Twenty years ago, the discovery of peptides able to cross cellular membranes launched a novel field in molecular delivery based on these non‐invasive vectors, most commonly called cell‐penetrating peptides (CPPs) or protein transduction domains (PTDs). These peptides were shown to efficiently transport various biologically active molecules inside living cells, and thus are considered promising devices for medical and biotechnological developments. Moreover, CPPs emerged as potential tools to study the prime mechanisms of cellular entry across the plasma membrane. This review is dedicated to CPP fundamentals, with an emphasis on the molecular requirements and mechanism of their entry into eukaryotic cells.

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 degrees C (endocytosis and translocation) and 4 degrees 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.

Cell‐penetrating peptides (CPPs) can cross the cell membrane and are widely used to deliver bioactive cargoes inside cells. The cargo and the CPP are often conjugated through a disulfide bridge with the common acceptation that this linker is stable in the extracellular biological medium and should not perturb the internalization process. However, with the use of thiol‐specific reagents combined with mass spectrometry (as a quantitative method to measure intracellular concentrations of peptides) and confocal microscopy (as a qualitative method to visualize internalized pep‐tides) analyses, we could show that, depending on the peptide sequence, thiol/disulfide exchange reactions could happen at the cell surface. These exchange reactions lead to the reduction of disulfide conjugates. In addition, it was observed that not only disulfide‐ but also thiol‐containing peptides could cross‐react with cell‐surface thiols. The peptides cross‐linked by thiolcontaining membrane proteins were either trapped in the membrane or further internalized. Therefore, a new route of cellular uptake was unveiled that is not restricted to CPPs: a protein kinase C peptide inhibitor that is not cell permeant could cross cell membranes when an activated cysteine (with a 3‐nitro‐2‐pyridine‐sulfenyl moiety) was introduced in its sequence.— Aubry, S., Burlina, F., Dupont, E., Delaroche, D., Joliot, A., Lavielle, S., Chassaing, G., Sagan, S. Cell‐surface thiols affect cell entry of disulfide‐conjugated peptides. FASEB J. 23, 2956–2967 (2009). www.fasebj.org

Deciphering the structural requirements and mechanisms for internalization of cell‐penetrating peptides (CPPs) is required to improve their delivery efficiency. Herein, a unique role of tryptophan (Trp) residues in the interaction and structuring of cationic CPP sequences with glycosaminoglycans (GAGs) has been characterized, in relation with cell internalization. Using isothermal titration calorimetry, circular dichroism, NMR, mass spectrometry, and phase‐contrast microscopy, we compared the interaction of 7 basic CPPs with 5 classes of GAGs. We found that the affinity of CPPs for GAGs increases linearly with the number of Trp residues, from 30 nM for a penetratin analog with 1 Trp residue to 1.5 nM for a penetratin analog with 6 Trp residues for heparin (HI); peptides with Trp residues adopt a predominantly β‐strand structure in complex with HI and form large, stable β‐sheet aggregates with GAGs; and in the absence of any cytotoxicity effect, the quantity of peptide internalized into CHO cells increased 2 times with 1 Trp residue, 10 times with 2 Trp residues, and 20 times with 3 Trp residues, compared with +6 peptides with no Trp residues. Therefore, Trp residues represent molecular determinants in basic peptide sequences not only for direct membrane translocation but also for efficient endocytosis through GAGs.—Bechara, C., Pallerla, M., Zaltsman, Y., Burlina, F., Alves, I. D., Lequin, O., Sagan S. Tryptophan within basic peptide sequences triggers glycosaminoglycan‐dependent endocytosis. FASEB J. 27, 738–749 (2013). www.fasebj.org