Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

Abstract

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.

Arginine‐rich cell‐penetrating peptides

“…The diffuse staining was not due to the release of peptide from endosomes as it appeared first and the punctate staining later, and it continued to occur at 4°C when endocytosis is inhibited. Several other studies have also reported cellular uptake when the cells have been incubated at 4°C [26,[51][52][53]. It has also been observed that blocking specific endocytotic pathways does not affect the ability of TAT peptide to enter cells.…”

“…The TAT protein is an 86 amino acid long protein that is released by infected cells and is an essential regulatory gene for HIV replication [8]. In 1997, Vives et al [9] found that a 11-amino acid sequence, TAT (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57), now known as the TAT peptide or TAT PTD, can not only enter cells but is more efficient than the full length protein. It was observed that the chirality of the peptide backbone has no effect on cellular uptake of TAT peptide; inverse and retro forms were able to enter cells as efficiently as the native peptide, suggesting uptake does not require a specific binding site.…”

Targeting antibodies to the cytoplasm

“…Internalization has consequently been suggested to occur via different routes of endocytosis, potentially including macropinocytosis, clathrin-and caveolin mediated endocytosis, as well as via a mode of entry by direct membrane penetration at high peptide concentrations. 125,[133][134][135] Although direct membrane penetration was proposed as a potential route of entry for PTDs alone, uptake of arginine-rich PTDs linked to macromolecular cargoes appears to occur solely by endocytosis. 127,134,136 Since routes of entry appear to be similar for most PTDs and delivery efficiency is subject to the same limitations, the potential applications of well-characterized PTDs are worth considering, rather than searching for the "best PTD."…”

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

“…The translocation can take place via the energy dependent or independent route and multiple factors have been suggested to control the balance between various internalization pathways [14][15][16]. Similarly, some reports suggest direct translocation to the cell of TP10 [17,18], whereas other studies suggest its endocytosis [12,19]. Thus, we aimed to check possible method of TP and TP10 penetration into CRC cells by determining their transport ratios after inhibition of endocytosis with the use of brefeldin A [20][21][22].…”