Abstract
Cell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from l- to d-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescence TAT as a model CPP to explore the broader consequences of l- to d-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the d-peptide, once in the endosome, is significantly more prone to escape than its l-counterpart. Moreover, the d-peptide is retained in the cytosol of cells for several days, whereas the l-peptide is degraded within hours. Notably, while the l-peptide is relatively innocuous to cells, the d-peptide exerts a prolonged anti-proliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties.
Highlights
We address these questions by using dimeric fluorescent TAT as a model Cell-penetrating peptides (CPPs). dfTAT is a disulfidebonded dimer of the prototypical CPP TAT. dfTAT is capable of escaping from endosomes with extremely high efficiency [27]
D-dfTAT Is Protease-resistant and Capable of Penetrating Cells—dfTAT (i.e. L-dfTAT) and D-dfTAT were prepared by synthesis of the peptide CK(TMR)RKKRRQRRRG or ck(TMR) rkkrrqrrrG, respectively, on solid phase followed by disulfide bond formation of the cysteine residue in solution
dominant negative Rab7 (DN-Rab7) inhibited cytosolic penetration of D-dfTAT, whereas wild type did not (Fig. 2B). These results indicate that DN-Rab7 blocks the endosomal escape of D-dfTAT and that, D-dfTAT escapes from endocytic organelles that are downstream of early endosomes in the endocytic pathway
Summary
DfTAT penetrates the cytosol of cells by escaping from late endosomes via interactions with the lipid BMP [30]. To further confirm that the membrane of these organelles is the site of peptide penetration, the activity of dfTAT and D-dfTAT toward liposomes mimicking late endosomes (i.e. BMP, PC, PE, cholesterol) was assessed in vitro. At any given concentration of peptide in the incubation medium, the number of cells positive for penetration was similar between the two peptides (with the exception of 10 M, where D-dfTAT is more active than dfTAT) (Fig. 3A).
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