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Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis

Nature Medicine volume 10, pages 310–315 (2004)Cite this article

Abstract

The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT–Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft–dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.

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Figure 1: Expression of EGFP requires transduction of TAT-Cre.
Figure 2: TAT-mediated transduction is lipid raft–dependent.
Figure 3: TAT-mediated transduction does not occur by caveolar- or clathrin-mediated endocytosis.
Figure 4: Inhibition of macropinocytosis prevents TAT-Cre–mediated recombination.
Figure 5: Enhancement of transduction by endosomal disruption.

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Acknowledgements

We thank E. Ruley and M. Bessler for providing cell lines; B. Meade, I. Kaplan and L. Gross for technical assistance; and M. Becker-Hapak for critical input. This work was supported by the Howard Hughes Medical Institute (to S.F.D.) and the National Institutes of Health (CA96098 to S.F.D. and HL065418 to R.V.S.).

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Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California San Diego School of Medicine, La Jolla, 92093-0686, California, USA

    Jehangir S Wadia & Steven F Dowdy

  2. Department of Cellular and Molecular Medicine, University of California San Diego School of Medicine, La Jolla, 92093-0686, California, USA

    Jehangir S Wadia, Radu V Stan & Steven F Dowdy

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  1. Jehangir S Wadia
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Correspondence to Steven F Dowdy.

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Wadia, J., Stan, R. & Dowdy, S. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10, 310–315 (2004). https://doi.org/10.1038/nm996

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