Tat Peptide Research Articles

Membrane binding of the insertion sequence of Proteus vulgaris L-amino acid deaminase stabilizes protein structure and increases catalytic activity

Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity.

Self-Assembling Lipid-Peptide Hybrid Nanoparticles of Phospholipid-Nonaarginine Conjugates for Enhanced Delivery of Nucleic Acid Therapeutics.

Despite potential applications of nucleic acid therapeutics, the lack of effective delivery systems hinders their clinical application. To overcome the barriers to nucleic acid delivery, we previously reported nanoparticles using phospholipid-polyethylenimine conjugates. However, toxicity of polyethylenimine remains as a problematic issue. Herein, we proposed to substitute the polyethylenimine with arginine-rich peptide to obtain a less-toxic carrier system. Nonaarginine was conjugated to the distal end of phospholipid hydrocarbon chains leading to phospholipid-nonaarginine conjugates (PL9R) and then lipid-peptide hybrid nanoparticles carrying oligonucleotide therapeutics (hNP) were constructed by self-assembly process. The hNP were further modified with cell penetrating Tat peptide (T-hNP) to enhance cellular uptake. The PL9R was less cytotoxic, and the hNP showed high loading capacity and colloidal stability. The T-hNP showed higher cellular uptake and transfection efficiency and effective accumulation to tumor tissue and silencing effect in tumor bearing mice. Altogether, T-hNP could provide a promising nanocarrier for nucleic acid therapeutics.

CRGD/TAT Dual-Ligand Reversibly Cross-Linked Micelles Loaded with Docetaxel Penetrate Deeply into Tumor Tissue and Show High Antitumor Efficacy in Vivo.

The application of cell-penetrating peptides like TAT for in vivo targeted delivery is limited because the penetration behavior is not cell-specific. Herein, we designed cRGD and TAT comodified cross-linkable micelles (cRGD/TAT CMs), in which the TAT peptide was shielded by relatively long poly(ethylene glycol) (PEG) chains. Docetaxel (DTX)-loaded cRGD/TAT CMs were very stable with minimal drug leakage under physiological conditions, whereas rapid DTX release took place in a reductive environment. Flow cytometry showed that the cRGD/TAT CMs with molar ratios of 20% cRGD and 10% TAT (cRGD20/TAT10 CMs) were selectively and efficiently taken up by ανβ3-overexpressing U87MG glioma cells, with 8.3-fold and 18.3-fold higher uptake than cRGD20 CMs and PEG CMs, respectively. DTX-loaded cRGD20/TAT10 CMs exhibited a high cytotoxicity in U87MG cells, leading to rapid apoptosis of the tumor cells. Uptake mechanism studies revealed that cRGD20/TAT10 CMs mainly employed the caveolae-mediated endocytotic pathway and efficiently escaped from the lysosomes. Notably, cRGD20/TAT10 CMs had a long circulating time of 6.25 h in vivo, due to cross-linking of the micelles and shielding of the TAT peptide. Moreover, DTX-loaded cRGD20/TAT10 CMs exhibited a significantly higher accumulation and deeper penetration in subcutaneous U87MG glioma tissue compared to cRGD20 CMs and PEG CMs, leading to superior antitumor efficacy in vivo. Therefore, this dual-ligand strategy provides an effective way to realize tumor-specific penetration and inhibition.

Multifunctional Gene Carriers with Enhanced Specific Penetration and Nucleus Accumulation to Promote Neovascularization of HUVECs in Vivo.

Recently, gene therapy has attracted much attention, especially for the treatment of vascular disease. However, it is still challenging to develop the gene carriers with high biocompatibility as well as highly efficient gene delivery to overcome multiple barriers. Herein, a frequently used cell-penetrating peptide PKKKRKV (TAT) was selected as a functional sequence of the gene carrier with distinctive cell-penetrating ability. REDV peptide with selectively targeting function for endothelial cells (ECs) and nuclear localization signals (NLS)were integrated with this TAT peptide to obtain a highly efficient gene delivery system with ECs specificity and nucleus accumulation capacity. Besides, the glycine sequences with different repeat numbers were inserted into the above integrated peptide. These glycine sequences acted as a flexible spacer arm to exert the targeting, cell-penetrating, and nucleus accumulation functions of each functional peptide. Three tandem peptides REDV-Gm-TAT-Gm-NLS (m = 0, 1, and 4) complexed with pZNF580 plasmid to form gene complexes. The results of hemocompatibility and cytocompatibility indicated that these peptides and gene complexes were nontoxic and biocompatible. The internalization efficiency and mechanism of these gene complexes were investigated. The internalization efficiency was improved as the introduction of targeting REDV and glycine sequence, and the REDV-G4-TAT-G4-NLS/pZNF580 (TP-G4/pZNF580) complexes showed the highest cellular uptake among the gene complexes. The TP-G4/pZNF580 complexes also presented significantly higher internalization efficiency (∼1.36 times) in human umbilical vein endothelial cells (HUVECs) than human umbilical artery smooth muscle cells. TP-G4/pZNF580 complexes substantially promoted the expression of pZNF580 by confocal live cell imaging, gene delivery efficiency, and HUVECs migration assay. The in vitro and in vivo revascularization ability of transfected HUVECs was further enhanced obviously. In conclusion, these multifunctional REDV-Gm-TAT-Gm-NLS peptides offer a promising and efficacious delivery option for neovascularization to treat vascular diseases.

Reducing Agent-Assisted Excessive Galvanic Replacement Mediated Seed-Mediated Synthesis of Porous Gold Nanoplates and Highly Efficient Gene-Thermo Cancer Therapy.

Porous Au nanoplates (pAuNPs) were manufactured by a reducing agent-assisted galvanic replacement reaction on Ag nanoplates using a seed-mediated synthetic approach. Two core additives, poly(vinylpyrrolidone) and l-ascorbic acid, prevented fragmentation and proceeded secondary growth. By controlling the concentration of the additives and the amount of replacing ion AuCl4-, various nanostructures including nanoplates with holes, nanoframes, porous nanoplates, and bumpy nanoparticles with unity and homogeneity were synthesized. The present synthetic method is advantageous, because it can be used to manufacture pAuNPs with ease, robustness, and convenience. The prepared pAuNPs exhibited a highly efficient photothermal conversion effect and cargo loading capacity on exposed surfaces by Au-thiol linkage. By using dual cargo mixed loading of the hepatitis C virus (HCV) targeting gene drug DNAzyme and cell-penetrating peptide TAT onto the surface of the pAuNPs and photothermal conversion-mediated hyperthermic treatment, successful gene-thermo therapy against HCV genomic human hepatocarcinoma cells were demonstrated.

Cell-Surface Interactions on Arginine-Rich Cell-Penetrating Peptides Allow for Multiplex Modes of Internalization.

One of the recent hot topics in peptide-related chemical biology research is the potential of cell-penetrating peptides (CPPs). Owing to their ability to deliver exogenous molecules into cells easily and effectively, their flexible design that allows transporters to comprise various chemical structures and functions, and their potential in chemical and cell biology studies and clinical applications, CPPs have been attracting enormous interest among researchers in related fields. Consequently, publications on CPPs have increased significantly. Although there are many types of CPPs with different physicochemical properties and applications, arginine-rich CPPs, which include the human immunodeficiency virus type 1 (HIV-1) TAT peptide and oligoarginines, are among the most extensively employed and studied. Previous studies demonstrated the importance of the guanidino group in arginine, which confers flexibility in transporter design. Therefore, in addition to peptides, various transporters rich in guanidino groups, which do not necessarily share specific chemical and three-dimensional structures, have been developed. Typically, cell-penetrating transporters have 6-12 guanidino groups. Since the pKa of the guanidino group in arginine is approximately 12.5, these molecules are highly basic and hydrophilic. Our group is interested in why these cationic molecules can penetrate cells. Understanding their mechanism of action should lead to the rational design of intracellular delivery systems that have high efficacy. Additionally, novel cellular uptake mechanisms may be elucidated during the course of these studies. Therefore, our group is trying to understand the basic aspects underlying the ability of these peptides to penetrate cells. Regarding the delivery of biopharmaceuticals including proteins and nucleic acids, achieving efficient and effective delivery to target organs and cells is one of the biggest challenges. Furthermore, when the target sites of these drug molecules are within cells, effective cell penetration becomes another obstacle. Cells are surrounded by a membrane that separates the inside of the cell from its outside. This barrier function is critical for keeping cellular contents inside cells, and without this, cells cannot function. Therefore, understanding the mechanism of action of CPPs is necessary to overcome these obstacles and will allow us not only to improve CPP-mediated delivery but also to create other types of intracellular delivery systems. In this Account, we summarize the current knowledge on the mechanisms of internalization of arginine-rich CPPs, from the viewpoints of both direct cell-membrane penetration (i.e., physicochemical aspects) and endocytic uptake (i.e., physiological aspects), and discuss the implications of this knowledge. We also discussed loosening of lipid packing as a factor to promote direct cell-membrane penetration.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

Gβγ subunit activation promotes dopamine efflux through the dopamine transporter.

The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ induces the release of DA through DAT. Specifically, a Gβγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ. A TAT peptide containing the Gβγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ-dependent mechanism. Collectively, our data show that Gβγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.

Glioma targeted delivery strategy of doxorubicin-loaded liposomes by dual-ligand modification

The blood-brain barrier (BBB) is the protective parclose of brain safety, but it is also the main obstacle of the drug delivery to cerebral parenchyma, which hamper therapy for brain diseases. In this work, a glioma targeted drug delivery system was developed through loading doxorubicin into Angiopep-2 and TAT peptide dual-modified liposomes (DOX-TAT-Ang-LIP). Low-density lipoprotein receptor-related protein-1 (LRP1) was one receptor overexpressed on both BBB and glioma cytomembranes. Angiopep-2, a specific ligand of LRP1, exhibited high LRP1 binding efficiency. Additionally, TAT could penetrate through cell membranes without selectivity via an unsaturated pathway. To avoid the receptor saturation of Angiopep-2, TAT was also conjugated on the surface of liposomes, providing that the liposomes not only have effective BBB penetrating effect, but also have the glioma targeting function. The prepared DOX liposomes appeared good stability and narrow dispersity in serum with a diameter of 90 nm, and exhibited sustained DOX release behaviors. The conjunctions of Angiopep-2 and TAT were confirmed by 1H NMR spectra. The BBB model, cellular uptake observations, antiproliferation study, and the cell ultrastructure analyses suggested that DOX-TAT-Ang-LIP could not only penetrate through BBB via transcytosis, but also concentrate in glioma, then enter into glioma cells and finally result in the necrosis of glioma cells.

HIV and HIV-Tat inhibit LPS-induced IL-27 production in human macrophages by distinct intracellular signaling pathways

Monocyte-derived Mϕs (MDMs) from HIV-infected patients and MDM infected in vitro with HIV exhibit a reduced ability to secrete various cytokines, including IL-12. Recently, IL-27, an IL-12 family cytokine, was shown to inhibit HIV replication in Mϕ. Whether HIV infection or HIV accessory protein(s) impact IL-27 production in Mϕs remains unknown. Herein, we show that in vitro HIV infection, as well as intracellular HIV-Tat (Tat) and Tat peptides, inhibit LPS-induced IL-27 production in human MDMs, suggesting impairment of the TLR4 signaling pathway. To understand the signaling pathways governing HIV or Tat-mediated inhibition of LPS-induced IL-27 production, we first demonstrated that p38 MAPK, PI3K, Src-homology region 2 domain-containing tyrosine phosphatase 1 (SHP-1), and Src kinases regulate LPS-induced IL-27 production in MDMs. Tat caused down-regulation of TNFR-associated factor (TRAF)-6 and inhibitor of apoptosis 1 (cIAP-1) and subsequently decreased phosphorylation of downstream PI3K and p38 MAPKs, which were implicated in LPS-induced IL-27 production. Whereas SHP-1 and Src kinases regulated LPS-induced IL-27 production, Tat did not inhibit these kinases, suggesting that they were not involved in Tat-mediated inhibition of LPS-induced IL-27 production. In contrast to Tat, in vitro HIV infection of MDM inhibited LPS-induced IL-27 production via inhibition of p38 MAPK activation. Overall, HIV and Tat inhibit LPS-induced IL-27 production in human macrophages via distinct mechanisms: Tat through the inhibition of cIAP-1-TRAF-6 and subsequent inhibition of PI3K and p38 MAPKs, whereas HIV through the inhibition of p38 MAPK activation.

Inhibition of highly pathogenic avian influenza (HPAI) virus by a peptide derived from vFLIP through its direct destabilization of viruses

The antiviral activities of synthesized Kα2-helix peptide, which was derived from the viral FLICE-like inhibitor protein (vFLIP) of Kaposi’s sarcoma-associated herpesvirus (KSHV), against influenza A virus (IAV) were investigated in vitro and in vivo, and mechanisms of action were suggested. In addition to the robust autophagy activity of the Kα2-helix peptide, the present study showed that treatment with the Kα2 peptide fused with the TAT peptide significantly inhibited IAV replication and transmission. Moreover, TAT-Kα2 peptide protected the mice, that were challenged with lethal doses of highly pathogenic influenza A H5N1 or H1N1 viruses. Mechanistically, we found that TAT-Kα2 peptide destabilized the viral membranes, depending on their lipid composition of the viral envelop. In addition to IAV, the Kα2 peptide inhibited infections with enveloped viruses, such as Vesicular Stomatitis Virus (VSV) and Respiratory Syncytial Virus (RSV), without cytotoxicity. These results suggest that TAT-Kα2 peptide is a potential antiviral agent for controlling emerging or re-emerging enveloped viruses, particularly diverse subtypes of IAVs.

Insights into the interactions of biomolecules with small gold clusters: a theoretical study from a DFTB perspective

In this study, we highlight the benefits and limits of using density-functional tight-binding (DFTB) with respect to standard density functional theory (DFT). The DFTB method is found to reliably reproduce the DFT adsorption energies and geometries. Overall, our results suggest that DFTB is a good reference method to set the correct chemical states and the initial geometries of proline interacting with gold cluster. The frontier orbitals energies suggest that in proline–gold cluster complex, the proline molecule can only act as an electron donor and the gold cluster as acceptor. The analysis of the electronic properties for arginine, arginine dipeptide, and TAT peptide which underline the role on the interaction of the s-like H states with the d-like Au states produces both bonding and anti-bonding occupied orbitals, and the process is well described by a model for the interaction of localized orbitals with narrow-band dispersive electron states. The bonding orbitals well below the metal Fermi level contribute to the adsorption of arginine, arginine dipeptide, and TAT peptide on gold.

Sphingosine kinase 2 activates autophagy and protects neurons against ischemic injury through interaction with Bcl-2 via its putative BH3 domain

Our previous findings suggest that sphingosine kinase 2 (SPK2) mediates ischemic tolerance and autophagy in cerebral preconditioning. The aim of this study was to determine by which mechanism SPK2 activates autophagy in neural cells. In both primary murine cortical neurons and HT22 hippocampal neuronal cells, overexpression of SPK2 increased LC3II and enhanced the autophagy flux. SPK2 overexpression protected cortical neurons against oxygen glucose deprivation (OGD) injury, as evidenced by improvement of neuronal morphology, increased cell viability and reduced lactate dehydrogenase release. The inhibition of autophagy effectively suppressed the neuroprotective effect of SPK2. SPK2 overexpression reduced the co-immunoprecipitation of Beclin-1 and Bcl-2, while Beclin-1 knockdown inhibited SPK2-induced autophagy. Both co-immunoprecipitation and GST pull-down analysis suggest that SPK2 directly interacts with Bcl-2. SPK2 might interact to Bcl-2 in the cytoplasm. Notably, an SPK2 mutant with L219A substitution in its putative BH3 domain was not able to activate autophagy. A Tat peptide fused to an 18-amino acid peptide encompassing the native, but not the L219A mutated BH3 domain of SPK2 activated autophagy in neural cells. The Tat-SPK2 peptide also protected neurons against OGD injury through autophagy activation. These results suggest that SPK2 interacts with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1 and activating autophagy. The observation that Tat-SPK2 peptide designed from the BH3 domain of SPK2 activates autophagy and protects neural cells against OGD injury suggest that this structure may provide the basis for a novel class of therapeutic agents against ischemic stroke.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10–800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.

The use of electronic-neutral penetrating peptides cyclosporin A to deliver pro-apoptotic peptide: A possibly better choice than positively charged TAT

Cell-penetrating peptides (CPPs) are increasingly important in transporting macromolecules across cell membranes, but their use remains confined to narrow clinical applications due to the systemic toxicity induced by their positive charges. Several newly discovered electronic neutral penetrating peptides are not attracting much attention because their penetrating capacity is normally far less powerful than cationic or amphiphilic CPPs. In this study, we found the electronic neutral cyclic peptide cyclosporin A (CsA) exhibited 5.6-fold and 19.1-fold stronger penetrating capacity, respectively, than two reported electronic neutral peptides PFVYLI (PFV) and pentapeptide VPTLQ (VPT) in MCF-7 human breast cancer cells. To systematically evaluate the efficiency and toxicity of CsA, we utilized CsA to deliver a membrane-impenetrable pro-apoptotic peptide (PAD) and compared this to the well-established cationic penetrating peptide TAT (RKKRRQRRR). By conjugating CsA to PAD, the internalization of PAD increased 2.2- to 4.7-fold in four different tumor cell lines, and that of CsA-PAD conjugate was significantly higher than TAT-PAD conjugate in MCF-7 and HeLa human cervical cancer cells. Cytotoxicity studies demonstrated that CsA-PAD exhibited a large increase in cell cytotoxicity compared to PAD in four different tumor cell lines, with the effect being similar or greater than the effect of TAT-PAD, depending upon the cell type. The mechanistic studies demonstrated that modifying CsA or TAT did not change the cytotoxicity mechanism of PAD, which occurred via mitochondrial membrane damage related to apoptosis. In vivo studies showed that CsA-PAD could achieve similar anti-tumor efficacy to TAT-PAD but with much lower systemic toxicity, especially to the heart and liver. In conclusion, our study demonstrates for the first time that the electronic-neutral penetrating peptide CsA can be used as a powerful tool to deliver peptide drugs with similar efficiency and less toxicity than the positively charged TAT peptide.

Self-Assembled DNA Dendrimer Nanoparticle for Efficient Delivery of Immunostimulatory CpG Motifs.

Dendrimer-like DNA nanostructures have attractive properties such as mechanical stability, highly branched nanostructure, customized sizes, and biocompatibility. In this study, we construct programmable DNA dendrimeric nanoparticles as efficient vehicles to deliver immunostimulatory cytosine-phosphate-guanosine (CpG) sequences for activation of the immune response. DNA dendrimers decorated with CpG-containing hairpin-loops triggered stronger immune response characterized by pro-inflammatory cytokines production, in contrast to DNA dendrimers loading linear CpG. After further modification with TAT peptide, a typical cell-penetrating peptide, on the surface of the nanocarriers, CpG loops-loaded DNA dendrimers showed the enhanced cell internalization and cytokines production. The TAT-DNA dendrimer-CpG loops constructs did not affect the viability of immune cells and no detectable cytotoxicity was observed. Our results demonstrate that the DNA dendrimers can serve as designable and safe vehicles for delivery of immune modulators and anticancer drugs.

A Glutamic Acid Analog Bearing an Ethylenediamine Moiety Promotes the Cytosolic Delivery of TAT Peptides

The stepwise protonation of ethylenediamine-containing peptides at low pH in endosomes is hypothesized to disrupt the endosomal membrane and enhance cytosolic delivery. Incorporation of a novel glutamic acid analog bearing an ethylenediamine moiety into TAT peptides markedly enhanced cytosolic delivery without cytotoxicity.

Expression, purification, stability and transduction efficiency of GST-SOD1-R9 fusion protein

The fusion of cell permeable peptide TAT and bifunctional antioxidant enzymes, GST (Glutathione sulfur transferase)-TAT-SOD1 (Cu, Zn superoxide dismutase), is an intracellular superoxide scavenger. Compared with SOD1-TAT, GST-TAT-SOD1 has better protective effect on oxidative damage but less transduction efficiency. A novel cell permeable bifunctional antioxidant enzymes with the fusion of GST, SOD1 and polyarginine R9 was constructed for higher transduction efficiency. The full nucleotide sequence of SOD1-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with the GST tag. After the successful construction of the prokaryotic expression vectors of GST-SOD1-R9, the recombinant vector was then transformed into Escherichia coli BL21 (DE3) and the GST-SOD1-R9 fusion protein was produced with the induction of IPTG. The soluble expression of GST-SOD1-R9 fusion protein was combining with the induction temperature and time. The best soluble expression was obtained with the induction temperature of 25 ℃ and the induction time of 11 h. The fusion protein was purified through the combination of 80% ammonium sulfate precipitation and affinity chromatography using glutathione agarose, and verified by SDS-PAGE and special enzymatic activity. The thermal and pH stability of GST-SOD1-R9 fusion protein were analyzed and the SOD and GST activity of fusion protein were proved to be well maintained under physiological conditions. Finally, the transduction efficiency of GST-SOD1-R9 fusion protein was proved to be better than GST-TAT-SOD1 fusion protein (P<0.05). These works establish a foundation for further study of the protective effect of GST-SOD1-R9 fusion protein against oxidative damage.

Using Tat-Cre to Generate Ex-Vivo Gene Knockouts in T-cells

Abstract We have found unanticipated effects using CD4-driven Cre expression to generate gene knockouts, due to changes in T cells that occur between gene deletion and harvest. To bypass this, we are developing a method to delete genes in T cells just prior to experimentation using brief ex vivo exposure to a Tat-Cre fusion protein, where the TAT peptide drives translocation of Cre to excise loxP-flanked genes. We first defined an optimal concentration and length of treatment by measuring production of a knockout band in DNA extracted from treated T cells. Maximal knockout occurred with 30 minutes of Tat-Cre incubation, while longer treatment times led to no further increase in gene deletion. In contrast, increasing concentrations of Tat-Cre correlated with increasing gene deletion in a dose-dependent manner. 4.6 uM was determined to be the optimal concentration balancing deletion efficacy and resource allocation. We next used T cells from td-Tomato (tdT)/green fluorescent protein (GFP) reporter mice, which express tdT at baseline, but delete tdT and turn on GFP when Cre is present, to determine if DNA deletions were measurable at the protein level. Immediately after Tat-Cre treatment there was no observable increase in GFP expression. However, by 2 hours post-treatment, 34% of treated cells expressed GFP, reaching 65% by 20 hours. We then sorted GFP+ T cells into a mixed leukocyte reaction, where they proliferated appropriately and where approximately 80% of treated T cells remained GFP+ while simultaneously losing tdT expression. Ultimately, this ex vivo method represents a promising approach for generating gene knockouts in T cells just prior to experimental manipulation avoiding changes that occur between deletion and harvest from donor animals.

Screening of commercial cyclic peptide conjugated to HIV-1 Tat peptide as inhibitor of N-terminal heptad repeat glycoprotein-2 ectodomain Ebola virus through in silico analysis

Ebola Hemorrhagic Fever (EHF) is a disease caused by viruses from genus Ebolavirus. Zaire ebolavirus (EBOV) is the deadliest species which has 76% case fatality rate. Up until now, there is no U.S. Food and Drug Administration (FDA) approved drugs to treat EHF. Antiviral drug based on EBOV N-terminal heptad repeat glycoprotein-2 (NHR GP2) Ectodomain inhibitor is one kind of treatment that has not well developed. NHR GP2 Ectodomain has an important role in the process of EBOV entry into the cell through endocytosis mechanism. In this study, we used in silico methods to investigate the activity of commercial cyclic peptide conjugated to Human Immunodeficiency Virus type 1 Trans-activator of the transcription (HIV-1 Tat) peptide as ligands which act as an inhibitor of EBOV NHR GP2 Ectodomain. The commercial cyclic peptides which we used in this study were obtained from the selected chemical companies. Conjugation of the commercial cyclic peptides to HIV-1 Tat peptide was done in order to accumulate it inside the endosome. The ligands which had the best inhibition properties were screened using molecular docking and molecular dynamics simulation. Prediction of pharmacological properties of the peptides was done to choose the best drug candidate. The result of screening processes shows that Ligand 023 has the highest potency as the drug lead. The ligand needs to undergo further analysis through in vitro, in vivo, and a clinical trial to ensure that this ligand has a therapeutic ability as an antiviral drug for Ebola virus infection.