Review
Peptide-mediated cell delivery: application in protein target validation

https://doi.org/10.1016/S1471-4892(02)00199-6Get rights and content

Abstract

Recent reports have suggested that conjugation of peptides, proteins and antisense to short highly basic peptides, such as TAT, antennapedia and transportan, results in their rapid translocation into cells. Importantly, these conjugates have been shown to exert actions in a number of animal models suggesting their general utility for the determination of protein function in vitro and in vivo.

Introduction

The complexity of biological interactions makes it increasingly difficult to predict gene and protein function as you proceed from the immediate metabolic pathway to the cellular and animal level. This phenomenon is partially responsible for the high attrition rate observed during drug development and necessitates early validation of protein function in cellular and/or animal models that are predicative of the disease. Classically, this is undertaken either through acute modulation using protein knockdown and/or inhibition, or using chronic models, such as transgenic and knockout mice.

In cellular systems, a variety of tools are employed to determine protein function, including antisense, peptide modulators and the overexpression of wild-type or dominant-negative protein. However, these studies are often limited by the inability to effectively deliver these validation tools. Typically, delivery is via lipids, electroporation or through viral vectors, but these have several severe limitations, including the inability to deliver to primary, non-dividing cells, the requirement for optimisation with each cell type, low transfection levels and cellular toxicity. Interestingly, recent studies have identified several short peptide sequences named protein transduction domains (PTDs) or cell penetrating peptides (CPPs), which appear to rapidly translocate into all cells both in vitro and in vivo. Importantly, conjugation of proteins, peptides and antisense to these PTDs has been shown to deliver these cargos effectively, allowing observation of biological action in several cell and animal models 1., 2.. In this review, we examine the use of PTDs as a novel and potentially universal delivery system for delineation of protein function and target validation.

Section snippets

Peptide transduction domains

PTDs were first identified while investigating the spontaneous cell entry of HIV TAT, which is subsequently localised to the nucleus and transactivates the viral long-terminal repeat promotor encoded within the human immunodefficiency virus [3]. Studies of the minimum translocation region identified a positively charged section between amino acids 47 and 57, which was previously associated with DNA binding [4]. Similar studies of antennapedia, a Drosophila homeodomain transcription factor,

Mechanism of cell entry

To date, studies of the mechanism of cell entry have largely examined the movement of labelled antennapedia and TAT across artificial membrane systems or into cells. Although this pathway is probably analogous to the situation during PTD-mediated delivery of small peptide cargos, it is likely that the mechanism will be different with larger macromolecules such as antisense and proteins. With this caveat in mind, the studies have identified several important characteristics, outlined below.

Peptide delivery

Both antennapedia and TAT have been used in peptide delivery and have been effectively employed to attenuate protein–protein and enzyme–substrate interactions involved in growth factor, cytokine and integrin signalling, apoptosis, and cell division (Table 1; [17••]). Examination of the literature shows that, compared with PTD–protein conjugates, substantially greater concentrations of PTD–peptide conjugates (>10 μM) were required for biological activity. The proteins are likely to have a

Protein delivery

Although antennapedia-mediated [20] and transportan-mediated [21] protein delivery has been reported, Steven Dowdy has pioneered this area using TAT-conjugated proteins [22•]. As with the peptides, this has been successfully employed to deliver several targets, including dominant-negative GTP-binding proteins (Rho, Rac, Cdc42), modulators of cyclin-dependent kinase (cdk) activation (p16INK4A and p27KIP1) and inhibitor of NFκB (IκB)-α (Table 2). Furthermore, as discussed earlier, TAT proteins

Antisense delivery

Although a limited number of studies have employed TAT, transportan and antennapedia for delivery of antisense, their general utility has been limited by the difficulty in chemically conjugating the PTD peptide and antisense oligonucleotide backbones (Table 3). However, this problem can be resolved through the use of peptide nucleic acid (PNA) antisense, which has been shown to attenuate protein expression and biological activity of a small number of targets, including protein tyrosine

In vivo delivery

To date, only a small number of studies have investigated the in vivo utility of PTD-mediated protein delivery. Considerable interest was first generated by a report demonstrating the presence of active TAT–β-galactosidase in all mice tissues (including the brain) at 4–8 h following intraperitoneal injection [30]. Since then, a study by Jo et al. [31••] has demonstrated Cre delivery to both mammalian cells and mouse tissue, using a PTD derived from Kaposi fibroblast growth factor (FGF)-4 [31••]

Conclusions

The recent completion of the first draft of the human genome has provided the scientific community with the majority of the basic building blocks responsible for human biology. The next phase, involving the identification of gene function, would be greatly facilitated by the ability to deliver tools that modulate protein function in vitro and in vivo. To this end, initial studies have suggested that PTDs may provide a valuable, possibly universal delivery tool for the acute in vitro and in vivo

Update

Continuing studies on the mechanism of PTD-mediated uptake have confirmed the importance of positively charged amino acids and demonstrated highly efficient delivery of proteins >500 kDa following conjugation to polylysine sequences [72]. Interestingly, a recent study using confocal microscopy identified the presence of labelled PTD–PNA conjugates in cytosolic vesicular compartments and suggested that delivery is mediated via a receptor-dependent endocytotic pathway [73••]. In both

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

References (73)

  • H. Hall et al.

    Inhibition of FGF-stimulated phosphatidylinositol hydrolysis and neurite outgrowth by a cell-membrane permeable phosphopeptide

    Curr Biol

    (1996)
  • D. Kato et al.

    Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts

    FEBS Lett

    (1998)
  • J.S. Wadia et al.

    Protein transduction technology

    Curr Opin Biotechnol

    (2002)
  • H.Y. Kwon et al.

    Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells

    FEBS Lett

    (2000)
  • L.H. Jin et al.

    Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells

    Free Radic Biol Med

    (2001)
  • M. Tyagi et al.

    Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans

    J Biol Chem

    (2001)
  • C.G. Ostenson et al.

    Overexpression of protein-tyrosine phosphatase PTPσ is linked to impaired glucose-induced insulin secretion in hereditary diabetic Goto-Kakizaki rats

    Biochem Biophys Res Commun

    (2002)
  • W.J. Bruyninckx et al.

    Phosphoinositide 3-kinase modulation of β3-integrin represents an endogenous ‘braking’ mechanism during neutrophil transmatrix migration

    Blood

    (2001)
  • K. Fujimoto et al.

    Inhibition of pRb phosphorylation and cell cycle progression by an antennapedia-p16(INK4A) fusion peptide in pancreatic cancer cells

    Cancer Lett

    (2000)
  • G.S. Liu et al.

    Protein kinase C-ε is responsible for the protection of preconditioning in rabbit cardiomyocytes

    J Mol Cell Cardiol

    (1999)
  • E.P. Holinger et al.

    Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases

    J Biol Chem

    (1999)
  • E.J. Williams et al.

    Evidence for and against a pivotal role of PI 3-kinase in a neuronal cell survival pathway

    Mol Cell Neurosci

    (1999)
  • D. Derossi et al.

    Stimulation of mitogenesis by a cell-permeable PI 3-kinase binding peptide

    Biochem Biophys Res Commun

    (1998)
  • E.J. Williams et al.

    Selective inhibition of growth factor-stimulated mitogenesis by a cell-permeable Grb2-binding peptide

    J Biol Chem

    (1997)
  • M. Rojas et al.

    Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor

    J Biol Chem

    (1996)
  • D.J. Hall et al.

    Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability

    Blood

    (2001)
  • G. Hjalm et al.

    Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase

    J Biol Chem

    (2001)
  • T. Fujiwara et al.

    Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein

    Biochim Biophys Acta

    (2001)
  • P.A. Klekotka et al.

    Mammary epithelial cell-cycle progression via the α2β1 integrin: unique and synergistic roles of the α2 cytoplasmic domain

    Am J Pathol

    (2001)
  • N. Soga et al.

    Rho family GTPases regulate VEGF-stimulated endothelial cell motility

    Exp Cell Res

    (2001)
  • M.A. Chellaiah et al.

    Rho-A is critical for osteoclast podosome organization, motility, and bone resorption

    J Biol Chem

    (2000)
  • A. Astriab-Fisher et al.

    Antisense inhibition of P-glycoprotein expression using peptide-oligonucleotide conjugates

    Biochem Pharmacol

    (2000)
  • J.C. Mai et al.

    Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate

    J Biol Chem

    (2002)
  • M. Lindgren et al.

    Translocation properties of novel cell penetrating transportan and penetratin analogues

    Bioconjug Chem

    (2000)
  • A. Joliot et al.

    Antennapedia homeobox peptide regulates neural morphogenesis

    Proc Natl Acad Sci USA

    (1991)
  • M. Pooga et al.

    Cell penetration by transportan

    FASEB J

    (1998)
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