Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles
Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles
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DOI：
10.1016/j.jconrel.2005.02.019
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发表时间：
2005-06-02
影响因子：
10.8
通讯作者：
Nakanishi, M
中科院分区：
文献类型：
--
作者：
Eguchi, A;Furusawa, H;Nakanishi, M
核膜是一道严密的屏障，阻止治疗性基因进入未分裂的组织细胞。借助多肽核定位信号(NLS)克服这一障碍对于提高合成基因输送载体的性能至关重要。在这篇文章中，我们研究了噬菌体颗粒的核转运，这些噬菌体颗粒表面展示了各种含有SV40T抗原最小NLS的肽。由于最小的NLS(PKKKRKV)是导入蛋白a的结合域，含有该肽的重组蛋白和分子偶联物可以有效地积聚到细胞核中。然而，我们发现，除了最小的NLS外，C-末端和N-末端的结构还深刻地影响噬菌体颗粒的核运输效率以及它们与导入蛋白α的结合能力：无论是C末端的几个氨基酸残基被截断，还是N末端被标志或c-myc标签所取代，这两种生物学活性都会被取消。优化后的NLS的结构不能从传统的蛋白质转运分析和电子计算机的结构分析中预测出来。我们的结果表明，当优化的NLS以足够的密度显示在它们的表面时，直径为50 nm的物体可以穿过核孔复合体。(C)2005爱思唯尔B.V.保留所有权利。
The nuclear membrane is a tight barrier against the delivery of therapeutic genes into non-dividing tissue cells. Overcoming this barrier with the aid of peptidic nuclear localization signals (NLS) is crucial for improving the performance of synthetic gene-delivery vehicles. In this article, we examine the nuclear transport of lambda phage particles displaying various peptides containing the minimum NLS of SV40 T antigen on their surface. As the minimum NLS (PKKKRKV) is a binding domain to importin a, recombinant proteins and molecular conjugates containing this peptide accumulate into the nucleus efficiently. However, we find that the C-terminal and N-terminal structures besides the minimum NLS profoundly affect the efficiency of the nuclear transport of the phage particles as well as their binding capacity to importin alpha: either truncation of a few amino acid residues from the C-terminus or the replacement of the N-terminus with a FLAG- or c-myc-tag abolish both of these biological activities. The structure of the optimized NLS is unpredictable from conventional protein transport assay and from the structural analysis in silico. Our results reveal that the objects with 50 nm in diameter can pass through the nuclear pore complex when the optimized NLS is displayed at a sufficient density on their surface. (c) 2005 Elsevier B.V All rights reserved.