Proteins are primary targets in drug discovery. However, with a few rare exceptions, they are unable to cross cell membranes, a limitation that prevents the full exploitation of their therapeutic potential. Major advances have been recently made through a novel approach of protein and peptide delivery into cells known as protein transduction or protein therapy. Proteins and peptides can be directly transferred to cells when covalently linked to protein transduction domains (PTD), small peptides that can freely cross cell membranes with low lytic activity (1–3). The mechanism of cellular translocation of PTD are currently poorly understood. Most of the PTD described in the literature have a high content of basic residues. It is believed that the interaction with the negative cell membrane environment has an important role in the translocation process, and the mechanism of cell internalization may differ for each of the PTD. Several PTD have been identified in naturally occurring proteins. The most commonly studied are homeodomain transcription factors such as antennapedia (4), the herpes simplex virus type 1 protein VP22 (5), and the human immunodeficiency virus (HIV) transactivator TAT protein (6–7). In addition, a new gamut of peptides with PTD capabilities have been recently identified. Some of these new peptides are derived from natural proteins, whereas others are synthetic peptides. The PTD included in these groups are described below, with emphasis on the TAT-PTD and its potential application in organ and cell transplantation. HOMEODOMAIN TRANSCRIPTION FACTORS Homeodomain proteins have a critical role in tissue architecture and morphogenesis. They exert their regulatory functions by binding to DNA through their homeodomain, a 60-amino acid domain composed of three α-helices separated by two β-turn motifs. Several PTD that form part of homeodomain proteins have been reported. The most widely studied PTD motif of this kind is known as pANTP or penetratin. It corresponds to 16-amino acid (RQIKIWFQNRRMKWKK) residues of the third helix of the antennapedia homeotic transcription factor (ANTP) from Drosophila (4). It was postulated that the positively charged pANTP associates with the charged phospholipids in the outer side of the cellular membrane. This is followed by destabilization of the membrane and formation of an inverted micelle that somehow penetrates the cytoplasmic compartment (8). However, recent studies performed on cells, rather than artificial phospholipid bilayers, indicate that adsorptive-mediated endocytosis has a role in translocation mechanism of pANTP into cells (9). pANTP has been mostly restricted to the delivery of small molecules such as peptides (10) and peptide nucleic acids (11–13). pANTP-mediated transduction of peptides has been successfully used to study the mechanism of RNA transport (14). In vivo applications of pANTP-fused molecules include the topical administration of the NH2-terminal peptide of α-smooth muscle actin. As this inhibits the contraction of rat wound granulation tissue, this approach could help develop new therapeutic strategies for fibrocontractive pathological situations (15). Moreover, the administration of pANTP fused to a 20-amino acid peptide (amino acids 84–103) from the p16 tumor suppressor protein suppressed pancreatic cancer growth and extended survival in mice (16). Inhibitors of the protein kinase C-ε fused to the antennapedia cell penetrating peptide were used to confirm the crucial role of this isozyme in the signaling pathway associated with protective heart ischemia preconditioning (17). Two other Drosophila homeodomain proteins, Fushitarazu and Engrailed, have similar transduction properties (18). Furthermore, a new PTD, pIsl1, with translocation ability similar to that of pANTP, was recently discovered (19). PIsl1 originates from rat protein homeodomain of islet-1, an insulin gene enhancer. It encompasses amino acid residues 45 to 60 (RVIVWFQNKKRCKDKK) from the third helix motif. Similarly, it was recently reported that pancreatic and duodenal homeobox-1, a key transcription factor for pancreatic development and insulin transcription, contains an antennapedia-like PTD (RHIKIWFQNRRMKWKK) in the third α-helix of its homeodomain. Pancreatic and duodenal homeobox-1 is capable of in vitro transduction of pancreatic ducts and islets (20). HERPES SIMPLEX VIRUS TYPE-1 VP22 PROTEIN The entire VP22 viral protein constitutes a PTD. In contrast to other proteins containing PTD, the short domain capable of cellular transduction when fused to a tandem protein has not yet been identified. However, VP22 has an additional feature: it participates in intercellular trafficking. Once expressed in the cell, this protein has the ability to spread to other nontransfected cells. The majority of published studies about transduction with the VP22 PTD have been essentially performed intercellularly. The VP22-fusion protein was generated from a gene transfected into cells and diffused to neighboring untransfected cells (Fig. 1). Conflicting reports have been published on the intercellular transduction of VP22-GFP fusion protein (21, 22). To overcome the difficulties associated with the detection of GFP and potential cell fixation artifacts (23), Benett et al. (24) demonstrated intercellular transport of VP22 by means of recombinase Flip fused to VP22. COS cells were transfected with a LacZ gene that was rendered nonfunctional with a transcription terminator sequence located between the CMV (cytomegalovirus) promoter and the LacZ gene. The transcription terminator sequence was flanked by Flip recombinase sites (frt sites). LacZ-COS transfected cells expressed β-galactosidase only if they were cultured together with cells expressing a VP22-Flip recombinase fusion protein, indicating that VP22-Flip translocated from one cell to another. Other studies showed biologic activity of several proteins fused to VP22 PTD on intercellular transduction, such as active thymidine kinase (25), the tumor suppressor factor p53 (26), and the Simian virus 40 large T antigen (27). Furthermore, the bystander effect achieved by the intercellular trafficking capability of VP22 has been exploited in a number of in vivo models (28–30).Figure 1: (green cargoes) Proteins and peptides. (orange dots) Generic PTD. (yellow, purple, and pink dots) TAT/PTD, VP22, and pANTP, respectively. Most of the proteins and peptides are unable to enter into cells unless fused to a PTD. The mechanism of PTD entrance remains for the most part unknown. For basic PTD, an electrostatic interaction between positively charged PTD residues and a negative cell membrane environment is apparently necessary as a first step (A). Endocytosis (B1) and subsequent escape from the endocytosis vesicle (B2) has been recently postulated for TAT/PTD and pANTP. VP22-fused cargos are generated from a gene (C1) and have intercellular trafficking properties (C2). All of the PTD have the potential to localize in the nuclei. TAT/PTD fusion proteins containing a mitochondrial localization signal enter into mitochondria, where the mitochondrial signal sequence is recognized and proteolyzed, resulting in the retention of the fused protein cargo in the mitochondria (D).TAT/HIV-PTD Green and Loewenstein (6) and Frankel and Pabo (7) first reported that HIV transactivator TAT protein (TAT/HIV) is capable of crossing cell membranes. Fawel et al. (31), later showed TAT-mediated delivery of chemically coupled heterologous proteins into cells. The TAT/PTD consists of 11 amino acids, YGRKKRRQRRR, from the basic domain of the TAT/HIV protein. The basic residues, K and R, have a critical role in transduction properties of TAT/PTD through an interaction with the heparan sulfate chains of cell membrane heparan sulfate proteoglycans (32). Although the mechanism of internalization is still poorly understood, there is evidence that endocytosis followed by endosome escape is partially involved (Fig. 1) (31, 33, 34). Substitution of any of these basic residues with alanine negatively affected internalization into cells (35). Furthermore, a 9-mer of arginine was reported to have a significantly greater transduction efficiency than that of the TAT/PTD (35). Schwarze et al. described a system to produce and purify recombinant TAT/ PTD-fusion proteins that have cell-transducing capabilities in vitro and in vivo and can even cross the hematoencephalic barrier (36). Although most of the cells are transduced by TAT/PTD, there are cases in which cell permeability is restricted. For example, extracellular matrix components can interfere with the TAT/PTD transduction to myofibers (37). Likewise, TAT/PTD was incapable of transducing in vivo bladder epithelium, as assessed by the lack of transepithelial permeation into other tissues after infusion of TAT/PTD conjugated to technetium-99 in the urinary bladder of living rats (38). It has been disputed that transduction and nuclear localization of TAT/PTD fusion proteins was the consequence of a fixative-induced artifact (22, 39). However, analysis by confocal microscopy of live, nonfixed cells showed efficient internalization of TAT/PTD derivatives (33, 40). Furthermore, the best possible indication of internalization is the biologic function of the full-length proteins and peptides delivered to many different target cells by means of TAT/PTD. They are included to date in more than 70 references in the biomedical database PubMed. A wide variety of full-length proteins fused to TAT/PTD were biologically active on delivery into cells (1). Some of the fusion proteins were biologically active in the nuclei such as the cell cycle regulator p21 (41) or in the cytoplasmic compartment such as the IκB, the nuclear factor (NF)-κB inhibitory protein. In addition, a significant number of TAT/PTD derivatives were biologically active when administered in vivo. Listed in Table 1 are proteins and peptides that have been delivered in vivo to different animal models by means of TAT/PTD protein transduction (42–51).Table 1: Proteins and peptides delivered in vivo by means of TAT/PTD protein transductionIn another interesting application of this technology, a mitochondrial signal sequence was included in the TAT/PTD fusion protein (52). On cell transduction and entrance into mitochondria, the mitochondrial signal is recognized and cleaved, releasing the cargo protein, which can be retained in the mitochondria for several days. Furthermore, when administered into pregnant mice, the fusion protein was capable of crossing the placenta and localizing in both the fetus and the newborn pups (52). Interestingly, it has been reported that in addition to transporting proteins through cell membranes, TAT/PTD is capable of transducing nanoparticles into lymphocytes, allowing for the visualization of lymphocytic infiltration in a diabetic mouse pancreas (53). If developed for humans, it has the potential for monitoring lymphocyte infiltration caused by autoimmune recurrence in pancreas and islets transplantation. OTHER PTD The broad interest in the fields of genomics and proteomics has led to new efforts and strategies to identify and design novel PTD. Examples of novel natural and synthetic PTD are as follows: MTS, a 12-residue hydrophobic peptide derived from hydrophobic core region of Kaposi fibroblast growth factor (54). HIV1 vpr, a 14-kDa protein from the HIV with protein-transducing properties (55). Syn B peptides, peptide vectors derived from natural peptides known as protegrins. Syn B peptides have an amphipathic structure with hydrophobic and positively charged residues in opposite sides of the structure, and are capable of mediating delivery of the antineoplastic drug doxorubicin to the brain in a mouse model (56). On the basis of the observation that arginine residues are crucial to the protein transduction capability of TAT/ PTD by means of their side chain guanidine groups, Rothbard et al. designed efficient protease-resistant molecular transporters consisting of polyguanidine peptoid derivatives (35). PTD-4, a synthetic peptide designed to have an optimized Arg alignment on one face of the helix and a strengthened α-helicity (57). PTD-5, a protein transduction domain selected from the M13 phage peptide display library. PTD-5 was reported to deliver a peptide inhibiting activation of NF-κB in pancreatic islets in vivo (58). Pep-1, a short amphipathic peptide consisting of three domains: a hydrophobic tryptophan-rich domain required for efficient transduction and interaction with protein hydrophobic pockets, a separator domain, and a nuclear localization signal domain from SV-40 T-large antigen. An advantage of Pep-1 is its capability to deliver proteins that are not bound to it covalently (59). Pep-1 was used to deliver active caspase-3 in vivo to simulate a mouse emphysema model (60). POTENTIAL APPLICATION TO CELL AND ORGAN TRANSPLANTATION PTD technology allows delivering proteins to different cell types and tissues with limited restrictions. These properties make protein transduction a potentially useful application for organ and cell transplantation. In particular, the ability to diffuse across the cellular membrane of most cells independent of specific membrane receptors may be greatly advantageous for ex vivo delivery of proteins to tissues, organs or cells. Another important and distinctive attribute of the PTD protein-peptide delivery system is the transient effect of the transduced protein within the cells. The duration of the expression of the fused PTD protein peptide is limited by its specific protein turnover. This is particularly important in the instance of antiapoptotic proteins, which might be protective and necessary at a particular stage of the transplantable organs or cells, but could be deleterious when expressed for a long period of time in unregulated fashion. Application of protein transduction in transplantation is most suitable for cells and organ preservation. The deleterious effects of prolonged ischemia after organ harvesting have been closely linked to apoptotic and necrotic events induced by oxidative stress and production of reactive oxygen species. Therefore, preservation of organs could be improved by delivery of cytoprotective, antiapoptotic, or antioxidative proteins or peptides by means of protein transduction at the time of harvesting or immediately after. Encouraging examples have been reported including studies describing reduction of ischemia-reperfusion injury in perfused heart by delivery of the caspase recruitment domain and BH4, the antiapoptotic Bcl-XL domain (61, 62). Delivery of cytoprotective proteins or peptides by means of PTD could be used to enhance viability of primary transplantable cells that are susceptible to anoikis, a programmed cell death induced by their detachment from the extracellular matrix, such as pancreatic islets and hepatocytes (63, 64). For instance, protein transduction technology has been successfully applied to improve the viability of isolated pancreatic islets (58). An inhibitor of NF-κB fused to the novel protein transduction domain PTD-5 was infused into pancreas, by means of the pancreatic duct, before islet isolation. Pancreatic islets are also susceptible to culture-induced damage, decreasing in number and losing function over time. This in part is caused by cell death induced by apoptosis or oxidative stress during isolation and in vitro culture. TAT/ PTD fusion proteins efficiently transduce pancreatic islets (65). Inclusion of TAT/PTD derivatives of antiapoptotic-anti-oxidative proteins, such as heme oxygenase-1, Bcl-XL, and BH4 in the culture media can improve islet viability (40, 66). Enhancing or preserving viability of islets in culture before transplantation could allow for the immunosuppressive preconditioning of recipients. Watanabe et al. (67) recently described an interesting application of protein transduction technology for the development of bioartificial organs. In particular, these investigators applied protein transduction to transiently immortalize human liver stellate cells (HSC), an important part of the hepatic sinusoid responsible for microcirculation and scavenger functions. HSC are capable of preserving hepatocyte liver-specific functions in co-culture. However, HSC have a short lifespan, thus limiting the applicability of this approach. To enhance the limited lifespan of HSC, the cells were transduced with a recombinant virus expressing human telomerase reverse transcriptase (hTERT). The hTERT gene was flanked by loxP sites, a sequence that is recognized and cleaved by the bacteriophage P1 Cre recombinase. Therefore, control of immortalization was carried out by expression of Cre recombinase. Efficient excision of the retrovirally transferred hTERT was achieved by TAT/PTD-mediated expression of the Cre recombinase, thus allowing reversible immortalization, a valuable safety feature for clinical application of genetically modified cells. CONCLUSION Currently in its infancy, protein transduction is an area of active research because of its potential to deliver proteins and peptides directly to cells and tissues. Numerous examples of biologically active full-length proteins and peptides have been delivered to cells and tissues, both ex vivo and in vivo. Delivery of cytoprotective proteins or peptides by means of PTD could protect cells or organs during a critical period of time before transplantation, without the requirement for genetic modifications. Growing numbers and a growing variety of PTD offer a great opportunity to delineate the real applicability of this technology in the field of organ and cellular transplantation. Acknowledgments. The authors thank Norman Klein for the preparation of the illustration used in Figure 1 and Antonello Pileggi and Alberto Pugliese for critical reading of the manuscript.
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