TY - JOUR
T1 - Cellular Uptake Mechanism of TCTP-PTD in Human Lung Carcinoma Cells
AU - Kim, Hyo Young
AU - Kim, Sabin
AU - Pyun, Hae Jun
AU - Maeng, Jeehye
AU - Lee, Kyunglim
N1 - Publisher Copyright:
© 2014 American Chemical Society.
PY - 2015/1/5
Y1 - 2015/1/5
N2 - 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.
AB - 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.
KW - endocytosis pathway
KW - lipid-rafts/caveolae
KW - mechanism
KW - protein transduction domain (PTD)
KW - subcellular fractionations
KW - translationally controlled tumor protein (TCTP)
UR - http://www.scopus.com/inward/record.url?scp=84920458187&partnerID=8YFLogxK
U2 - 10.1021/mp500547f
DO - 10.1021/mp500547f
M3 - Article
C2 - 25423047
AN - SCOPUS:84920458187
SN - 1543-8384
VL - 12
SP - 194
EP - 203
JO - Molecular Pharmaceutics
JF - Molecular Pharmaceutics
IS - 1
ER -