Evidence that membrane transduction of oligoarginine does not require vesicle formation |
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Authors: | Zaro Jennica L Shen Wei-Chiang |
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Affiliation: | University of Southern California, School of Pharmacy, Department of Pharmaceutical Sciences, 1985 Zonal Avenue, PSC 404B, Los Angeles, CA 90033-1039, USA. |
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Abstract: | The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 degrees C as compared to the 37 degrees C control, and was only partially inhibited at 4 degrees C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine. |
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Keywords: | α-MEM, α-minimum essential medium AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride CHO, Chinese hamster ovary CPP, cell-penetrating peptide E-64, (2S,3S)-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-methylbutyl}carbamoyl)-oxirane-2-carboxylic acid EDTA, ethylene-di-amine-tetra-acetic acid E-64, trans-(epoxy-succinyl)- smallcaps" >l-leucylamino-(4-guanidino)butane FITC, fluorescein 5′-isothiocyanate HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV-1, human immunodeficiency virus type 1 MTP, membrane transduction peptide NH4Cl, ammonium chloride PBS, phosphate-buffered saline PMSF, phenyl-methylsulfonyl fluoride RFU, relative fluorescent units |
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