Design and intracellular delivery of peptides for transcription regulation

一种来源于人类免疫缺陷病毒(HIV)-1Tat的碱性多肽被报道具有通过细胞膜转运和将外源蛋白带入细胞的能力。我们已经证明，这些特征在许多富含精氨酸的多肽中都可以观察到，包括那些具有支链结构的多肽。基于这些发现，提出了富含精氨酸的多肽普遍存在的内化机制。我们还证明了这些特征也适用于具有支链结构的多肽。合成了4个支链上含有精氨酸残基的多肽(Rn)4[n(精氨酸残基个数)=0~6]。荧光显微镜观察表明，(R2)4肽的易位效率最高。支链多肽的转位效率和细胞定位也依赖于精氨酸残基的数量，就像在线性多肽中所看到的那样。我们已经证明，含有富含精氨酸片段的核糖核酸酶S的非共价蛋白组装被成功地导入细胞中，表现出抗艾滋病毒的活性。制备了与转录因子NF-κB激活相关的I-κB的磷酸化和泛化位点对应的多肽。通过与膜通透性精氨酸肽的偶联将这些多肽引入细胞内，可抑制NF-κB的激活。然而，抑制程度的差异是有意义的。我们还发现转录因子Sp1的转录被Sp1的DNA识别片段所产生的多肽所抑制。

A basic peptide derived from the human immunodeficiency virus (HIV)-1 Tat has been reported to have the ability to translocate through the cell membranes and to bring exogenous proteins into the cells. We have demonstrated that these features were observable among many arginine-rich peptides including those having a branched chain structure. Based on these findings, the presence of a ubiquitous internalization mechanism for the arginine-rich peptides has been suggested. We have also demonstrated that these features are also applicable to the peptides having branched-chain structures. Peptides that have arginine residues on four branched-chains (R_n)_4 [n (number of arginine residues) = 0〜6] were prepared. Fluorescence microscopic observation revealed that the (R_2)_4 peptide showed the most efficient translocation. Dependence on the number of arginine residues for the translocation efficiency and cellular localization was also observed for the branched-chain peptides as was seen in the linear peptides. We have shown that a non-covalent protein assembly of Rnase S bearing arginine-rich segment was successfully introduced into cells to exhibit an anti-HIV activity. Peptides corresponding to the phosphorylation and ubiquitilation sites of IκB, which is involved in the activation of transcription factor NF-κB, were prepared. Introduction of these peptides into cells by conjugation with the membrane-permeable arginine peptide resulted in the inhibition of NF-κB activation. However, significance of the difference in the extent of inhibition was observed. We have also shown that the transcription by transcription factor Sp1 was inhibited by the peptide derived from DNA recognition segment of Sp1.