Nuclear Localization of Cell-Penetrating Peptides Is Dependent on Endocytosis Rather Than Cytosolic Delivery in CHO Cells

Abstract

The nuclear localization of various cell penetrating peptides (CPPs), including Tat [47–57], YG(R)9, YG(K)9, and Model Amphipathic Peptide (MAP), was examined and correlated with the endocytosis and cytosolic transfer efficiency in CHO cells. The results showed that the internalization of the amphipathic peptide, MAP, was much higher than the other cationic CPPs tested. During subcellular fractionation analysis, MAP was only found in the vesicular fraction and was not detectable in the cytosol, similar to the intracellular localization of YG(K)9 as previously determined. This localization pattern differs greatly from the cationic CPPs oligoarginine and Tat, which were previously found primarily in the cytosol. Both quantitative and qualitative analysis of MAP showed high nuclear localization, with staining in perinuclear vesicles. On the other hand, YG(R)9 was found to be excluded from the nucleus. Lysosomotropic amines altered the nuclear localization of the CPPs tested, and the change was correlated with the release of degradation products from the treated cells. These results suggest that highly endocytosed CPPs such as MAP may be more suitable for nuclear drug delivery applications than peptides such as Tat and YG(R)9 that are efficiently delivered to the cytosol.

A Cell-penetrating Antibody Fragment against HIV-1 Rev Has High Antiviral Activity

“…There are reports for some other proteins that the Tat peptide position is not important (38) but this has not been our experience in this instance. Of the several other cell-penetrating peptides that have been described (21,39), the most promising would be those that have been demonstrated to significantly enhance nuclear import (40,41).…”

Characterization of a novel cell penetrating peptide derived from human Oct4

“…sulfated sugars present in the extracellular matrix [50,51]. Predominant cyotosolic and nuclear staining has been observed before with other CPPs or CPP-containing proteins in different cell lines [52][53][54]. Also, for lactoferrin-derived CPPs conjugated to BiotinStreptavidin, nuclear localization in live cells was dependent on concentration of the peptide-cargo complex as well as the origin, since only peptide derived from human but not rat lactoferrin was detected in the nuclei [52].…”

PKA-Type I Selective Constrained Peptide Disruptors of AKAP Complexes

“…64 Limitations of these aforementioned techniques include variability in cell penetration, 65 lack of reinforcement of the peptide secondary structure, susceptibility to proteolysis, 66,67 and potential mislocalization caused either by the CPP sequence or chemical moiety within the cellular environment. 65 Other chemical modifications including lactam bridges were shown to stabilize the secondary structural fold of helices. 68 However, peptides bearing this modification may still have limited cell permeability and susceptibility to cellular degradation.…”