Efficient translocation of hybrid peptides through cell membrane for the control of transcription

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Several methods for the introduction of peptides and proteins into mammalian cells have been developed with the hopes of understanding intracellular signal transudation mechanisms and applying these techniques for therapeutic uses. Among them, methods using basic peptides as carriers seem promising. Two peptides derived from HIV-1 Tat protein (48-60) and Antennapedia homeodomain protein (43-58) have been reported as cell permeable peptides. We have compared the translocation activity of these peptides using mouse macrophage RAW 264.7 cells. Cellular uptake and intracellular distribution of the peptides labeled with maleimido fluorescein diacetate were observed by fluorescence microscopy, and the former peptide was judged to have a superior activity. Other related peptides, such as the D-substituted analog and the Arg-substituted analogs of the Tat peptide were also found to be cell-permeable, which implied the applicability of these peptides to the peptide carriers. As an application of Tat (48-60) to the intracellular delivery of synthetic peptides, a hybrid peptide of Tat (48-60) and IκBα(15-49) was synthesized. IκBα is an inhibitory protein of a transcription factor NF-κB. Phosphorylation in its N-terminal domain leads the activation of NF-κB. The hybrid peptide was efficiently delivered into the cells within 3 h, indicative of the usefulness of Tat (48-60) as a peptide carrier. Intracellular delivery mediated by other Tat-related peptides was also investigated.