Translocation and Endocytosis for Cell-penetrating Peptide Internalization

Abstract

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 proteoglycandeficient 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) 3 that share the activity of cellular entry are usually short peptides of less than 20 amino acids highly enriched in basic residues. Among them, Antp, Tat-(48 -60), and oligoarginine peptides are the most intensively studied. Despite the wide use of these CPPs as macromolecular delivery devices, the internalization mechanism of these peptides in cells still remains largely controversial. The energy dependence of the internalization mechanism is unique because all endocytotic pathways are inhibited at low temperature. Consequently, at low temperature, internalization likely reflects a direct translocation mechanism. Early studies proposed that Antp enters cells by an energy-independent membrane translocation mechanism (1). This first analysis was then contradicted by other studies that suggested, with the use of inhibitors, the involvement of endocytosis in the cellular internalization of cell-penetrating peptides (2-4).The hypothesis that endocytosis was the only internalization mechanism of CPP resulted from studies examining whether the temperature or the binding to cell-surface glycosaminoglycans (GAGs) were critical for peptide internalization. Most of these interpretations resulted from fluorescence microscopy data. For instance, it was reported that Antp, Tat, and oligoarginine peptides were not efficiently internalized in the Chinese hamster ovary (CHO) mutant pgsA-745 cell line, which does not produce cell-surface heparan sulfate or chondroitin sulfate (5, 6). However, recent data indicates that Tat-mediated transduction occurs in the absence of heparan sulfate and chondroitin sulfate (7). The discrepancies observed between studies may be explained in part by different incubation conditions (peptide/cells ratio and peptide concentration) (8), limits in fluorescence imaging, such as quenching (9), or fluorophore-dependent intracellular trafficking (10), as...

Membrane Oxidation Enables the Cytosolic Entry of Polyarginine Cell-penetrating Peptides

“…This low temperature condition only led to a minor reduction in cell penetration ( Fig. 2A) (15,17,18). In addition, cytosolic penetration was achieved in a relatively high percentage of cells within 1 or 3 min of incubation, making a process involving endocytosis and endosomal escape upon slow endocytic maturation unlikely (typical time frame varies from 5 to 30 min) (Fig.…”

“…1B). This staining is common for arginine-rich peptides, and because cytosolic entry presumably precedes nucleolar staining, it indicates that CPPs reach the intracellular cytosolic/nuclear milieu (15)(16)(17)(18). Importantly, this specific staining was observable at ϫ20 magnification for D-CPPs but not for L-CPPs (Fig.…”

“…In some instances, cytosolic penetration appears to involve direct plasma membrane translocation (15)(16)(17)(18). Alternatively, a two-step process of endocytosis followed by escape from endosomes has also been observed (19 -22).…”

Cell-penetrating Peptides with Intracellular Actin-remodeling Activity in Malignant Fibroblasts

“…These cell-penetrating peptides are more or less amphipathic peptides generally rich in lysyl and arginyl residues. It is now well accepted that these peptides can enter cells through direct translocation and endocytosis pathways and thus that they can end up into the cytosol of the cell (5,6). In addition, cell-penetrating peptides are widely used as conjugates to vehiculate inside bioactive molecules of the cell such as peptides, proteins, or nucleic acids (7,8).…”

Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies

“…It has also been demonstrated that CPPs have the capacity to shuttle bioactive cargoes inside eukaryotic cells. The final localization in the cell of the CPP/cargo conjugates or complexes is undoubtedly associated with the mechanism of entry; however, the mechanism is still an ongoing matter of debate [7][8][9][10][11][12][13][14][15][16][17]. The capacity for CPPs to deliver a bioactive molecule inside of cells has opened avenues for intracellular pharmacology, and recently, significant advances have been accomplished for siRNA delivery and also for specific Laurence Dutot and Pascaline Lécorché contributed equally Electronic supplementary material The online version of this article (doi:10.1007/s12154-009-0031-9) contains supplementary material, which is available to authorized users.…”