TY - JOUR
T1 - The uptake of HIV Tat peptide proceeds via two pathways which differ from macropinocytosis
AU - Ben-Dov, Nadav
AU - Korenstein, Rafi
N1 - Publisher Copyright:
© 2014 Elsevier B.V.All rights reserved.
PY - 2015
Y1 - 2015
N2 - Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15°C corresponds to Q10 = 1.1, proceeding through a prompt (< 5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10 = 1.44, accompanied by activation energy Ea = 4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10 = 2.2 and Ea = 7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~ 15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence.
AB - Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15°C corresponds to Q10 = 1.1, proceeding through a prompt (< 5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10 = 1.44, accompanied by activation energy Ea = 4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10 = 2.2 and Ea = 7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~ 15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence.
KW - Cell penetrating peptide
KW - Endocytosis
KW - Membrane translocation
KW - Plasma membrane
KW - Tat peptide
UR - http://www.scopus.com/inward/record.url?scp=84937129484&partnerID=8YFLogxK
U2 - 10.1016/j.bbamem.2014.12.015
DO - 10.1016/j.bbamem.2014.12.015
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AN - SCOPUS:84937129484
SN - 0005-2736
VL - 1848
SP - 869
EP - 877
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 1
ER -