)] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the alpha-helical structure of the peptides. The R53A/K57A and R52A/K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.     

Protease-activated receptor-1 down-regulation: a mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by a proteolytic mechanism, then internalized and degraded in lysosomes. The latter is critical for temporal fidelity of thrombin signaling. Toward understanding PAR1 down-regulation, we first investigated the pathway of PAR1 internalization. Activated PAR1 was rapidly recruited to clathrin-coated pits, where it colocalized with transferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization. Blockade of PAR1 internalization with dynamin K44A also inhibited activation-dependent PAR1 degradation. Thus activated PAR1 internalizes via clathrin-coated pits together with receptors that recycle and is then sorted away from such receptors and delivered to lysosomes. In the course of these studies we identified a mutant HeLa cell line, designated JT1, that was defective in PAR1 internalization. PAR1 signaled robustly in JT1 cells but was not phosphorylated or recruited to clathrin-coated pits after activation. Internalization of TfnR was intact in JT1 cells and internalization of beta(2)-adrenergic receptor, a GPCR that internalizes and recycles, was present but perhaps reduced. Taken together, these studies suggest that PAR1 is internalized in a dynamin- and clathrin-dependent manner like TfnR and beta(2)-adrenergic receptor but requires a distinct gene product for recruitment into this pathway.     

Physico-chemical requirements for cellular uptake of pAntp peptide. Role of lipid-binding affinity.

The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptor-independent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide-lipid interaction in this mechanism and the physico-chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala-Scan mapping. The peptides were labeled with a fluorescent group (7-nitrobenz-2-oxo-1,3-diazol-4-yl-; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process. To further investigate which are the physico-chemical requirements for phospholipid-binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane-mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.     

Translocation and Endocytosis for Cell-penetrating Peptide Internalization

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

Ubiquitylation of MHC class I by the K3 viral protein signals internalization and TSG101-dependent degradation

The Kaposi's sarcoma-associated herpes virus gene product K3 (KK3) subverts the MHC class I antigen presentation pathway by downregulating MHC class I from the plasma membrane. We now show that KK3 associates with MHC class I molecules and promotes ubiquitylation of class I after export from the endoplasmic reticulum. Ubiquitylation requires the KK3 N-terminal plant homeodomain and provides the signal for class I internalization at the plasma membrane. Once internalized, ubiquitylated MHC class I is targeted to the late endocytic pathway, where it is degraded. Depletion by small interfering RNA of TSG101, a ubiquitin enzyme 2 variant protein involved in late endosomal sorting, prevents class I degradation and preserves cell surface class I expression in KK3-expressing cells. These results suggest a mechanism by which the KK3-induced class I ubiquitylation provides a signal for both internalization and sorting to the late endosomal pathway for degradation. KK3 is the first viral gene product that subverts the trafficking of a host protein via the ubiquitin-dependent endosomal sorting machinery.