A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus
A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus
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DOI：
10.1074/jbc.272.25.16010
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发表时间：
1997-06-20
影响因子：
4.8
通讯作者：
Lebleu, B
中科院分区：
文献类型：
--
作者：
Vives, E;Brodin, P;Lebleu, B
达特是一种参与人类免疫缺陷病毒1型（HIV-1）复制的86个氨基酸的蛋白质。几项研究表明，外源性达特蛋白能够通过质膜易位并到达细胞核以反式激活病毒基因组。达特蛋白的以碱性氨基酸簇为中心的区域被分配给这种易位活性，最近的数据表明，Tat衍生肽的化学偶联（从残基37延伸至72）与几种蛋白质连接，允许其功能性内化到几种细胞系或组织中。该相同结构域的一部分可以折叠成具有两亲特性的α-螺旋结构。这种螺旋结构被认为是通过融合或内吞作用摄取几种包膜病毒的关键决定因素。在本研究中，我们已经划定了达特易位所需的主要决定因素在这个序列中合成几个肽覆盖焦油结构域从残基37至60。出乎意料的是，从氨基酸37延伸至47的结构域（其对应于α-螺旋结构）对于细胞摄取和核转位不是必需的。通过用荧光素直接标记或通过使用针对达特碱性簇的单克隆抗体间接免疫荧光来评估肽内化。这两种方法都确定了含有碱性结构域的所有肽在低至100 nM的浓度下在不到5分钟内被细胞吸收。相反，具有完整α-螺旋但具有截短的碱性氨基酸簇的肽不被细胞摄取。内化过程不涉及内吞途径，因为在4 ℃下没有观察到摄取的抑制。已经报道了来自于拟南芥同源结构域的富含碱性氨基酸的肽的类似观察结果（1）。允许有效易位通过质膜的短肽可以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药理学感兴趣的肽）的有用载体。
Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shows 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 Tar 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.