Protein Transduction Domain Peptide Research Articles

Establishment of Stable and Secretable TATκ-GFP Recombinant Protein: A Preliminary Report of Promoter Methylation in 293T Cell Line

Induced pluripotent stem cells (iPSC) is a novel technology useful for therapeutic and research applications. To date, iPSCs is produced through genetic modification that can promote mutation; making it harmful for therapeutic use. Therefore, application of non-genetic modification through direct delivery of recombinant proteins aided by protein transduction domain (PTD) enable a safer production of iPSC. This study is aimed to establish a stable production of secretable recombinant protein via recombination of green fluorescence protein (GFP) and a novel PTD peptide, namely TATκ-GFP. 293Tcell line was transfected with 20 μg/ml of TATκ-GFP plasmid and the stably transfected 293T cells were then cultured for 54 days to determine the stability of expression and secretion of TATκ-GFP recombinant protein in prolonged culture. Methylation at the CMV promoter of the TATκ-GFP plasmid was investigated following treatment of transfected cells with 3 μM/mL of demethylation agent, namely 5-Azacytidine for 72 h in three cycles. Flow cytometry analysis demonstrated a transfection efficiency of 9.33% and successful secretion of TATκ-GFP proteins into the culture medium as analysed by Western blot at 72 h post-transfection. However, the transfected cells exhibited a decreasing level of GFP expression and secretion following prolonged culture with notable stability that only sustained for two weeks. 5-Azacytidine-treated cells showed a slight increase of GFP expression compared to non-treated control, suggesting possible promoter methylation which could cause instability of TATκ-GFP expression. Conclusively, promoter methylation should be considered for future establishment of iPSCs as it could inhibit stable expression and secretion of recombinant proteins.

A Pilot Trial of WT1 Peptide-Loaded Allogeneic Dendritic Cell Vaccine and Donor Lymphocyte Infusion for WT1-Expressing Hematologic Malignancies and Post-Transplant Relapse

Abstract 3055 Background:Treatment of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) remains challenging. The Wilms' tumor 1 (WT1) gene product is a tumor-associated antigen that is expressed in acute leukemia and other hematologic malignancies with limited expression in normal tissues. This pilot trial incorporates antigen-specific immunotherapy and allogeneic adoptive cell transfer for pediatric and adult patients with relapsed hematologic malignancies after alloHSCT. The primary objectives are to assess safety and feasibility of a peptide-loaded donor-derived dendritic cell (DC) vaccine and donor lymphocyte infusion (DLI) designed to enhance the graft-vs -leukemia effect. Secondary objectives are to determine if immunologic and clinical responses to WT1 specific peptides can be generated by this novel vaccine strategy after alloHSCT. Design:HLA-A2+ patients with WT1-expressing hematologic malignancies that have relapsed after alloHSCT are eligible. Donor-derived DC vaccines are given every 2 weeks for 6 doses and DLI every 4 weeks for 3 doses. Peripheral blood monocyte-derived DCs are loaded with a combination of three HLA-A2 binding WT1 peptides (WT1 37–45; WT1 126–134; WT1 187–195) linked to the 11-mer HIV TAT protein transduction domain peptide (47–57) designed to enhance antigen presentation. The DCs are generated from donor peripheral blood monocytes that are separated from DLI collected by apheresis. Monocytes are incubated with GM-CSF and IL-4 followed by maturation with LPS and IFN-g. WT1 expression is assessed using immunohistochemistry and/or quantative RT-PCR. Study endpoints included toxicity, feasibility, and antigen-specific immune and clinical responses. Results:4 patients, aged 9–19 years have been treated to date, 3 with acute lymphoblastic leukemia (ALL) and one with Hodgkin lymphoma (HL). Donors were matched siblings in 3 cases and a 10/10 HLA matched father in one case. Vaccines were successfully produced from all donors. All patients tolerated vaccine and DLI administrations well. The most common adverse events were mild, reversible pain and pruritus at vaccine administration and delayed type hypersensitivity (DTH) skin test sites. One patient developed Grade I skin GVHD that did not require treatment. Immune responses were observed in all 3 patients with ALL. ELISPOT was considered positive when it was at least two times greater than and had at least 10 spots more than background. 3 of 4 (75%) patients had positive ELISPOT responses to WT1 peptides. 3 of 4 (75%) patients had positive DTH responses to the keyhole limpet hemocyanin (KLH) control and 2 of 4 (50%) to the WT1 peptides. Median overall survival was 12 months. 1 patient remains in remission 12 months after initiation of therapy and 3 have died of disease. All donors tolerated apheresis well and there were no complications from donor procedures. Conclusions:This novel allogeneic immunotherapy regimen is feasible, well-tolerated and can induce immune responses in the allogeneic setting. Accrual is ongoing.Table:Summary of DTH and ELISPOT ResponsesPatient #DiagnosisKLH DTH ResponseWT1 DTH ResponseWT1 ELISPOT Response1ALL+++2HL---3ALL+-+4ALL+++ Disclosures:Off Label Use: This is a pilot clinical trial of an investigational cancer vaccine.

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A protein transduction domain peptide enhances the cellular uptake of Foxp3 protein for the treatment of autoimmune and allergic disorders.

Revised Role of Glycosaminoglycans in TAT Protein Transduction Domain-mediated Cellular Transduction

Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.

Gene delivery using a derivative of the protein transduction domain peptide, K-Antp

Due to their intracellular permeability, protein transduction domains (PTDs) have been widely used to deliver proteins and peptides to mammalian cells. However, their performance in gene delivery has been relatively poor. To improve the efficiency of PTD-mediated gene delivery, we synthesized a new peptide, KALA-Antp (K-Antp), which contains the sequences for PTD of the third α-helix of Antennapedia (Antp) homeodomain and the fusogenic peptide KALA. In this configuration, Antp is designed to provide the cell permeation capacity and nuclear localization signal, while the KALA moiety to promote cellular entry of the peptide–DNA complex. An optimal K-Antp/DNA formula was nearly 400–600 fold more efficient than Antp or poly-lysine-Antp (L-Antp) in gene delivery, and comparable or superior to a commercial liposome. The K-Antp-mediated plasmid DNA transfection not only exhibited temperature sensitivity, reflecting the involvement of an endocytosis-mediated gene transfer mechanism similar to other known PTDs, but also temperature insensitivity, suggesting the role of an energy-independent mechanism. Incorporation of an endosomolytic polymer polyethylenimine (PEI) into the system or treatment with chloroquine further increased the efficiency of K-Antp-mediated gene delivery. These results demonstrate the potential of the combinatorial use of KALA, Antp and PEI in the development of efficient PTD-derived gene carriers.

Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila melanogaster

The phenomenon of protein transduction represents internalization of short peptides known as protein transduction domains (PTD) by cells. It is widely used in the development of new preparations for treatment of various brain disorders. However, the drug discovery process is limited by lack of simple and reliable models of blood brain barrier (BBB). These models should meet two main criteria: they should be applicable for testing of large numbers of samples simultaneously reproduce the physiological and functional characteristics of mammalian (including) human BBB. The major goal of this study was to estimate the BBB-crossing ability of known PTD-peptides using Drosophila melanogaster BBB as the model. We demonstrate here that after abdominal administration the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein can cross Drosophila and deliver the apoE mimetic peptide exhibiting neuroprotective properties.

PTD-modified ATTEMPTS system for enhanced asparaginase therapy: A proof-of-concept investigation

Macromolecular drugs such as proteins and gene products are presumably the most desirable therapeutic agents due to their unmatched substrate specificity and reaction efficiency. Yet, clinical use of these drugs has met with limited success, primarily due to the impermeable nature of the cell membrane that restricts cellular drug uptake to only small (< 600 Da) and hydrophobic molecules. The recent discovery of the protein transduction domain (PTD) membrane-penetrating peptides, such as HIV-TAT, has finally offered the possibility of resolving this cell-membrane barrier for macromolecular drug delivery. Via covalent linkages, these PTD peptides have been shown to ferry the attached macromolecular species across membranes of all cell types, both in vitro and in vivo. Nevertheless, the lack of selectivity for PTD-mediated internalization restricts the application of this cell uptake method in clinical practice, due to concerns of inducing systemic toxicity caused by the carried drugs. Presented herein is a modified version of our previously established “ATTEMPTS” approach in delivery of macromolecular drugs, which integrates the cell-penetrating PTDs into a heparin/protamine-regulated delivery system. In vitro findings using asparaginase (ASNase) as a model macromolecular anti-tumor agent were able to validate the feasibility of this delivery system. The chemically constructed TAT-ASNase conjugates not only were able to translocate into the MOLT-4 cells and elicit the cytotoxic effects, but also this PTD-mediated intracellular drug uptake could be regulated (with on/off control) by the addition of heparin and protamine. This modified ATTEMPTS system therefore presents a new avenue of treatment of various types of cancers and other diseases with macromolecular drugs. In vitro characterization and a preliminary proof-of-concept animal investigation that demonstrates the feasibility of this PTD-mediated ASNase therapeutic system is subsequently described.

Prevention of chemotherapy-induced alopecia by the anti-death FNK protein

Many anticancer drugs attack rapidly dividing cells, including not only malignant cells but also hair follicle cells, and induce alopecia. Chemotherapy-induced alopecia (CIA) is an emotionally distressing side effect of cancer chemotherapy. There is currently no useful preventive therapy for CIA. We have previously constructed anti-death rFNK protein from rat Bcl-x L by site-directed mutagenesis to strengthen cytoprotective activity. When fused to the protein transduction domain (PTD) of HIV/Tat, the fusion protein PTD (TAT)-rFNK successfully entered cells from the outside in vitro and in vivo to exhibit anti-death activity against apoptosis and necrosis. Here, we show that topical application of FNK protected against CIA in a newborn rat model. The protective activity against hair-loss was observed in 30–1000 nM TAT-rFNK administrative groups in a dose-dependent manner. Furthermore, a human version of FNK (hFNK) fused to other PTD peptides exhibited a protective ability. These results suggest that PTD-FNK possesses protective activity against CIA and is not restricted to a sequence of PTD peptides or species of FNK. Thus, PTD-FNK represents potential to develop a useful method for preventing CIA in cancer patients.

A Heat Shock Protein 90 Binding Domain in Endothelial Nitric-oxide Synthase Influences Enzyme Function

Previous reports suggest heat shock protein 90 (hsp90) associates with endothelial nitric-oxide synthase (eNOS) to increase nitric oxide (*NO) generation. Ansamycin inhibition of chaperone-dependent activity increases eNOS generation of superoxide anion (O(2)(*)) upon enzyme activation. In the present study we identify where hsp90 binds to eNOS using overlapping decoy peptides based on the amino acid (aa) sequence of eNOS (291-420). B1, B2, and B3 peptides inhibited hsp90 association with eNOS in cell lysates from proliferating bovine aortic endothelial cells. B2 (aa 301-320), common to both B1 and B3, decreased stimulated *NO production and hsp90 association in bovine aortic endothelial cells. The B2/B3 peptide was redesigned to TSB2 that includes a TAT protein transduction domain and shortened to 14 aa. TSB2 impaired vasodilation of isolated facialis arteries in vitro and in vivo and increased eNOS-dependent O(2)(*) generation in native endothelial cells on mouse aortas, whereas a control peptide, TSB(Ctr), which has the four glutamic acids in TSB2 substituted with alanine, showed no such effects. Site-directed mutagenesis of eNOS at 310, 314, 318, and 323 Glu to Ala yields an eNOS mutant that exhibited reduced hsp90 association and generated O(2)(*) rather than *NO upon activation. Together, these data demonstrate that hsp90 associates with eNOS at aa 310-323. Moreover, a decoy peptide based on this sequence is sufficient to displace hsp90 from eNOS and uncouple eNOS activity from *NO generation. Thus, Glu-310, Glu-314, Glu-318, and Glu-323 in eNOS, although each does not do much by itself, synergistically they increase "cooperativity" in the association step that is critical for maintaining hsp90-eNOS interactions and promoting coupled eNOS activity. Such chaperone-dependent signaling may play an important role in modulating the balance of *NO and O(2)(*) generation from eNOS and, therefore, vascular function.

An intracellular delivery method for siRNA by an arginine-rich peptide

RNA interference has recently become a useful research tool for the studies of gene functions, regulations, and therapies. The double-stranded RNA is utilized to induce the sequence-specific gene silencing. To achieve this goal of specific gene silencing, a proper delivery system of siRNA is highly demanded. A number of approaches for delivering siRNA have been explored over the last few years. In the present study, we demonstrated a simple peptide-based siRNA delivery system in mammalian cells. A GC-EGFP cell line stably expressing enhanced green fluorescent protein was established from stable transfection of human gastric carcinoma cells. The synthetic nona-arginine peptide, an arginine-rich intracellular delivery peptide, or called protein transduction domain peptide, could noncovalently form stable complexes with EGFP siRNA and deliver these mixtures into cells. After entry, siRNA appeared to stay in perinuclear regions within cell, and ultimately fulfilled its targeted egfp gene silencing. These data were in consonance with that RNA-induced silencing complex components could be also localized to these perinuclear regions, creating a focal point for RNA interference factories. In the future, this non-toxic peptide may be proved to be a useful tool for the delivery of exogenous siRNA in RNA interference research.

Characterisation of cell‐penetrating peptide‐mediated peptide delivery

Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be critical in determining the most effective sequence. In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine=transportan>antennapedia>TAT. However, examination of cellular toxicity showed that antennapedia<TAT<transportan< intersectionpolyarginine, with antennapedia-peptide conjugates having no significant toxicity even at 100 microM. Confocal studies of the mechanism of antennapedia- and TAT-peptide uptake showed that the time course of uptake and their cellular distribution did not correlate with transferrin, a marker of clathrin-mediated endocytosis. In contrast, the peptides co-localised with a marker of lipid rafts domains, cholera toxin, which was attenuated following the disruption of these domains using methyl-beta-cyclodextrin. Overall, comparison of the uptake and toxicity suggests that antennapedia provides the optimal cell-penetrating peptide for peptide delivery in vitro and that both antennapedia- and TAT-mediated peptide delivery occurs predominantly via lipid raft-dependent but clathrin-independent endocytosis.

Nontoxic membrane translocation peptide from protamine, low molecular weight protamine (LMWP), for enhanced intracellular protein delivery: in vitro and in vivo study

Naturally derived, nontoxic peptides from protamine by the authors, termed low molecular weight protamines (LMWPs), possess high arginine content and carry significant sequence similarity to that of TAT, by far the most potent protein transduction domain peptide. Therefore, it was hypothesized that these LMWPs would also inherit the similar translocation activity across the cell membrane, which enables any impermeable species to be transduced into the cells. LMWPs were prepared by enzymatic digestion of protamine, examined their capability of transducing an impermeable protein toxin into the tumor cells by chemical conjugation, and determined cytotoxicity of transduced protein toxin (e.g., gelonin) against cancer cell lines and a tumor-bearing mouse. In vitro results showed that LMWPs could indeed translocate themselves into several mammalian cell lines as efficiently as TAT, thereby transducing impermeable gelonin into the cells by chemical conjugation. In vivo studies further confirmed that LMWP could carry an impermeable gelonin across the tumor mass and subsequently inhibit the tumor growth. In conclusion, the presence of equivalent cell translocation potency, absence of toxicity of peptide itself, and the suitability for low-cost production by simple enzymatic digestion could expand the range of clinical applications of LMWPs, including medical imaging and gene/protein therapies.

879. Transduction Enhancement by the Adenovirus/Polymer Complexes Derivatized with Protein Transduction Domain Peptides

Top of pageAbstract Background: Cellular uptake of group C adenoviruses critically depends on the availability of the cognate Coxsackie Adenovirus Receptors (CARs) on the cell surface. In cardiovascular tissues paucity of CARs is a limiting factor for the successful application of adenoviral vectors. Additionally, immune responses elicited by Ad infection diminish transgene expression due to the elimination of free Ad and transduced cells. Polymer modification of adenoviral surface potentially addresses these problems by masking Ad particles from neutralizing antibodies and providing a convenient platform for the conjugation of ligands possessing transduction-facilitating properties. We report here that a covalent modification of Ad surface with a photoactivatable polymer derivatized with protein transduction domains (PTD), TAT and Antp, increases transduction efficiency. Methods: A positively charged, thiol-reactive, photoactivatable polyallylamine (PAA) derivative was coupled with TAT-Cys and Antp-Cys. Ad samples suspended in water or PBS of different ionic strength (x1 and x5) were admixed with PTD-derivatized and unmodified polymers to obtain final polymer/Ad ratios ranging from 1:100 to 1:5 (w/w). Initial charge-based polymer/virus attachment was rendered covalent by 15 sec UV light (350 nm) illumination. Surface charge and size of the Ad/polymer complexes were measured by zeta-potentiometry and dynamic light scattering, respectively. Ad surface modification was examined by transmission electron microscopy (TEM) employing colloidal anionic gold. Transduction of a rat aortic smooth muscle cell line (A10) and primary bovine aortic endothelial cells (BAEC) was assessed by the fluorescence microscopy and the fluorimetry of cell lysates. In some experiments the cells were pretreated with the recombinant soluble knob protein (5 |[mu]|g/ml) prior to the Ad infection. Results: Association of Ad with unmodified polymer increased surface charge of the vector from |[minus]|31.67 mv to |[minus]|11.89 mv, while the interaction with the Antp- and TAT-modified polymers inverted the surface charge to +3.75 mv and +12.09 mv, respectively. Recharging of the Ad surface was confirmed by the TEM demonstrating the attraction of 5 nm anionic gold particles to the surface of the Ad modified with TAT-derivatized polymer, but not to the surface of naive Ad. The association of Ad-GFP with PTD-derivatized polymers resulted in 3-7-fold increase of GFPexpression in cells, whereas unmodified polymer-derivatized virus demonstrated the same efficiency as the naive Ad. Pretreatment of A10 cells with knob protein caused 86% transduction inhibition with free and unmodified polymer-derivatized Ad, while the transgene expression of the PTD/polymer-modified Ad was not affected. Optimization of the transduction protocol revealed maximal enhancement of transgene expression at a polymer/Ad ratio of 1:10. Also, complexes prepared in x5 PBS achieved higher GFP expression than those prepared in water or x1 PBS, which correlated with smaller size of complexes (330 nm vs 1360 and 1060 nm diameter, respectively). Conclusion: Covalent attachment of PDT-decorated polymers to the Ad surface increases transduction in vitro.

HIV-1 Tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes

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.

Peptide-derivatized shell-cross-linked nanoparticles. 1. Synthesis and characterization.

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell-cross-linked (SCK) nanoparticles is reported as a method to facilitate cell surface binding and transduction of SCK nanoparticles. Attaching increasing numbers of peptide sequences to SCK nanoparticles in a global solution-state functionalization strategy has been devised as a method for increasing the efficiency of the cell-penetrating process. The numbers of peptides per SCK were controlled through stoichiometric balance and measured experimentally by two independent methods, UV-visible spectroscopy and phenylglyoxal analysis. PTD was conjugated in (0.005, 0.01, and 0.02) molar ratios, relative to the acrylic acid residues in the shell, to the SCK nanoparticles resulting in SCK populations possessing nominally 52, 104, and 210 (41, 83, and 202 as measured by phenylglyoxal analysis) PTD peptides per particle, respectively. The methodologies for the block copolymer and nanoparticle syntheses, peptide derivatization, and characterization of peptide-functionalized SCK nanoparticles are reported and the feasibility and efficiency of intracellular internalization of the respective SCKs were quantified.

Transduction of anti-apoptotic proteins into chondrocytes in cartilage slice culture

Peptides of the protein transduction domain (PTD) mediate the introduction of passenger proteins into cells in vitro and in vivo, where the domains are positively charged. This unusual ability can be exploited for medical applications in protein therapeutics. Chondrocytes are embedded in a dense extracellular matrix, whose components are highly negatively charged. We examined whether PTD mediates the delivery of functional proteins into chondrocytes through the matrix using the super anti-apoptotic protein FNK fused with Tat/PTD peptide (PTD–FNK), the FNK protein being constructed from anti-apoptotic Bcl-x L to enhance its activity. The PTD–FNK protein labeled with a fluorescent dye was incorporated into chondrocytes through the matrix and immunostaining confirmed the transduction into the cells. The PTD–FNK protein protected chondrocytes from cell death induced by Fas antibody and nitrogen oxide (NO). Thus, the PTD peptide has the ability to deliver passenger proteins into chondrocytes by penetrating the extracellular matrix of cartilage.