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

Fretz, Marjan M., Penning, Neal Aart, Al-Taei, Saly, Futaki, Shiroh, Takeuchi, Toshihide, Nakase, Ikuhiko, Storm, Gert and Jones, Arwyn Tomos ORCID: https://orcid.org/0000-0003-2781-8905 2007. Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochemical Journal 403 (2) , pp. 335-342. 10.1042/BJ20061808

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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 °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 °C incubations. At temperatures between 12 and 30 °C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 °C. Small increases in the extracellular peptide concentration in 37 °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-b-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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Pharmacy
Subjects:	R Medicine > R Medicine (General)
Uncontrolled Keywords:	Cell-penetrating peptide ; Endocytosis ; fluorescence microscopy ; KG1a cell line ; Leukaemia ; Octa-arginine.
Publisher:	Portland Press Limited
ISSN:	1470-8728
Last Modified:	01 Dec 2022 09:59
URI:	https://orca.cardiff.ac.uk/id/eprint/973

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