The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

Human disorders caused by inborn errors of cholesterol biosynthesis are characterized by dysmorphogenesis of multiple organs. This includes limb malformations that are observed at high frequency in some disorders, such as the Smith-Lemli-Opitz syndrome, indicating a pivotal role of cholesterol in limb morphogenesis. Recently, it has been demonstrated that cholesterol can modulate the activity of the Hedgehog proteins, that act as morphogens to regulate the precise patterning of many embryonic structures, among which the developing limbs. To provide insight in the functions of cholesterol during limb development and in the potential role of Hedgehog signaling in the genesis of limb defects, we developed an in vivo rat model of cholesterol deficiency. We show here that treatment with Triparanol, a distal inhibitor of cholesterol biosynthesis, induced patterning defects of the autopod at high frequency, including pre-axial syndactyly and post-axial polydactyly, thus reproducing limb anomalies frequently observed in humans. Using in situ hybridization, we show that these malformations originate from a modification of Sonic Hedgehog signaling in the limb bud at 13 days post-coitum, leading to a deficiency of the anterior part of the limb. This deficiency results in an imbalance of Indian Hedgehog expression in the forming cartilage, ultimately leading to reduced interdigital apoptosis and syndactyly. Our study thus unravels the molecular mechanisms underlying the genesis of limb defects associated with cholesterol deficiency in rodents, and most probably in humans.

Histiotrophic nutrition is essential during the peri-implantation development in rodents, but little is known about receptors involved in protein and lipid endocytosis derived from the endometrium and the uterine glands. Previous studies suggested that cubilin, a multiligand receptor for vitamin, iron, and protein uptake in the adult, might be important in this process, but the onset of its expression and function is not known. In this study, we analyzed the expression of cubilin in the pre- and early post-implantation rodent embryo and tested its potential function in protein and cholesterol uptake. Using morphological and Western blot analysis, we showed that cubilin first appeared at the eight-cell stage. It was expressed by the maternal-fetal interfaces, trophectoderm and visceral endoderm, but also by the future neuroepithelial cells and the developing neural tube. At all these sites, cubilin was localized at the apical pole of the cells exposed to the maternal environment or to the amniotic and neural tube cavities, and had a very similar distribution to megalin, a member of the LDLR gene family and a coreceptor for cubilin in adult tissues. To analyze cubilin function, we followed endocytosis of apolipoprotein A-I and HDL cholesterol, nutrients normally present in the uterine glands and essential for embryonic growth. We showed that internalization of both ligands was cubilin dependent during the early rodent gestation. In conclusion, the early cubilin expression and its function in protein and cholesterol uptake suggest an important role for cubilin in the development of the peri-implantation embryo.

Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.

Cell-penetrating peptides (CPPs) internalization occur both by endocytosis and direct translocation through the cell membrane. These different entry routes suggest that molecular partners at the plasma membrane, phospholipids or glycosaminoglycans (GAGs), bind CPPs with different affinity or selectivity. The analysis of sequence-dependent interactions of CPPs with lipids and GAGs should lead to a better understanding of the molecular mechanisms underlying their internalization. CPPs are short sequences generally containing a high number of basic arginines and lysines and sometimes aromatic residues, in particular tryptophans. Tryptophans are crucial residues in membrane-active peptides, because they are important for membrane interaction. Membrane-active peptides often present facial amphiphilicity, which also promote the interaction with lipid bilayers. To study the role of Trp and facial amphiphilicity in cell interaction and penetration of CPPs, a nonapeptide series containing only Arg, Trp or D-Trp residues at different positions was designed. Our quantitative study indicates that to maintain/increase the uptake efficiency, Arg can be advantageously replaced by Trp in the nonapeptides. The presence of Trp in oligoarginines increases the uptake in cells expressing GAGs at their surface, while it compensates for the loss of charge interactions from Arg and maintains similar peptide uptake in GAG-deficient cells. In addition, we show that facial amphiphilicity is not required for efficient uptake of these nonapeptides. Thermodynamic analyses point towards a key role of Trp that highly contributes to the binding enthalpy of complexes formation. Density functional theory (DFT) analysis highlights that salt bridge-π interactions play a crucial role for the GAG-dependent entry mechanisms.

Annexin A2 (AnxA2) is a Ca(2+)- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca(2+) binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca(2+)-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)(2), the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)(2) heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In "homotypic" membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in "homotypic" bridged membranes in the presence of Ca(2+).

Cell-penetrating peptides (CPPs) internalization can occur both by endocytosis and direct translocation through the cell membrane. These different entry routes suggest that molecular partners at the plasma membrane, phospholipids or glycosaminoglycans (GAGs), bind CPPs with different affinity or selectivity. The analysis of sequence-dependent interactions of CPPs with lipids and GAGs should lead to a better understanding of the molecular mechanisms underlying their internalization. CPPs are short sequences generally containing a high number of basic arginines and lysines and sometimes aromatic residues, in particular tryptophans. Tryptophans are crucial residues in membrane-active peptides, because they are important for membrane interaction. Membrane-active peptides often present facial amphiphilicity, which also promote the interaction with lipid bilayers. To study the role of Trp and facial amphiphilicity in cell interaction and penetration of CPPs, a nonapeptide series containing only Arg, Trp or D-Trp residues at different positions was designed. Our quantitative study indicates that to maintain/increase the uptake efficiency, Arg can be advantageously replaced by Trp in the nonapeptides. The presence of Trp in oligoarginines increases the uptake in cells expressing GAGs at their surface, when it only compensates for the loss of Arg and maintains similar peptide uptake in GAGdeficient cells. In addition, we show that facial amphiphilicity is not required for efficient uptake of these nonapeptides. Thermodynamic analyses point towards a key role of Trp that highly contributes to the binding enthalpy of complexes formation. Density functional theory (DFT) analysis highlights that salt bridge-π interactions play a crucial role for the GAG-dependent entry mechanisms. Peptide synthesisAll peptides were synthesized using standard Boc solid phase peptide synthesis. Boc-l-Arg(Tos), Boc-l-Trp(For), Boc-Gly, d-Biotin, MBHA Resin (0.53 mmol/g loading) and HBTU were purchased from Iris Biotech GmbH. Boc-(2,2-D 2 )-Gly was obtained from Cambridge Isotope Laboratories. Boc-d-Trp was purchased from Sigma-Aldrich. d-Biotin was fully oxidized to d-Biotin sulfone (Biot(O 2 ), Figure S1) by 4 days treatment with 30% H 2 O 2 in H 2 O and used without further purification. This avoids further oxidation of the peptide throughout time. Peptides were synthetized by hand on a 0.1 mmol scale for non-deuterated peptides and 0.01 mmol for deuterated peptides. Amino acid (5 eq) activation was performed by HBTU (4.5 eq) in the presence of excess DIEA (12 eq), and Boc deprotection was performed in neat TFA (2× 1 min). Trp side chains were deprotected prior to cleavage by treatment with 10% piperidine in DMF (1, 2, 5, 10, 30 and 60 min successive incubations). Peptides were cleaved from the resin by anhydrous HF (2h, 0°C) in the presence of anisole and dimethylsulfide.

Annexin A2 (AnxA2) is a Ca 2؉ -and acidic phospholipidbinding protein involved in many cellular processes. It undergoes Ca 2؉ -mediated membrane bridging at neutral pH and has been demonstrated to be involved in an H ؉ -mediated mechanism leading to a novel AnxA2-membrane complex structure. We used fluorescence techniques to characterize this H ؉ -dependent mechanism at the molecular level; in particular, the involvement of the AnxA2 N-terminal domain. This domain was labeled at Cys-8 either with acrylodan or pyrene-maleimide fluorescent probes. Steady-state and time-resolved fluorescence analysis for acrylodan and fluorescence quenching by doxyl-labeled phospholipids revealed direct interaction between the N-terminal domain and the membrane. The absence of pyrene excimer suggested that interactions between N termini are not involved in the H ؉ -mediated mechanism. These findings differ from those previously observed for the Ca 2؉ -mediated mechanism. Protein titration experiments showed that the protein concentration for half-maximal membrane aggregation was twice for Ca 2؉ -mediated compared with H ؉ -mediated aggregation, suggesting that AnxA2 was able to bridge membranes either as a dimer or as a monomer, respectively. An N-terminally deleted AnxA2 was 2-3 times less efficient than the wild-type protein for H ؉ -mediated membrane aggregation. We propose a model of AnxA2-membrane assemblies, highlighting the different roles of the N-terminal domain in the H ؉ -and Ca 2؉ -mediated membrane bridging mechanisms.