Abstract
During the past 15 years, a variety of peptides, known as protein transduction domains (PTDs), or cell-penetrating peptides (CPPs), have been characterized for their ability to translocate into live cells. There are now numerous examples of biologically active full-length proteins and peptides that have been successfully delivered to cells and tissues, both in vitro and in vivo. One of the principal mechanisms of protein transduction is via electrostatic interactions with the plasma membrane, subsequent penetration into the cells by macropinocytosis, and release into the cytoplasm and nuclei by retrograde transport. Recent reports have also now shown that some of the limitations of protein transduction technology have been overcome. In particular, the use of ubiquitination-resistant proteins has been demonstrated to be a more effective strategy for transduction because the half-life of these molecules is significantly increased. Moreover, the use of the NH2-terminal domain of the influenza virus hemagglutinin-2 subunit (HA2) or photosensitive PTDs has been shown to specifically enhance macropinosome escape. Hence, these and other recent advances in protein transduction technologies have created a number of possibilities for the development of new peptide-based drugs.
Keywords
Introduction
Proteins have been evolutionarily selected to perform specific functions and therefore any ability to deliver a wide variety of full-length, functional proteins to a specific cellular target is thus considered to have a tremendous potential as a biological tool for studying the cellular processes, as well as for developing novel and potentially very specific therapeutic agents. However, the hydrophobic nature of lipids prevents the vast majority of proteins/peptides from crossing the membrane barrier. A significant development in overcoming this barrier has recently been described however, and is referred to as protein transduction technology. This technology enables proteins and peptides to be directly internalized in cells by covalent linkage to protein transduction domains (PTDs), which are also known as cell-penetrating/permeable peptides (CPPs). PTDs facilitate the transduction of cargo across the membrane, and thus allow the proteins to accumulate within the cell. The most commonly studied PTDs are homeodomain transcription factors such as Antennapedia (Antp) (19, 24, 31, 34, 36, 44), the human immunodeficiency virus (HIV-1) transactivator TAT protein (1, 8, 20, 49), and the herpes simplex virus (HSV) type 1 protein VP22 (7). Recently, poly-arginine (polyR) and poly-lysine have been shown to exhibit even a greater efficiency in terms of the delivery of several peptides and proteins (11, 25, 33, 35, 37, 39). To date, a growing number of transducible proteins, covering a wide range of sizes and functional classes, have been successfully used to study intracellular mechanisms. PTDs have been shown to deliver proteins in excess of 100 kDa into cultured cells and most cell types in mammalian model systems (49). These PTD fusion proteins can be found in both the cytoplasm and the nucleus. Subsequently, several reports have described the delivery of peptides and proteins in vivo (20, 37). Moreover, several studies have also now demonstrated these protein transduction systems to have opened up a number of possibilities for the development of new peptide/protein drugs (20, 30–33, 35, 37–41, 53).
The most impressive aspect of PTD-mediated delivery and its therapeutic potential is its size independence. The ability of PTDs to deliver cargo into cells is not limited to proteins or peptides as they have also been shown to mediate the efficient intracellular accumulation of various biologically active substances such as antisense oligonucleotides (29), peptide nucleic acids (PNA) (42, 43), short interfering RNAs (siRNAs) (50), iron nanoparticles (22), liposomes (52), and plasmids (12, 47). These data may have significant implications for the future delivery of other large molecules in vitro and in vivo. This report reviews some of the most recent advances in this rapidly expanding area of research.
Mechanisms of Protein Transduction
Several studies have shown the internalization mechanisms of protein transduction. It is likely that TAT, polyR, Antp-PTD, transportan, etc., are not all internalized by a single mechanism. Moreover, some peptides are internalized by more than one mechanism working in parallel and the presence of a tag or cargo could favor one mechanism among several. One of the principal mechanisms of protein transduction is via electrostatic interactions with the plasma membrane, subsequent penetration into the cells by macropinocytosis, and release into the cytoplasm and nuclei by retrograde transport. Previous studies have described protein transduction in real time, while also showing that TAT-PTD, 11R-PTD, and the BETA2/NeuroD transcription factor containing an arginine- and lysine-rich PTD, penetrate cells by macropinocytosis, a type of endocytosis, and are homogeneously released from the endosome into the cytoplasm and nuclei by retrograde transport (30, 36, 41). Antp-PTD and PDX-1 protein, which have Antp-like PTD, are also internalized in cells by this mechanism (34, 36, 53). Recently, the role of endosomal acidification and retrograde transport for the uptake of Antp-PTD, TAT-PTD, and 9R-PTD has also been reported (9). A number of well-characterized toxins reach the cytosol of eukaryotic cells after binding to the cell surface, and then undergoing endocytosis and retrograde transport to the Golgi apparatus and endoplasmic reticulum (23, 32, 48). The arginine-rich motif of 8–10 amino acids in the A subunits of these toxins, reported to be transported by means of retrograde transport, is similar to those of TAT-PTD, Antp-PTD (9), and PDX-1-PTD.
Protein transduction technologies have had some important limitations in the past. To overcome these problems, a series of recent advances in this technology have now been developed.
Ubiquitination-Resistant Protein Transduction
One of the previous limitations of protein transduction technology was that the transduced proteins only have a limited active half-life, so that their effects were only transient. However, ubiquitination-resistant proteins, p53 with polyR, are delivered into cells and have a long intracellular half-life (27). Previous studies have also shown that the COOH-terminal region of p53 modulates the susceptibility of the protein to Mdm2-mediated degradation (46). A mutant p53 protein, in which multiple lysine residues in the COOH-terminal are replaced by arginine, is resistant to ubiquitin-proteosome-mediated degradation and its transcriptional activity is higher than that of the wild-type p53 (46). Therefore, a mutant PTD-p53 protein was generated by substituting multiple lysine residues with arginine. The resulting mutant proteins were effectively delivered into glioma cells and were resistant to Mdm2-mediated ubiquitination. Moreover, these mutant p53 proteins displayed higher transcriptional regulatory activity than their wild-type counterpart and induced a potent inhibition of glioma cell proliferation (27). These findings suggest that ubiquitination-resistant proteins may provide a more effective strategy for protein transduction technology in the future (Fig. 1).
Recent advances in protein transduction technology. One of the principal mechanisms of protein transduction involves an electrostatic interaction with the plasma membrane, penetration into cells by macropinocytosis, and release to the cytoplasm and nuclei by retrograde transport. (A) Ubiquitination-resistant protein transduction. The activity of mutant proteins that are resistant to ubiquitin-proteosome-mediated degradation is higher than that of their wild-type counterparts. (B) Photoacceleration of protein release from endosomes. Exposure to light at 480 nm stimulates endosomal release of transduced FITC-PTDs or FITC-PTD-proteins. (C) The acceleration of protein release from endosomes by HA2. The pH-sensitive HA2 peptide markedly and specifically enhances macropinosome escape of its fused proteins/peptide molecules. (D) Tissue-specific transduction. Tissue-specific peptides can facilitate the internalization of large marker protein complexes into specific cells in culture and in vivo in a cell type-specific manner. (E) Organelle-specific delivery. The incorporation of a nuclear localization signal or a mitochondrial signal sequence into the PTD allows processing and localization of exogenous proteins/peptides in the nucleus or mitochondria, respectively. (F) Transvascular delivery to brain. RVG-9R can cross the BBB and enables the transvascular delivery of siRNA to the brain.
When using chemically synthesized peptides, some studies have suggested that the use of D-amino acid transduction domains is preferable to the corresponding L-isomers, as the D-peptides are not naturally occurring and display a higher level of stability in serum medium (55). However, a decreased degradation does not account for all of the increase in the cellular accumulation of the D-enantiomers (13). In contrast, some studies have suggested that there is a decreased stability of D-peptides in serum medium (5) and less impact of the corresponding cargo in comparison to the L-enantiomers (1).
Acceleration of Protein Release From Endosomes
The principal mechanism of protein transduction involves penetration into cells by macropinocytosis, and release to the cytoplasm and nuclei by retrograde transport. Although PTDs or PTD-fusion proteins can be transduced into cells by macropinocytosis, such proteins are often subsequently entrapped inside the macropinosomes and prevented from release. Therefore, high-dose concentrations of PTDs or PTD-fused proteins are often needed for this delivery technology to function effectively. To overcome this problem, methods for enhancing endosome escape using a photosensitive PTD were developed (24). Exposure to light at 480 nm stimulates the endosomal release of transduced FITC-polyR-PTD, TAT-PTD, Antp-PTD, and polyR-p53 protein (Fig. 1B). Moreover, a nuclear localization signal fused to FITC-conjugated polyR was found to localize to nuclei after exposure to light, suggesting that transduced peptides released from the endosome are not degraded and remain functional (24).
Several viruses have evolved an endosomal escape mechanism that takes advantage of the drop in pH that occurs in mature endosomes (51). The NH2-terminal domain of influenza virus hemagglutinin-2 subunit (HA2) is a pH-dependent fusogenic peptide that induces the lysis of membranes at low pH levels (51). Groups have fused proteins and peptides to HA2 (28, 54) and shown the pH-sensitive HA2 peptide to markedly and specifically enhance the macropinosome escape (Fig. 1C) (54). In addition, HA2- and polyarginine-fused p53 induces p21(WAF1) transcriptional activity and inhibits the growth of cancer cells more effectively than a polyarginine-fused p53 protein (28). Therefore, these techniques have the capability of enhancing the efficiency of protein transduction systems.
Tissue-Specific Transduction
Cationic PTDs, such as TAT, Antp, and polyR, transduce a wide variety of cells and mediate highly efficient transduction both in vitro and in vivo. However, the lack of tissue specificity of the cationic PTDs limits their in vivo utility, especially for the delivery of apoptotic agents. A study by Mi et al. screened an M13 peptide phage display library for synovial-specific transduction peptides (26). Using this approach, the synovial cell type-specific peptides, HAP-1 and HAP-2, were identified (Fig. 1D). These peptides were demonstrated to facilitate the internalization of large marker protein complexes in synovial cells in culture and in vivo in a synovial cell type-specific manner (26). The results further demonstrate the feasibility of identifying tissue-specific transduction peptides capable of mediating efficient transduction in vivo.
Organelle-Specific Delivery
Another strategy for increasing the efficacy of protein delivery is to cleave the transduction domain by including an organelle-specific cleavage recognition site. Therefore, the PTD-linked cargo would be expected to accumulate in the respective compartment. The SV40 nuclear localization signal (NLS) was the first such site to be used to restrict the delivered peptides to the nucleus. PolyR-NLS and polyR-NLS-PKI (PKA inhibitory peptide) molecules were synthesized and these peptides were localized specifically in the nucleus, whereas polyR or polyR-PKI without an NLS exhibited a diffuse fluorescent signal throughout the cytoplasm, nucleus and cellular processes (24, 25) (Fig. 1E).
In addition, the incorporation of a mitochondrial signal sequence into a PTD facilitates the processing and localization of exogenous proteins/peptides in mitochondria (3). Accordingly, a TAT-mitochondrial malate dehydrogenase signal sequence (mMDH)-enhanced green fluorescent protein (EGFP) fusion protein was found to be localized in mitochondria of multiple cell types in vitro and in vivo.
Transvascular Delivery to Brain
A major impediment in the treatment of neurological diseases is the presence of the blood–brain barrier (BBB), which precludes the entry of therapeutic molecules from the blood to the brain. Kumar et al. showed that a short peptide derived from rabies virus glycoprotein (RVG)-9R enables the transvascular delivery of siRNA to the brain (21). Because neurotropic viruses, such as rabies virus, do cross the BBB to infect brain cells, they chose the 29-amino acid peptide of RVG, which specifically binds to the acetylcholine receptor expressed by neuronal cells. Because short, positive charged PTDs bind negative charged nucleic acids by charge interaction (4, 6, 15), they also used 9R peptide to enable siRNA binding. The intravenous treatment with RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice without the induction of inflammatory cytokines or antipeptide antibodies. Therefore, RVG-9R could provide a safe and noninvasive approach for the delivery of siRNA and potentially other therapeutic molecules across the BBB (Fig. 1F).
Conclusions
The initial discovery of PTDs originated from the unexpected observation that certain full-length proteins or protein domains have the ability to translocate across the plasma membrane. This was first shown for the HIV TAT transactivator (10, 14) and for the homeodomain of the Drosophila melanogaster transcription factor Antennapedia (18), but it has since been shown to include “nonnatural” peptides that share this property. In the case of homeodomain- and TAT-derived peptides, and in contrast with many other PTDs, the uptake seems to reflect the associated in vivo biological process. In support of this, the translocating activity of these peptides correlates with that of their parental full-length proteins (2). This strongly suggests that, in some circumstances, these proteins may have paracrine activities as part of a physiological process, in that they are released by one kind of cell and then are internalized by other cells. Accordingly, the full-length proteins are also secreted, probably by unconventional secretion pathways that do not involve a secretion signal sequence (16, 17). Although the exact function of this intercellular transfer is not yet clear, recent data on transcription factors, and homeoproteins in particular, suggest that after transfer, the transcription factor can regulate the transcription and translation in the recipient cell, thus acting as a “messenger protein” (44, 45). As the homeoprotein expression is regionally specified, it has therefore been speculated that this corresponds to the passage of positional information between cells (44, 45).
The present protein transduction systems have a low toxicity and a high yield of delivery. The use of this technology thus opens up interesting therapeutic perspectives. Moreover, the current interest in protein translocation across plasma membranes is not limited to PTDs and their use as a tool with which to investigate cell biology, or conversely, to their biotechnological applications.
Footnotes
Acknowledgments
This work was supported in part by the Juvenile Diabetes Research Foundation International (JDRFI); the Ministry of Education, Science and Culture, the Ministry of Health, Labour and Welfare; and All Saints Health Foundation.