Cellular Uptake of Arginine-Rich Peptides: Roles for Macropinocytosis and Actin Rearrangement

Abstract

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.

A novel concept in antiangiogenic and antitumoral therapy: multitarget destabilization of short-lived mRNAs by the zinc finger protein ZFP36L1

“…This observation is in agreement with previous studies showing that some TAT-fusion proteins are still active at least up to 24 h (Cai et al, 2006). It is worth mentioning that we used low concentrations of purified PTD-ZFP36L1 (100 nM), which is in contrast to previous studies using micromolar concentrations of PTD fusion proteins to transduce different cell types (Richard et al, 2003;Nakase et al, 2004;Duchardt et al, 2007). We demonstrated that R7-and R9-ZFP36L1 induced a 40% decrease in VEGF protein levels compared with a 20% decrease obtained with TAT-ZFP36L1, thus confirming that arginine-rich ).…”

Arginine‐rich cell‐penetrating peptides

“…FACS analysis following trypsin treatment indicated a relatively slow rate of uptake, comparable to that of classical markers of endocytosis. Since then, many other studies have also observed inhibition of cellular uptake at 4°C and with chemical means that induce energy depletion, indicating an energy-dependent process as the major route for the internalization of cell-penetrating peptides [27][28][29][30][31][32][33].…”

Tumour lineage-homing cell-penetrating peptides as anticancer molecular delivery systems

“…To reveal the primary physiological pathway of incorporation, two representative CPPs, colon cancer-homing CPP2 and myeloid leukaemia-homing CPP44, were chosen as examples. First, their internalization was examined using 5-(N-ethyl-N-isopropyl) amiloride (EIPA) 10 , Dynamin inhibitor I (DI; Dynasore) 11 , and chlorpromazine (CPZ) 12 . Pretreatment of the cells with EIPA, a macropinocytosis inhibitor, prevented the entry of polyarginine (r9) 13 in a dose-dependent manner; however, the entry of CPP44 into primary AML cells was not impeded.…”