The cationic cell-penetrating peptide CppTAT derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts:: Optical, biophysical, and metabolic evidence
The cationic cell-penetrating peptide CppTAT derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts:: Optical, biophysical, and metabolic evidence
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DOI：
10.1021/bi0491604
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发表时间：
2005-01-11
期刊：
影响因子：
2.9
通讯作者：
Seelig, J
中科院分区：
文献类型：
--
作者：
Ziegler, A;Nervi, P;Seelig, J
细胞穿透肽（CPP）是阳离子肽，当其与基因、蛋白质或纳米颗粒连接时，促进这些实体穿过细胞膜的运输。尽管它们在基因转移和药物输送方面有潜在的用途，但CPP的作用方式仍然是神秘的。甚至有人认为，观察到的跨细胞膜转运是由细胞的化学固定引起的人为现象，这是显微镜观察的常见制备方法。在这里，我们已经合成了一种荧光衍生物的HIV-1达特蛋白转导域[Fg-CPPTAT（PTD）]，并观察到其摄取到nonfixed活成纤维细胞与延时共聚焦显微镜，消除了固定的需要。我们观察到，Fg-CPPTAT（PTD）进入细胞质和细胞核的非固定的成纤维细胞在几秒钟内，反对建议的人工制品的细胞固定。使用微分干涉相差显微镜，在细胞表面上检测到致密的聚集体。几项观察结果表明，这4种聚集体由与膜相关硫酸乙酰肝素（HS）结合的Fg-CPPTAT（PTD）组成。聚集体与Fg-CPPTAT（PTD）摄取平行，仅在显示Fg-CPPTAT（PTD）摄取的成纤维细胞上检测到。这些观察结果类似于早期报道的细胞表面分子与极化内吞流相结合的“盖帽”。酶法去除细胞外HS降低了Fg-CPPTAT（PTD）摄取和聚集体形成的速率，表明HS参与了摄取机制。用测量细胞外酸化速率（ECAR）的细胞传感器微生理仪研究了成纤维细胞在CPP摄取过程中的功能。短时间暴露（2.5分钟）的CPP降低ECAR，然而，这是可逆的再灌注后，仅用缓冲液。相比之下，在重复暴露于CPP后没有观察到恢复到基线值，这表明CPP在长期应用中是有毒的。
Cell-penetratin peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPPTAT(PTD)] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPPTAT(PTD) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these 4 aggregates consist of Fg-CPPTAT(PTD) bound to membrane-associated heparan sulfate (HS). The aggregates arow in parallel with Fg-CPPTAT(PTD) uptake and are detected only on fibroblasts showing, Fg-CPPTAT(PTD) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPPTAT(PTD) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.