Effects of Na+/H+ exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV-TAT peptide and octaarginine
Effects of Na+/H+ exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV-TAT peptide and octaarginine
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DOI：
10.1016/j.jconrel.2006.07.009
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发表时间：
2006-11-28
影响因子：
10.8
通讯作者：
Jones, Arwyn T.
中科院分区：
文献类型：
--
作者：
Fretz, Marjan;Jin, Jing;Jones, Arwyn T.
蛋白质转导结构域，如来自HIV蛋白TAT的蛋白质转导结构域，作为将治疗实体（如基因和蛋白质）输送到细胞中的载体具有巨大的潜力。广泛的研究表明，大部分被研究的变异是通过内吞机制进入细胞的。然而，围绕确切的摄取机制和是否使用特定途径存在争议。研究表明，在离子转运抑制剂如阿米洛利及其更有效的类似物5-（n-乙基- n-异丙基）阿米洛利（EIPA）的存在下，蛋白质转导域的摄取受到抑制，这表明肽内化与巨噬细胞增多症之间存在联系。在这项研究中，通过对内吞途径的早期和晚期组分进行免疫标记，我们发现EIPA和阿米洛利在较小程度上影响了早期、晚期内体和溶酶体的形态和亚细胞位置。eipa处理的细胞早期和晚期内吞结构增大，这些细胞器聚集在核周区域。研究EIPA对荧光辛精氨酸分布影响的实验结果与免疫定位研究一致。在HIV-TAT肽和八精氨酸的荧光偶联物存在下，用EIPA处理CD34（+）白血病细胞系KG1a，与未处理的细胞相比，显示出明显的囊泡染色，但EIPA处理的细胞还具有细胞质中肽定位增加的特征。EIPA的水平先前在其他细胞系中显示抑制HIV-TAT肽和八精氨酸的摄取，但在KG1a细胞中对这两种肽的摄取没有主要影响。(c) 2006 Elsevier B.V.版权所有
Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34(+) leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV-TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV-TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells. (c) 2006 Elsevier B.V. All rights reserved.