Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide - Role for cell-surface proteoglycans
Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide - Role for cell-surface proteoglycans
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DOI：
10.1074/jbc.m205395200
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发表时间：
2002-10-11
影响因子：
4.8
通讯作者：
Belting, M
中科院分区：
文献类型：
--
作者：
Sandgren, S;Cheng, F;Belting, M
基于基因转移和蛋白质递送的新疗法需要更好地理解大分子膜转运的基本机制。我们已经研究了细胞摄取的大分子聚阴离子，即DNA和糖胺聚糖，和一个多元的HIV-Tat衍生肽（GRKKRRQRRRPPQC）使用荧光辅助细胞分选和共聚焦荧光显微镜。HIV转录的反式激活因子（达特）肽以时间、浓度和温度依赖性方式刺激细胞对DNA和硫酸乙酰肝素的摄取。肽聚阴离子复合物积累在大，酸性，细胞质囊泡从头形成。这是其次是转移的聚阴离子进入核室和随后消失的内溶酶体囊泡。在不存在聚阴离子的情况下，达特肽在核隔室中显示快速积累。然而，在聚阴离子的存在下，肽几乎完全保留在细胞质囊泡中。细胞表面蛋白聚糖在摄取复合物中发挥了关键作用，表现出相对较高的肽与聚阴离子的比例，对应于复合物的净正电荷。摄取聚阴离子本身或复合物与相对较低的肽聚阴离子的比例有利于蛋白聚糖缺乏症的受体细胞，表明存在不同的运输机制。此外，全长HIV-Tat的表达以及HIV-Tat肽的外源性添加导致内源性蛋白聚糖的细胞积累。我们的结论是，HIV-Tat衍生肽有效地靶向外源DNA和糖胺聚糖的核室和蛋白多糖在这些过程中发挥调节作用，这可能有影响的定向基因和药物在体内的交付。
New therapies based on gene transfer and protein delivery require a better understanding of the basic mechanisms of macromolecular membrane transport. We have studied cellular uptake of macromolecular polyanions, i.e. DNA and glycosaminoglycans, and a polybasic HIV-Tat derived peptide (GRKKRRQRRRPPQC) using fluorescence assisted cell sorting and confocal fluorescence microscopy. The transactivator of HIV transcription (Tat) peptide stimulated cellular uptake of both DNA and heparan sulfate in a time-, concentration-, and temperature-dependent manner. Peptide-polyanion complexes accumulated in large, acidic, cytoplasmic vesicles formed de novo. This was followed by transfer of polyanion into the nuclear compartment and subsequent disappearance of the endolysosomal vesicles. In the absence of polyanion the Tat peptide displayed rapid accumulation in the nuclear compartment. However, in the presence of polyanion the peptide was almost exclusively retained in cytoplasmic vesicles. Cell-surface proteoglycans played a pivotal role in the uptake of complexes exhibiting a relatively high peptide to polyanion ratio, corresponding to a net positive charge of the complexes. Uptake of polyanions per se or complexes with a relatively low peptide to polyanion ratio was favored by proteoglycan deficiency in the recipient cells, indicating the existence of distinct transport mechanisms. Moreover, expression of full-length HIV-Tat as well as exogenous addition of HIV-Tat peptide resulted in cellular accumulation of endogenous proteoglycans. We conclude that an HIV-Tat derived peptide efficiently targets extraneous DNA and glycosaminoglycans to the nuclear compartment and that proteoglycans serve a regulatory role in these processes, which may have implications for directed gene and drug delivery in vivo.