Cellular Uptake Mechanism of TCTP-PTD in Human Lung Carcinoma Cells
- 주제(키워드) endocytosis pathway , lipid-rafts/caveolae , mechanism , protein transduction domain (PTD) , subcellular fractionations , translationally controlled tumor protein (TCTP)
- 등재 SCIE, SCOPUS
- 발행기관 American Chemical Society
- 발행년도 2015
- 총서유형 Journal
- URI http://www.dcollection.net/handler/ewha/000000114131
- 본문언어 영어
- Published As http://dx.doi.org/10.1021/mp500547f
- 저작권 이화여자대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
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.
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