ReviewPeptide-mediated cell delivery: application in protein target validation
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
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