Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1768, Issue 7, Pages 1769-1776Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2007.03.029
Keywords
cell-penetrating peptide; protein transduction domains; uptake; degradation; endocytosis inhibitor
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Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated. (c) 2007 Elsevier B.V. All rights reserved.
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