Design and intracellular delivery of peptides for transcription regulation

一种源自人类免疫缺陷病毒(HIV)-1 Tat的碱性肽已被报道具有通过细胞膜转运并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肽中都可以观察到，包括那些具有支链结构的肽。基于这些发现，已经提出了富含精氨酸肽的普遍内化机制。我们还证明了这些特征也适用于具有支链结构的肽。制备了四支链上有精氨酸残基的肽（R_n）_4 [n（精氨酸残基数）= 0 ~ 6]。荧光显微镜观察发现（R_2）_4肽的易位效率最高。与线性肽一样，支链肽的易位效率和细胞定位也依赖于精氨酸残基的数量。我们已经证明，携带富含精氨酸片段的Rnase S的非共价蛋白组装成功地引入细胞，以显示抗hiv活性。制备了参与转录因子NF-κB活化的i -κB磷酸化和泛素化位点对应的多肽。将这些多肽与可穿透膜的精氨酸肽结合引入细胞，可抑制NF-κB的活化。然而，观察到抑制程度差异的显著性。我们还发现转录因子Sp1的转录被Sp1的DNA识别片段衍生的肽抑制。

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.