Vivès E, Brodin P, Lebleu B
Institut de Génétique Moléculaire de Montpellier, CNRS-UMR 5535, BP5051, 1919 route de Mende, 34033 Montpellier cedex 1, France.
J Biol Chem. 1997 Jun 20;272(25):16010-7. doi: 10.1074/jbc.272.25.16010.
Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.
Tat是一种由86个氨基酸组成的蛋白质，参与1型人类免疫缺陷病毒（HIV-1）的复制。多项研究表明，外源性Tat蛋白能够穿过质膜并到达细胞核，从而反式激活病毒基因组。Tat蛋白中以碱性氨基酸簇为中心的区域被认为与这种转运活性有关。最近的数据表明，将一段Tat衍生肽（从第37位氨基酸延伸至第72位氨基酸）化学偶联到几种蛋白质上，可使其功能性内化到多种细胞系或组织中。该结构域的一部分可以折叠成具有两亲性特征的α-螺旋结构。这种螺旋结构被认为是几种包膜病毒通过融合或内吞作用进入细胞的关键决定因素。在本研究中，我们通过合成覆盖Tat结构域第37位至第60位氨基酸的几种肽段，确定了该序列内Tat转运所需的主要决定因素。出乎意料的是，从氨基酸37延伸至47的结构域，即对应于α-螺旋结构的区域，对于细胞摄取和核转运并非必需。通过用荧光素直接标记或使用针对Tat碱性簇的单克隆抗体进行间接免疫荧光来评估肽的内化。两种方法均证实，所有含有碱性结构域的肽在浓度低至100 nM时，均可在不到5分钟的时间内被细胞摄取。相比之下，具有完整α-螺旋但碱性氨基酸簇被截断的肽不会被细胞摄取。内化过程不涉及内吞途径，因为在4℃下未观察到摄取受到抑制。对于源自触角足同源结构域的富含碱性氨基酸的肽也有类似的观察结果（1）。能够有效穿过质膜进行转运的短肽可能是用于细胞内递送各种非渗透性药物（包括反义寡核苷酸和具有药理学意义的肽）的有用载体。
