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。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。这些复合物的作用位点位于病毒进入细胞和逆转录之间的阶段，这表明细胞中这些基本肽可能识别病毒RNA。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。对于传递分子与细胞蛋白之间形成交联，评估了使用光诱导交联剂和醛基团的可行性。然而，由于交联形成的效率不令人满意，我们现在正在寻求解决这一问题的替代制度。

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. 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. The site of action for these complexes resides in the stages between the viral entry into the cells and reverse transcription, suggesting the possibility of the recognition of viral RNA by these basic peptides in the cells. 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. As for cross-linking formation between delivered molecules and cellular proteins, the feasibility of employment of light-induced cross-linker and aldehyde moieties was assessed. However, since the efficiency of cross-linking formation was not satisfactory, we are now seeking alternative systems to resolve this problem.