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Specific protein domains known as protein transduction domains (PTDs) can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs), we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP) in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT), to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

Protein transduction domains (PTDs) have been shown to promote the delivery of therapeutic proteins or peptides into the living cells. In a previous study, we showed that the double mutant of TCTP-PTD 13, TCTP-PTD 13M2, was more effective in the delivery of insulin than the wild-type TCTP-PTD 13. In this study, we applied this approach to the nasal delivery of a different peptide, exendin-4, using as carriers, several modified TCTP-PTDs, such as TCTP-PTD 13M1, 13M2, and 13M3. Nasal co-administration of TCTP-PTD 13M2 with exendin-4 showed the highest exendin-4 uptake among the three analogs in normal rats, and also decreased blood glucose levels by 43.3% compared with that of exendin-4 alone and by 18.6% compared with that of exendin-4 plus TCTP-PTD 13 in diabetic mice. We also designed an additional covalently linked conjugate of TCTP-PTD 13M2 and exendin-4 and evaluated its hypoglycemic effect after subcutaneous or intranasal delivery. Subcutaneous administration of exendin-4 that its C-terminus is covalently linked to TCTP-PTD 13M2 showed hypoglycemic effect of 42.2% compared to that in untreated group, whereas intranasal delivery was not successful in diabetic mice. We conclude that a simple mixing TCTP-PTD 13M2 with peptide/protein drugs can be potentially a generally applicable approach for intranasal delivery into animals. [ABSTRACT FROM AUTHOR]

Abstract: Previously, we have reported that a 10-amino acid peptide (MIIYRDLISH) derived from the NH2-terminus of the human translationally controlled tumor protein (TCTP) functions as a protein transduction domain (PTD). In this study, we evaluated the transduction ability of SOD fused to TCTP-PTD (TCTP-SOD) into various cell lines. We also evaluated its ability to protect cells against paraquat-induced cell damage, in vitro and its neuroprotective effect in vivo against kainic acid-induced neuronal damage in an animal model. TCTP-SOD was transduced into various cell lines in a dose- and time-dependent manner without cytotoxic effect. Furthermore, TCTP-SOD showed cytoprotective activity in SH-SY5Y cells, and intraperitoneally, injected TCTP-SOD was delivered into the mouse brain and protected the cells in the hippocampal region against the damage induced by kainic acid. We propose TCTP-SOD as a potential candidate drug for treatment of brain diseases. [Copyright &y& Elsevier]

Abstract: Background: Protein transduction domains (PTDs) can be fused to a protein to render it cell-permeable. The delivery efficiencies of PTDs are, however, often poor because PTD-protein conjugates cannot escape from endosomes. A potential solution to this problem consists in adding HA2 analogs to the PTD-protein construct as these peptides can cause endosomal lysis upon acidification of the endosomal lumen. To date, however, the utility of HA2-based PTDs has not been clearly established. Methods: We investigate the biophysical and cellular properties of the glutamate-rich HA2 analog E5 fused to the model protein TAT-mCherry. Results: E5-TAT-mCherry causes the release of fluorescent dextrans trapped with the protein inside endosomes. Yet, E5-TAT-mCherry itself is not released in the cytosol of cells, indicating that the protein remained trapped inside endosomes even after endosomal lysis takes place. Cytosolic delivery of the protein could be achieved, however, by insertion of a disulfide bond between E5 and its cargo. Conclusions: These results show that E5 causes the retention of its fused protein inside endosomes even after lysis takes place. General significance: These data establish that HA2 analogs might not be useful PTDs unless cleavable linkers are engineered between PTD and protein cargo. [Copyright &y& Elsevier]

Abstract: Protein transduction domains (PTDs) are short amino acid sequences that promote their own translocation across the cell plasma membrane and have been studied for possible use in drug delivery and gene therapy. However, no direct method to quantify transduction is available. Here, using a new luciferase-tagged human PTD, we show that cellular uptake levels can be determined in a reliable manner. Furthermore, we show that enhanced in vivo tracking by human PTD can be quantified in a mouse model. This is the first report on the direct quantification of PTD transduction in vitro and in vivo, which will be necessary for studying its possible therapeutic application in drug delivery and gene therapy. [Copyright &y& Elsevier]

Abstract: Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of florescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization. [Copyright &y& Elsevier]

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein. The resulting fusion protein (TAT-CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT-CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT-CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20–30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT-CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine-like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Abstract: Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191–206, 2.23kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1μM, and appeared to plateau above a concentration of 1 μM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins. [Copyright &y& Elsevier]

Crossing of the plasma membrane for all macromolecules without energy, receptors or any artificial methods was thought to be difficult. Our previous studies demonstrated that arginine-rich intracellular delivery (AID) peptides are able to deliver macromolecules, such as proteins, RNAs and DNAs, into either animal or plant cells. Cellular internalization could be mediated by effective and nontoxic AID peptides in either a covalent or noncovalent protein transduction (NPT) manner. AID peptides were so versatile that the procedure seemed to replace the current artificial transfection methods. However, the utilization of AID peptides has been limited to animal or plant systems so far. None has proposed that AID peptides could work in other species. Here, we select some representative organisms to screen whether NPT mediated by AID peptides works in them. They include cyanobacteria, bacteria, archaea, algae, fungi and yeasts. The results reveal that not all living beings possess this capability of protein transduction. Interestingly, all species of prokaryotes tested, which were thought to be highly diverse from the animal and plant systems, appear to be capable of NPT. The mechanism of AID-mediated NPT in cyanobacteria is in a classical endocytosis- and energy-independent pathway and may involve macropinocytosis. In contrast, green algae and multicellular fungi of the eukaryotes are impermeable to protein passage. Our results bring an interesting clue to the reexamination of the phylogeny of both algae and fungi. [ABSTRACT FROM AUTHOR]

The anti-apoptotic Bcl-xL is a promising agent to prevent neurodegeneration in Parkinson’s disease, which is characterized by a demise of dopaminergic neurons. We linked Bcl-xL to a peptide that allows its delivery across biological membranes and the blood–brain barrier. We tested the fusion protein in two models of Parkinson’s Disease. Cell-permeable Bcl-xL protected neuroblastoma cells from the selective neurotoxin 1-methyl-4-phenylpyridinium. Furthermore, its systemic application in aged mice protected dopaminergic neurons following administration of MPTP as revealed by counting of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta. Hence, we present that a cell-permeable form of an anti-apoptotic protein can be delivered to CNS neurons through its systemic application, and we provide the proof that the delivery of this protein to the CNS neurons effectively prevents neuronal cell death in models of chronic neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Plasma membranes of animal cells are generally impermeable to macromolecules. Protein transduction mediated by protein transduction domains (PTDs) covalently cross-linked to cargoes for cellular internalization has previously been demonstrated. Peptides with PTDs could be an effective way to deliver proteins into living cells or tissues in vitro. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to facilitate the delivery of proteins into animal cells and to penetrate skin tissues rapidly. This cellular internalization and transdermal delivery of proteins is mediated by non-toxic AID peptides in a non-fusion protein and non-conjugation dependent manner. The efficiency of intracellular transport is further increased in the presence of chemical enhancer oleic acid. The mechanism of the AID-mediated cellular entry may involve macropinocytosis and actin rearrangement. Thus, we confirm that direct delivery of bioactive proteins into living cells and tissues mediated by non-covalent actions of AID peptides represents a useful strategy in pharmaceutics, therapeutics and cosmetics. [ABSTRACT FROM AUTHOR]

Abstract: Tat peptides are useful carriers for delivering biologic molecules into the cell for both functional analysis of intracellular disease-related proteins and treatment of refractory diseases. Most internalized Tat-fused cargos (Tat-cargos) are trapped within the endosome, however, which limits the biologic function of the cargo. In this study, we demonstrated that Tat-fused HA2 peptide (HA2Tat), an endosome disrupted peptide, enhanced the endosome-escape efficiency of Tat-cargos. In cells treated with a mixture of fluorescein isothiocyanate-labeled Tat and HA2Tat, widespread fluorescence was observed throughout the cytosol. In addition, this HA2Tat-mediated cytosolic delivery technique led to enhanced cytotoxicity of Tat-fused anti-cancer peptides, specifically shepherdin. Thus, we improved the function of the delivered molecules by co-treating with HA2Tat and propose that this is a useful method for the delivery of therapeutic macromolecules into the cytosol. [Copyright &y& Elsevier]

Many lines of evidence suggest that α-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of α-synuclein, and the mechanistic features of the phenomenon. We first showed that not only α-synuclein but also β- and γ-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of α-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. α-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of α-synuclein at 4°C was comparable with the import observed at 37°C. Furthermore, membrane translocation of α-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of α-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis. [ABSTRACT FROM AUTHOR]

Abstract: Protein transduction domains (PTDs) offer an exciting therapeutic opportunity for the treatment of many diseases. An 11-amino acid fragment of human immunodeficiency type 1 (HIV-1) TAT-protein can transduce large, biologically active proteins into mammalian cells; recent evidence has shown an in vivo PTD for the 116kDa β-galactosidase protein. However, there is little information on the in vivo distribution of the TAT fusion protein to define the viability of PTDs for human studies. In this study we examined the tissue kinetics and tissue distribution of the PTD-transduced TAT fusion protein in mice. Low (100μg) or high (500μg) doses of TAT-β-galactosidase fusion protein were administrated to mice through four routes (portal vein, i.v., i.p., and oral). Tissues were harvested 15min, 1h, 6h, 10h, and 24h after treatment. Distribution of β-galactosidase in various tissues was analysed by in situ staining, enzymatic activity assay, and Western blot analysis. β-Galactosidase enzyme activity was observed in all tissues (liver, kidney, spleen, lung, bowel, and brain). β-Galactosidase activity peaked at 15min in most tissues after portal vein, i.v., and i.p. administration and at 1h after oral dosing in all tissues. β-Galactosidase activity in the liver at 15min after portal vein injection (67milliunits [mU]/mg) was higher than after i.v. (9.8mU/mg), i.p. (4.4mU/mg), and oral (0.3mU/mg) dosing. In situ staining and Western blot results correlated closely with β-galactosidase enzyme activity assay. The median initial half-life for activity was 2.2h, ranging from 1.2h to 3.4h (coefficient of variation=28.9%). The bioavailability of β-galactosidase activity after an orally administered PTD was 24%. This study details the kinetics and tissue distribution of delivering of a model TAT fusion protein into the mouse via PTD. These data allow rational selection of delivery route and schedules for therapeutic PTD and will aid the use of TAT fusion protein transduction in the development of protein therapies. [Copyright &y& Elsevier]

With the elucidation of the human genome, exhaustive analysis of genomic data related to gene transcription and the structure and function of translated protein products has progressed rapidly. Delivery of proteins and their functional domains or inhibitory peptides directly into the cell is ideal to use this protein information and analyze associated physiological functions. Protein transduction technology, which controls cell function via direct delivery of a desired protein into the cell, involves fusing the protein with a special peptide sequence consisting of 10-20 amino acids, referred to as the protein transduction domain. The recent discovery that the protein transduction domain can also be inserted into various macromolecules heightens expectations in terms of development of novel advanced experimental tools and clinical reagents [ABSTRACT FROM AUTHOR]

Abstract: Human Period1 (hPer1) has been proved to be able to translocate into cells in a protein transduction manner. The segment of amino acids 830–845 of hPer1 is its protein transduction domain (PTD). In order to explore the membrane penetrating mechanism of hPer1-PTD and the physico-chemical properties necessary in the process, Ala scanning mutation method was used to investigate the variation in the peptide internalization. To further investigate the related physico-chemical requirements, the three dimensional structures of hPer1-PTD and its mutant analogs were simulated by Rosetta method. The electrostatic potentials and energies of these structures were calculated using the Delphi algorithm to solve Poisson–Boltzman equation. The hydrophobicity was assessed by the percentage of the nonpolar area in SAS (solvent accessible surface (SAS)). It has been proved that the Arg836 was the key residue for peptide internalization. When this Arg mutated into Ala, the peptide could not cross the membrane. The large enough area with positive charge was the decisive factor for hPer1-PTD. The alpha-helical structure seemed to play an assistant role so as to enable the positive charge connected in spatial arrangement. [Copyright &y& Elsevier]

Protection of cells from necrosis would be important for many medical applications. Here, we show protein transduction domain (PTD)-FNK therapeutics based on protein transduction to prevent necrosis and acute hepatic injury with zonal death induced by carbon tetrachloride (CCl4). PTD-FNK is a fusion protein comprising the HIV/Tat PTD and FNK, a gain-of-function mutant of anti-apoptotic Bcl-xL. PTD-FNK protected hepatoma HepG2 from necrotic death induced by CCl4, and additionally, increased the apoptotic population among cells treated with CCl4. A concomitant treatment with a pan-caspase inhibitor Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), which alone could not prevent the necrosis, protected these cells from the apoptosis. When pre-injected intraperitoneally, PTD-FNK markedly reduced zonal liver necrosis caused by CCl4. Moreover, injection of PTD-FNK accompanied by Z-VAD-FMK suppressed necrotic injury even after CCl4 administration. These results suggest that PTD-FNK has great potential for clinical applications to prevent cell death, whether from apoptosis or necrosis, and organ failure.Cell Death and Differentiation (2005) 12, 384-394. doi:10.1038/sj.cdd.4401569 Published online 4 February 2005 [ABSTRACT FROM AUTHOR]

The protein transduction domain (PTD) of the HIV-1 Tat protein can facilitate the cellular and nuclear uptake of macromolecular particles. Here, we demonstrate that incorporation without covalent linkage of a 17-amino acid PTD peptide into gene delivery lipoplexes improves gene transfer. Tat/Liposome/DNA (TLD) transfection, as evaluated by Fluorescence Activated Cell Scan analysis of a Green Fluorescence Protein expression plasmid, enabled transfection of highly recalcitrant primary cells in the form of air/liquid interface cultures of sheep tracheal epithelium. Treatment with chloroquine increased, and incubation at low temperature decreased, TLD transfection, suggesting that the endocytosis uptake pathway is involved. [Copyright &y& Elsevier]

The protein transduction system has been employed for delivery of bioactive proteins into cells via an endocytotic mechanism. However, trapping of endocytosed proteins in the endosome may significantly attenuate biological actions in cells. The present investigation demonstrated that endosomal release of transduced protein could be artificially accelerated by exposure to fluorescent light. Exposure to light at 480 nm stimulated endosomal release of transduced FITC-11 arginine-protein transduction domain (11R-PTD), TAT-PTD and Antennapedia-PTD. Moreover, FITC-11R-p53 protein was released from endosomes following stimulation with light. These data suggest that photo-acceleration is a more efficient strategy in terms of the protein transduction system. [Copyright &y& Elsevier]

Protein transduction domains (PTDs) are small cationic peptides that can facilitate the uptake of large, biologically active molecules into mammalian cells. Recent reports have suggested that PTDs may be able to mediate the delivery of cargo to tissues throughout a living organism. Such technology could eliminate the size restrictions on usable drugs, enabling previously unavailable large molecules to modulate in vivo biology and alleviate disease. In this article, we review the evidence that PTDs can be used both to deliver active molecules to pathological tissue in vivo and to treat models of disease such as ischemia, inflammation, and cancer. [ABSTRACT FROM AUTHOR]

The lipid bilayer of a cell presents a significant barrier for the delivery of many molecular imaging reagents into cells at target sites in the body. Protein translocation domains (PTDs) are peptides that breach this barrier. Conjugation of PTDs to imaging agents can be utilized to facilitate the delivery of these agents through the cell wall, and in some cases, into the cell nucleus, and have potential for in vitro and in vive applications. PTD imaging conjugates have included small molecules, peptides, proteins, DNA, metal chelates, and magnetic nanoparticles. The full potential of the use of PTDs in novel in vivo molecular probes is currently under investigation. Cells have been labeled in culture using magnetic nanoparticles derivatized with a PTD and monitored in vive to assess trafficking patterns relative to cells expressing a target antigen. In vivo imaging of PTD-mediated gene transfer to cells of the skin has been demonstrated in living animals. Here we review several natural and synthetic PTDs that have evolved in the quest for easier translocation across biological barriers and the application of these peptide domains to in vive delivery of imaging agents. [ABSTRACT FROM AUTHOR]