Cell-penetrating Peptide TAT Research Articles

The synthesis and characterisation of a novel dendritic system for gene delivery

There has been increasing interest in recent years in gene delivery. We report the synthesis of non-viral delivery systems composed of variations of the cell penetrating peptide TAT, a nuclear localisation signal peptide and dendritic polylysine. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilising gel shift analysis, isothermal titration calorimetry, particle size analysis, zeta potential and transmission electron microscopy. These techniques indicated the successful formation of complexes between the peptide dendrimer and DNA.

Time-course of c-Jun N-terminal kinase activation after cerebral ischemia and effect of D-JNKI1 on c-Jun and caspase-3 activation

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The d-retro-inverso form of c-Jun N-terminal kinase-inhibitor (D-JNKI1), a cell-permeable inhibitor of JNK, powerfully reduces neuronal death induced by permanent and transient ischemia, even when administered 6 h after the ischemic insult, offering a clinically relevant window. We investigated the JNK molecular cascade activation in rat cerebral ischemia and the effects of D-JNKI1 on this cascade. c-Jun activation starts after 3 h after ischemia and peaks at 6 h in the ischemic core and in the penumbra at 1 h and at 6 h respectively. The 6 h c-Jun activation peak correlates well with that of P-JNK. We also examined the activation of the two direct JNK activators, MAP kinase kinase 4 (MKK4) and MAP kinase kinase 7 (MKK7). MKK4 showed the same time course as JNK in both core and penumbra, reaching peak activation at 6 h. MKK7 did not show any significant increase of phosphorylation in either core or penumbra. D-JNKI1 markedly prevented the increase of P-c-Jun in both core and penumbra and powerfully inhibited caspase-3 activation in the core. These results confirm that targeting the JNK cascade using the TAT cell-penetrating peptide offers a promising therapeutic approach for ischemia, raising hopes for human neuroprotection, and elucidates the molecular pathways leading to and following JNK activation.

Lung Delivery Studies Using siRNA Conjugated to TAT(48−60) and Penetratin Reveal Peptide Induced Reduction in Gene Expression and Induction of Innate Immunity

The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48-60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.

Delivery of 2-5A cargo into living cells using the Tat cell penetrating peptide: 2-5A-tat

2′,5′-Oligoadenylate tetramer (2-5A) has been chemically conjugated to short HIV-1 Tat peptides to provide 2-5A-tat chimeras. Two different convergent synthetic approaches have been employed to provide such 2-5A-tat bioconjugates. One involved generation of a bioconjugate through reaction of a cysteine terminated Tat peptide with a α-chloroacetyl derivative of 2-5A. The second synthetic strategy was based upon a cycloaddition reaction of an azide derivative of 2-5A with a Tat peptide bearing an alkyne function. Either bioconjugate of 2-5A-tat was able to activate human RNase L. The union of 2-5A and Tat peptide provided an RNase L-active chimeric nucleopeptide with the ability to be taken up by cells by virtue of the Tat peptide and to activate RNase L in intact cells. This strategy provides a valuable vehicle for the entry of the charged 2-5A molecule into cells and may provide a means for targeted destruction of HIV RNA in vivo.

The Cell‐Penetrating Peptide TAT(48‐60) Induces a Non‐Lamellar Phase in DMPC Membranes

Cell-penetrating peptides (CPPs) are short polycationic sequences that can translocate into cells without disintegrating the plasma membrane. CPPs are useful tools for delivering cargo, but their molecular mechanism of crossing the lipid bilayer remains unclear. Here we study the interaction of the HIV-derived CPP TAT (48-60) with model membranes by solid-state NMR spectroscopy and electron microscopy. The peptide induces a pronounced isotropic (31)P NMR signal in zwitterionic DMPC, but not in anionic DMPG bilayers. Octaarginine and to a lesser extent octalysine have the same effect, in contrast to other cationic amphiphilic membrane-active peptides. The observed non-lamellar lipid morphology is attributed to specific interactions of polycationic peptides with phosphocholine head groups, rather than to electrostatic interactions. Freeze-fracture electron microscopy indicates that TAT(48-60) induces the formation of rodlike, presumably inverted micelles in DMPC, which may represent intermediates during the translocation across eukaryotic membranes.

Cargo‐dependent mode of uptake and bioavailability of TAT‐containing proteins and peptides in living cells

Cell-penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy-independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well-studied CPP TAT fused to a protein (>50 amino acids) or peptide (<50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21(WAF/Cip) and DNA Ligase I measured in living cells.

Tat-Conjugated PAMAM Dendrimers as Delivery Agents for Antisense and siRNA Oligonucleotides

PAMAM G5 dendrimer (P) was conjugated to Tat peptide (T), a cell penetrating peptide, in search of an efficient cellular delivery vehicle for antisense and siRNA oligonucleotides. PAMAM G5 dendrimer was reacted with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt (BODIPY) for visualization to yield the conjugate BP. Bifunctional sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT) was then used to conjugate primary amino groups of BP to cysteine derivatized Tat peptide to give the designed conjugate, BPT. This conjugate was complexed with antisense and siRNA oligonucleotides designed to inhibit MDR1 gene expression. NIH 3T3 MDR cells were used for the evaluation of biological activity of the conjugate. Both antisense and siRNA readily formed complexes with the synthesized BPT, introduced into NIH 3T3 MDR cells, and primarily accumulated in intracellular vesicles. MDR1 gene expression was partially inhibited by the antisense-BPT complex and weakly inhibited by the siRNA-BPT complex when both were tested at nontoxic levels of dendrimer. Conjugation with Tat peptide did not improve the delivery efficiency of the dendrimer. Dendrimer-oligonucleotide complexes were moderately effective for delivery of antisense and only poorly effective for delivery of siRNA. Conjugation of the dendrimer with the Tat cell penetrating peptide failed to further enhance the effectiveness of the dendrimer.

Synthesis of doxorubicin–peptide conjugate with multidrug resistant tumor cell killing activity

Cell penetrating peptide TAT was introduced into doxorubicin structure. Synthesized doxorubicin–TAT conjugate showed different intracellular distribution pattern and cell killing activity from those of free doxorubicin. Unlike free doxorubicin, doxorubicin–TAT conjugate was highly permeable to drug-resistant cells and was able to kill drug-resistant tumor cells efficiently.

Strategies for cytosolic delivery of liposomal macromolecules

Potential approaches to achieve cytosolic delivery of liposomal macromolecules are presented. These approaches include: (1) the co-encapsulation of fusogenic peptides into targeted drug-containing liposomes (2) coupling of the HIV-1-derived cell-penetrating peptide TAT to the surface of liposomes and (3) photochemical internalization, based on photochemically inducible permeabilization of endocytic vesicles.

Translocation of the cell-penetrating Tat peptide across artificial bilayers and into living cells.

The ability of a short, charged peptide to penetrate synthetic DOPC (1,2-dioleoyl-sn-3-glycerophosphocholine) liposomes was investigated by fluorescence confocal microscopy. The peptide, termed Tat (trans-activating transcription factor), was a 14-mer derived from the region of the HIV-1 Tat protein responsible for cellular internalization. This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-iodoacetamidofluorescein) or A568 (Alexa Fluor 568). The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx. 258C) in the absence of pH gradient, ATP or other energy source. The uptake of the Tat-A568 conjugate in unfixed, live HeLa cells was found to be via endocytosis, as expected. In contrast, when the peptide was attached to an IAF-labelled 25 kDa protein corresponding to the catalytic domain of Clostridium botulinum C3 exotoxin, this larger, Tat-C3-IAF construct was not able to enter liposomes, although it localized similarly to Tat-A568 in live cells. The data suggest that Tat peptide can cross synthetic bilayers spontaneously in vitro, but that size and type of cargo may limit this behaviour.

601. HIV-1 TAT Protein Transduction Domain-Mediated Enhancement of HSV-TK/Ganciclovir Suicide Gene Therapy

Top of pageAbstract INTRODUCTION Suicide gene therapy using herpes simplex thymidine kinase gene combined to prodrug ganciclovir (GCV) is one of the most prominent approaches in cancer gene therapy. However, efficacy of the therapy is often insufficient due to the poor rate of gene transfer and due to the limited bystander effect. Recent studies have shown that a protein transduction domain from HIV-1 transactivator protein (TAT-PTD) can translocate through plasmamembrane and deliver proteins into cell. Aim of this study was to make intercellularly spreading HSV-TK protein with the aid of cell penetrating peptide TAT and thus enhance the cytotoxic effect of HSV-TK/GCV gene therapy.METHODS We constructed a novel triple fusion protein containing TAT protein transduction domain (TAT-PTD), HSV-TK suicide gene and GFP marker gene (TAT-TK-GFP) to study effect of TAT-PTD on HSV-TK/GCV-suicide gene therapy. Sensitivity to ganciclovir was tested in different cell lines by comparing cells expressing TAT-TK-GFP to cells expressing TK-GFP. Biodistribution of TAT-TK-GFP from transfected cell to surrounding cells was analyzed by direct fluorescence microscopy or by immunofluorescence staining.RESULTS We found that TAT-peptide enhances cytotoxic effect of HSV-TK/GCV-suicide gene therapy in rapidly dividing cells e.g. in BTC4 rat glioma cell line or in SKOV3.ip1 ovarian cancer cell line. However, enhancement was abolished after serum deprevation. Intercellular spreading of TAT-TK-GFP-protein was not visualized by direct fluorescence microscopy or immunofluorescence staining. Control fusion protein, VP22-GFP, contrast to TAT-fusion protein showed spreading from transfected cell to adjacent cells after methanol fixation.CONCLUSION This study shows that the triple fusion gene TAT-TK-GFP is useful tool for investigating the utility of TAT fusion proteins in suicide gene therapy. Our data with TAT-TK-GFP demonstrate that HSV-TK/GCV cytotoxicity can be enhanced in rapidly growing cancer cells, despite the fact that intercellular spreading cannot be visualized.

Tat cell-penetrating peptide has the characteristics of a poly(proline) II helix in aqueous solution and in SDS micelles.

Tat cell-penetrating peptide (GRKKRRQRRRPPQG) is able to translocate and carry molecules across cell membranes. Using CD spectroscopy the conformation of this synthetic peptide was studied in aqueous and membrane-mimicking, micellar SDS solutions at different temperatures. The CD spectrum of the Tat cell-penetrating peptide in SDS micellar solution was virtually unchanged from that in aqueous solution, and at low temperature it was close to that of a poly(proline) II helix.

Quantitative Assessment of the Cell Penetrating Properties of RI-Tat-9: Evidence for a Cell Type-Specific Barrier at the Plasma Membrane of Epithelial Cells

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.

No entry for TAT(44–57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism.We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb3+-containing liposomes. Upon chelation of Tb3+ by an aromatic carboxylic acid, the fluorescence of Tb3+ increases many fold. The CPP TAT(44–57)-SAM and TAT(37–53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol.In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44–57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44–57).Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44–57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.

Influence of antioxidant therapy on islet graft survival

Subcutaneous site is ideal for clinical islet transplantation because it has the advantage of simple operation procedure under local anesthesia and can be biopsied when needed. However, the transplantation outcomes at subcutaneous site have been disappointing due to hypoxia-induced oxidative stress by poor vascularization. We hypothesized that subcutaneously transplanted islets would have hypoxia resistance by using internalization of metallothionein (MT), an antioxidant scavenging enzyme, which was mediated by fusion between MT and cell penetrating Tat peptide. The Tat-MT was dose-dependently transduced into islets without any damage. Tat-MT-treated islets could be protected from oxidative stress induced by intracellular nitric oxide donor, sodium nitroprusside (SNP). When Tat-MT-treated islets were subcutaneously transplanted into diabetic nude mice, they normally controlled the blood glucose levels without severe fluctuation (median survival time (MST): > 30 days), whereas most untreated islets were rejected (MST 17 days). From the intraperitoneal glucose tolerance test 5 days after posttransplantation, glucose responsiveness of Tat-MT-treated islets was similar to that of normal healthy mice, while untreated islets had delayed glucose responsiveness. From the results of immunohistochemical stain, Tat-MT-treated islets had strong anti-insulin positive cells and lower anti-HIF-1α positive cells. However, untreated islets had rare anti-insulin positive cells and strong anti-HIF-1α-positive cells. Collectively, these findings demonstrated that Tat-MT delivery into islet could offer a new strategy for successful islet transplantation under subcutaneous space.

Evaluation of strategies for the intracellular delivery of proteins.

The intracellular delivery of functionally active protein represents an important emerging strategy for laboratory investigation and therapeutic applications. Although a number of promising approaches for protein delivery have been developed, thus far there has been no attempt to compare the merits of the various deliver technologies. This issue is addressed in the current study. In this study we utilize a sensitive luciferase reporter gene assay to provide unambiguous and quantitative evaluation of several strategies for the intracellular delivery of a biologically active protein comprised of the Gal4 DNA binding domain and the VP16 transactivating domain. Both a cationic lipid supramolecular complex and a poly meric complex were able to effectively deliver the chimeric transcription factor to cultured cells. In addition, protein chimeras containing the Tat cell penetrating peptide, but not those containing the VP22 peptide, were somewhat effective in delivery. Both supramolecular protein-carrier complexes and protein chimeras with certain cell penetrating peptides can support intracellular delivery of proteins. In the cell culture setting the supramolecular complexes are more effective, but their large size may present problems for in vivo applications.

Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat.

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. A promising strategy to overcome this problem consists in the chemical conjugation (or fusion) to cell penetrating peptides (CPP) derived from proteins able to cross the plasma membrane. A large number of different cargo molecules such as oligonucleotides, peptides, peptide nucleic acids, proteins or even nanoparticles have been internalized in cells by this strategy. One of these translocating peptides was derived from the HIV-1 Tat protein. The mechanisms by which CPP enter cells remain unknown. Recently, convincing biochemical and genetic findings has established that the full-length Tat protein was internalized in cells via the ubiquitous heparan sulfate (HS) proteoglycans. We demonstrate here that the short Tat CPP is taken up by a route that does not involve the HS proteoglycans.