Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells
Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells
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DOI：
10.1096/fj.05-5523com
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发表时间：
2006-09-01
期刊：
影响因子：
4.8
通讯作者：
Cardoso, M. Cristina
中科院分区：
文献类型：
--
作者：
Tuennemann, Gisela;Martin, Robert M.;Cardoso, M. Cristina
细胞穿透肽（CPP）能够将各种各样的货物引入活细胞。对内化过程的描述从不依赖能量的细胞膜穿透到内吞摄取。为了阐明CPP构建体的进入机制是否可能受到货物性质的影响，我们使用活哺乳动物细胞的时间推移共聚焦显微镜分析来直接比较充分研究的与蛋白质（> 50个氨基酸）或肽（< 50个氨基酸）货物融合的CPP达特的摄取。我们还分析了各种结构的亚细胞分布和流动性后的内化事件。达特融合蛋白主要进入胞质囊泡，而融合到达特的肽以依赖于膜电位的快速方式进入细胞。尽管它们在核仁中积累，达特融合肽的光漂白揭示了它们的流动性。测试了内化达特肽的生物利用度，并通过在活细胞中测量的对源自肿瘤抑制因子p21（WAF/Cip）和DNA连接酶I的两种达特融合肽的细胞周期进展的强抑制作用来证实。Tunnemann，G.，马丁河，巴西-地M.，奥普特，Patsch，C.，Edenhofer，F.，Cardoso，M. C.活细胞中含TAT蛋白质和肽的摄取和生物利用度的货物依赖性模式。
Cell-penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy-independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well-studied CPP TAT fused to a protein (> 50 amino acids) or peptide (< 50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21(WAF/Cip) and DNA Ligase I measured in living cells.-Tunnemann, G., Martin, R. M., Haupt, S., Patsch, C., Edenhofer, F., Cardoso, M. C. Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells.