Elsevier

Journal of Controlled Release

Volume 102, Issue 1, 20 January 2005, Pages 247-253
Journal of Controlled Release

Cationic TAT peptide transduction domain enters cells by macropinocytosis

https://doi.org/10.1016/j.jconrel.2004.10.018Get rights and content

Abstract

Naturally occurring and synthetic short arginine containing protein transduction domains (PTDs), including HIV1 TAT, poly-Arg and Antp, have been used to deliver a wide variety of macromolecular, biologically active therapeutic cargo into cells, including peptides, proteins, antisense oligonucleotides and liposomes, in vitro and to treat pre-clinical models of cancer and stroke. PTDs enter cells in a rapid, receptor-independent fashion. Recently, large TAT-fusion proteins (in excess of 30,000 Da) were shown to transduce into cells by fluid-phase macropinocytosis, a specialized form of endocytosis that is independent of caveloe, clathrin and dynamin. However, it remains controversial as to whether or not PTD peptides (1000–5000 Da) enter cells via macropinocytosis and/or through an unknown alternative mechanism. Due to strong ionic interactions with the cell surface, previous measurements of PTD peptide internalization were inaccurate. Cationic PTD peptides containing variable numbers of arginine residues and conditions entered cells exclusively through macropinocytosis. In addition, no PTD peptide was found to enter cells at 4 °C, a long held assumption of transduction. Taken together, these observations provide a solid scientific basis for the development of novel biologically active transducible anticancer PTD peptide therapeutics.

Introduction

In 1988, Green and Frankel [1], [2] independently observed the ability of the 86 amino acid human immunodeficiency virus type 1 (HIV-1) TAT protein to enter cells in culture and activate transcription. Subsequently, it was found that the basic region of the TAT protein, comprising residues 47–57, was responsible for the ability of TAT to enter cells [3], [4], [5]. Recent work by many groups in cell culture has shown the utility of the TAT protein transduction domain (PTD) as a delivery vehicle for a wide variety of macromolecular, biologically active anti-cancer therapeutic cargo, including synthetic molecules, peptides, proteins and antisense oligonucleotides [6], [7]. Moreover, several groups, including ours, have demonstrated the in vivo efficacy of treating mouse models of cancer by delivering anti-cancer peptides with PTDs [8], [9], [10], [11]. However, the mechanism that the TAT PTD peptide enters cells has remained elusive.

Due to strong cell surface binding by cationic poly-Arg containing PTDs, such as TAT, peptide redistribution occurred upon fixation and resulted in experimental artifacts that incorrectly concluded the transduction mechanism to be both temperature-independent and energy-independent. However, using a live cell genetic reporter assay, recent work determined that large TAT-fusion proteins (>30,000 Da) enter cells by lipid raft-dependent macropinocytosis [12], a specialized form of fluid phase endocytosis [13]. However, the mechanism that PTD peptides (1000–5000 Da) utilize to enter cells has remained controversial. To determine if TAT PTD peptides enter cells via macropinocytosis or through an alternative mechanism, an assay system was designed that through extensive washing removes all external cell surface bound PTD peptide in live cells and thereby allowed for an accurate measurement of the PTD peptide transduction mechanism.

Section snippets

Peptide synthesis

All peptides were synthesized with L-amino acids using solid-phase FMOC chemistry on an Applied Biosystems 433A synthesizer. Peptides were fluorescently labeled by coupling 5-carboxyfluorescein (Sigma) to the N-terminus followed by a GlyGlyGly linker. The resin was washed with 1 M K2CO3 prior to cleavage in order to remove any unbound fluorescein. Peptides were cleaved and deprotected in TFA/water/thioanisole/phenol/EDT (82.5:5:5:5:2.5%) for 5 h prior to ether precipitation. Peptides were then

TAT peptide enters cells by an energy-dependent mechanism

Early PTD mechanistic reports suggested that peptide transduction occurs by an energy independent mechanism [14] that could function at 4 °C. However, recent studies have questioned these early observations and demonstrated that PTD peptides bound to cell surface become redistributed during cell fixation resulting in inaccurate measurements [15]. To avoid these potential pitfalls, a stringent washing protocol was developed that removes external cell surface bound PTD peptides and thereby allows

Discussion

PTDs have now been used to deliver a variety of experimental anti-cancer cargo, including small molecules, peptides and proteins, into cells in culture and to treat pre-clinical tumor models in vivo [3], [5]. Although originally discovered in 1988, the mechanism that the TAT PTD enters cells has remained elusive, hindering the field from making logical improvements to the delivery efficacy. However, as demonstrated here and consistent with recent work on TAT-fusion proteins [12], TAT peptides

Conclusion

In summary, our observations are most consistent with a mechanism whereby both TAT-fusion proteins (>30,000 Da) and TAT PTD peptides (1000–5000 Da) enter cells by macropinocytosis. Although the mechanistic details have yet to be elucidated, cationic PTD peptides require a specific total charge to initiate macropinocytosis at the cell membrane. Further understanding the process of transduction and macropinocytic escape will greatly enhance the development of novel macromolecular transducible

Acknowledgements

This work was supported by the National Institutes of Health (CA96098). S.F.D. is an investigator of the Howard Hughes Medical Institute.

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