title: Cellular uptake mechanism of TCTP-PTD in human lung carcinoma cells.
abstract: We reported previously that human translationally controlled tumor protein (TCTP) contains, at its NH2-terminus, a protein transduction domain (PTD), which we called TCTP-PTD, with the amino acid sequence MIIYRDLISH. In this report we describe how TCTP-PTD penetrates A549 human lung cancer cell membranes and promotes protein internalization. Cellular uptake of fluorescent TCTP-PTD and a recombinant fusion protein consisting of TCTP-PTD and GFP (green fluorescent protein) was analyzed by confocal fluorescence microscopy and flow cytometry. Inhibitor assays using several agents that perturb the internalization process revealed that TCTP-PTD transduces the cells partly via lipid-raft/caveola-dependent endocytosis and partly by macropinocytosis in a dynamin/actin/microtubule-dependent pathway. To trace the pathway followed by the penetration of TCTP-PTD, the localization of PTDs was investigated in the lipid-raft, subcellular, and ER fractions. We found that, after entry, TCTP-PTD is localized in the cytoplasm and cytoskeleton, but not in the nucleus, and is transported into endoplasmic reticulum (ER). Expression levels of caveolin-1 in A549 and HeLa cells are different, and these differences appear to contribute to the sensitivity of TCTP-PTD uptake inhibition, against lipid-raft depleter, nystatin. This elucidation of the underlying mechanism of TCTP-PTD translocation may help the design of approaches that employ TCTP-PTD in the cellular delivery of bioactive molecules.
authors.affiliation: Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University , Seoul 120-750, Republic of Korea.
authors.forename: Hyo Young
authors.initials: HY
authors.lastname: Kim
authors.forename: Sabin
authors.initials: S
authors.lastname: Kim
authors.forename: Hae Jun
authors.initials: HJ
authors.lastname: Pyun
authors.forename: Jeehye
authors.initials: J
authors.lastname: Maeng
authors.forename: Kyunglim
authors.initials: K
authors.lastname: Lee
mesh_terms: D014408:Biomarkers, Tumor / Q000737:chemistry* / Q000493:pharmacokinetics*; D002277:Carcinoma / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry; D018613:Microscopy, Confocal; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D000090324:Tumor Protein, Translationally-Controlled 1
chemical_list: D014408:Biomarkers, Tumor; D010455:Peptides; D011993:Recombinant Fusion Proteins; C000715647:TPT1 protein, human; D000090324:Tumor Protein, Translationally-Controlled 1; D049452:Green Fluorescent Proteins
keywords: endocytosis pathway; lipid-rafts/caveolae; mechanism; protein transduction domain (PTD); subcellular fractionations; translationally controlled tumor protein (TCTP)