RSSEngineering and identifying supercharged proteins for macromolecule delivery into Mammalian cells.
Abstract
Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells.
Copyright © 2012 Elsevier Inc. All rights reserved.
- PMID:
- 22230574
- [PubMed - in process]
A rapid generation of adenovirus vector with a genetic modification in hexon protein.
Source
Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an 710062, PR China.
Abstract
The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of theTat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy.
Copyright © 2011. Published by Elsevier B.V.
Efficient Intracellular Delivery of Nucleic Acid Pharmaceuticals Using Cell-Penetrating Peptides.
Source
Institute for Chemical Research, Kyoto University , Uji, Kyoto 611-0011, Japan.
Abstract
Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic acid pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPs. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core-shell nanoparticle with NAPs, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates "programmed packaging" to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.
Revisiting G3BP1 as a RasGAP Binding Protein: Sensitization of Tumor Cells to Chemotherapy by the RasGAP 317-326 Sequence Does Not Involve G3BP1.
Source
Department of Physiology, University of Lausanne, Lausanne, Switzerland.
Abstract
RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317-326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptidecontaining the 317-326 sequence of RasGAP (TAT-RasGAP(317-326)), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP(317-326) did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP(317-326) was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.
Protein targeting constructs in alpha therapy.
Source
Crump Institute for Molecular Imaging, Dept. of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; tolafsen@mednet.ucla.edu.
Abstract
The progress in the field of targeted α-particle therapy (TAT) has to a great extent been enhanced by developments in both recombinant DNA technology and radionuclide labeling chemistry. Advances in genomics and proteomics have promoted an increase in the identification of novel targets and molecules that can define different diseases, such as cancer. In radioimmunotherapy (RIT), the primary goal is to improve delivery to and therapeutic efficacy of the cancer cells, whilst minimizing toxicity. Different approaches have been investigated to achieve this, such as reducing the size of the carrier, pretargeting, multidosing, locoregional administration and using a cocktail of radiolabeled monoclonal antibodies for targeting multiple antigens simultaneously. Some of these approaches have been encouraging, but translation of TAT into the clinic has been slow, in part because of the limited availability and the short physical half-lives of some of the available α-particle emitters. The clinical studies carried out to date have been promising, although many challenges remain in order to make TAT safe and economically feasible. In this paper a number of different targeting constructs used hitherto that may be promising carriers for TAT in the future are presented and discussed. The constructs include enzymatic cleaved antibody fragments (Fab and F(ab˙)2 fragments); genetically engineered antibody fragments (scFv monomer, dimer (i.e. diabody) and tetramer, CH2 domain deleted antibody fragments); other protein targeting constructs such as affibodies and peptides as well as liposomal delivery.
- PMID:
- 22201709
- [PubMed - in process]
P38 MAPK is involved in enhanced NMDA receptor-dependent excitotoxicity in YAC transgenic mouse model of Huntington disease.
Source
Graduate Program in Neuroscience, University of British Columbia, Vancouver, BC, Canada; Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada; Brain Research Centre, University of British Columbia, Vancouver, BC, Canada.
Abstract
Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Previous studies have shown enhanced N-methyl-d-aspartate (NMDA)-induced excitotoxicity in neuronal models of HD, mediated in part by increased NMDA receptor (NMDAR) GluN2B subunit binding with the postsynaptic density protein-95 (PSD-95). In cultured hippocampal neurons, the NMDAR-activated p38 Mitogen-activated Protein Kinase (MAPK) death pathway is disrupted by a peptide (Tat-NR2B9c) that uncouples GluN2B from PSD-95, whereas NMDAR-mediated activation of c-Jun N-terminal Kinase (JNK) MAPK is PSD-95-independent. To investigate the mechanism by which Tat-NR2B9c protects striatal medium spiny neurons (MSNs) from mutant htt (mhtt)-enhanced NMDAR toxicity, we compared striatal tissue and cultured MSNs from presymptomatic yeast artificial chromosome (YAC) mice expressing htt with 128 polyQ (YAC128) to those from YAC18 and/or WT mice as controls. Similar to the previously published shift of GluN2B-containing NMDARs to extrasynaptic sites, we found increased PSD-95 localization as well as elevated PSD-95-GluN2B interactions in the striatal non-PSD (extrasynaptic) fraction from YAC128 mice. Notably, basal levels of both activated p38 and JNK MAPKs were elevated in the YAC128 striatum. NMDA stimulation of acute slices increased activation of p38 and JNK in WT and YAC128 striatum, but Tat-NR2B9c pretreatment reduced only the p38 activation in YAC128. In cultured MSNs, p38 MAPK inhibition reduced YAC128 NMDAR-mediated cell death to WT levels, and occluded the Tat-NR2B9c peptide protective effect; in contrast, inhibition of JNK had a similar protective effect in cultured MSNs from both WT and YAC128 mice. Our results suggest that altered activation of p38 MAPK contributes to mhtt enhancement of GluN2B/PSD-95 toxic signaling.
Copyright © 2011. Published by Elsevier Inc.
Engineering Antibody Fitness and Function Using Membrane-Anchored Display of Correctly Folded Proteins.
Source
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Abstract
A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Ras-related GTPases, Rap1 and RhoA collectively induce the phagocytosis of serum-opsonized zymosan particles in macrophages.
Source
Hallym University, Korea, Republic of.
Abstract
Phagocytosis primarily occurs through two main processes in macrophages: the Fcγ receptor- and the integrin αMβ2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin αMβ2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA), suggesting that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Si-profilin suppressed the phagocytosis, suggesting profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTPγS rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.
Cellular uptake of an α-AApeptide.
Source
Department of Chemistry, University of South Florida, 4202 E. Fowler Ave, Tampa, FL 33620, USA. Jianfengcai@usf.edu.
Abstract
Some short and cationic peptides such as the Tat peptide can cross the cell membrane and function as vectors for intracellular delivery. Here we show that an α-AApeptide is able to penetrate the membranes of living cells from an extracellular environment and enter the endosome and cytoplasm of cells. The efficiency of the cellular uptake is comparable to a Tat peptide (48-57) of the same length and is unexpectedly superior to an α-peptide with identical functional groups. The mechanism of uptake is similar to that of the Tat peptide and is through endocytosis by an energy-dependent pathway. Due to the easy synthesis of the α-AApeptides, their resistance to proteolytic hydrolysis, and their low cytotoxicity, α-AApeptides represent a new class of transporters for the delivery of drugs.
Targeting human epidermal growth factor receptor 2 by a cell-penetratingpeptide-affibody bioconjugate.
Source
Centre for Cellular and Molecular Biology, Council for Scientific and Industrial Research, Uppal Road, Hyderabad 500007, Andhra Pradesh, India.
Abstract
Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide,TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVβ-gal, resulted in enhanced β-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of β-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Molecular partners for interaction and cell internalization of cell-penetratingpeptides: how identical are they?
Source
UPMC Univ Paris 06, UMR 7203, LBM, 75005 Paris, France.
Abstract
Cell-penetrating peptides are short basic peptide sequences that might display amphipathic properties. These positively charged peptides internalize into all cell types, albeit with different efficiency. Cell-penetrating peptides use all routes of pinocytosis to internalize, in addition to direct membrane translocation that requires interaction with lipid membrane domains. These differences in internalization efficiency according to the peptide sequence and cell type suggest that the cell-penetrating peptides interact with different molecular partners at the cell surface. This review will first report on data that describe the molecular interaction of the most popular cell-penetrating peptides (penetratin, Tat and oligoarginine) with carbohydrates and lipids. The second part of the review will be dedicated to cell studies that have reported how cell surface composition influences cell internalization. Discussion will focus on the gap between in vitro and in cellulo studies, and more specifically to which extent the interaction with molecules found in membranes reflect the internalization efficiency of the peptides.
Effect of silk fibroin peptide derived from silkworm Bombyx mori on the anti-inflammatory effect of Tat-SOD in a mice edema model.
Source
Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea sychoi@hallym.ac.kr.
Abstract
We investigated whether silk fibroin peptide derived from the silkworm, Bombyx mori, could inhibit inflammation and enhance the anti-inflammatory activity of Tat-superoxide dismutase (Tat-SOD ), which was previously reported to effectively penetrate various cells and tissues and exert anti-oxidative activity in a mouse model of inflammation. Inflammation was induced by topical treatment of mouse ears with 12-O-tetradecanoylphorbol- 13-acetate (TPA ). Histological, Western blot, and reverse transcription-polymerase chain reaction data demonstrated that silk fibroinpeptide or Tat-SOD alone could suppress elevated levels of cyclooxygenase-2, interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha induced by TPA. Moreover, silk fibroin peptide significantly enhanced the anti-inflammatory activity of Tat-SOD, although it had no influence on in vitro and in vivo transduction of Tat-SOD. Silk fibroin peptideexhibited anti- inflammatory activity in a mice model of inflammation. Therefore, silk fibroin peptide alone or in combination with Tat-SOD might be used as a therapeutic agent for various inflammatory diseases. [BMB reports 2011; 44(12): 787-792].
Effects of the TAT peptide orientation and relative location on the protein transduction efficiency.
Source
Department of Biochemistry and Molecular Biology, China Medical University Key Laboratory of Medical Cell Biology, Ministry of Education Shenyang, China, 110001.
Abstract
To understand the protein transduction domain (PTD)-mediated protein transduction behavior and to explore its potential in deliverying biopharmaceutic drugs, we prepared four TAT-EGFP conjugates : TAT(+)-EGFP, TAT(-)-EGFP, EGFP-TAT(+) and EGFP-TAT(-), where TAT(+) and TAT(-) represent the original and the reversed TAT sequence, respectively. These four TAT-EGFP conjugates were incubated with HeLa and PC12 cells for in vitro study as well as injected intraperitoneally to mice for in vivo study. Flow cytometric results showed that four TAT-EGFP conjugates were able to traverse HeLa and PC12 cells with almost equal transduction efficiency. The in vivo study showed that the TAT-EGFP conjugates could be delivered into different organs of mice with different transduction capabilities. Bioinformatic analyses and CD spectroscopic data revealed that the TAT peptide has no defined secondary structure and conjugating the TAT peptide to the EGFP cargo protein would not alter the native structure and the function of the EGFP protein. These results conclude that the sequence orientation, the spatial structure, and the relative location of the TAT peptidehave much less effect on the TAT-mediated protein transduction. Thus, the TAT-fused conjugates could be constructed in more convenient and flexible formats for a wide range of biopharmaceutical applications. © 2011 John Wiley & Sons A/S.
© 2011 John Wiley & Sons A/S.
Comparison of four different peptides to enhance accumulation of liposomes into the brain.
Source
Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University , Chengdu, P.R . China.
Abstract
The cell penetrating peptide TAT, which appears to enter cells with alacrity, can pass through the BBB efficiently. It has been indentified to enhance the brain delivery of the liposome. However, little was known about its mechanism. TATcontains a basic region consisting of six arginine and two lysine residues. These eight basic amino acids seem to be the key to its highly efficient membrane translocation and brain delivery. In this study, four selected peptides are synthesized. (1) TAT peptide with terminal Cysteine (Cys-AYGRKKRRQRRR). (2) TAT peptide with disordered sequence (Cys-RKARYRGRKRQR). (3) Glycine and glutamic acid substituted TAT peptide (Cys-AYGGQQGGQGGG). (4) R8 (Cys-RRRRRRRR). Liposomes were chosen as the delivery vehicle. The peptide was covalently bonded with the liposome. We compare four peptides for their brain targeting potential, and investigate their ability to target liposomes to the brain in vitro and in vivo. The cellular uptake of these four liposomes by brain capillary endothelial cells (BCECs) of rats and C6s and the mechanism of the pathway of endocytosis were explored. Biodistribution in vivo was also investigated qualitatively and quantitatively. The results showed that the charge of the peptide played an important role in enhancing its brain delivery. The sequence had little to do with its membrane translocation and brain delivery indicated there might be no specific receptor or transporter for the Tat peptide.
Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity.
Abstract
pH-sensitive PEGylated (with PEG-PE) long-circulating liposomes (HSPC:cholesterol and Doxil®), modified with cell-penetrating TAT peptide (TATp) moieties and cancer-specific mAb 2C5 were prepared. A degradable pH-sensitive hydrazone bond between a long shielding PEG chains and PE (PEG(2k)-Hz-PE) was introduced. TATp was conjugated with a short PEG(1k)-PE spacer and mAb 2C5 was attached to a long PEG chain (2C5-PEG(3.4k)-PE). The "shielding" effect of TATp by long PEG chains was investigated using three liposomal models. At normal pH, surface TATp moieties are "hidden" by the long PEG chains. Upon the exposure to lowered pH, this multifunctional carrier exposes TATp moieties after the degradation of the hydrazone bond and removal of the long PEG chains. Enhanced cellular uptake of the TATp-containing immunoliposomes was observed in vitro after pre-treatment at lowered pH (using flow cytometry and fluorescence microscopy techniques). The presence of mAb 2C5 on the liposome surface further enhanced the interaction between the carrier and tumor cells but not normal cells. Furthermore, multifunctional immuno-Doxil® preparation showed increased cellular cytotoxicity of B16-F10, HeLa and MCF-7 cells when pre-incubated at lower pH, indicating TATp exposure and activity. In conclusion, a multifunctional immunoliposomal nanocarrier containing a pH-sensitive PEG-PE component, TATp, and the cancer cell-specific mAb 2C5 promotes enhanced cytotoxicity and carrier internalization by cancer cells and demonstrates the potential for intracellular drug delivery after exposure to lowered pH environment, typical of solid tumors.
Copyright © 2011 Elsevier B.V. All rights reserved.
A novel self-assembled nanoparticle vaccine with HIV-1 Tat(49-57)/HPV16 E7(49-57) fusion peptide and GM-CSF DNA elicits potent and prolonged CD8(+) T cell-dependent anti-tumor immunity in mice.
Source
Department of Dermatology, Southwestern Hospital, Third Military Medical University, Chongqing 400038, China; Department of Dermatology, the 105(th) Hospital of PLA, Hefei 230001, China.
Abstract
Peptide-based vaccines derived from the E7 protein of human papillomavirus (HPV) type 16 were developed to induce effective T cell responses against established cervical cancer, but have met with limited clinical success. It is necessary to develop novel peptide-based strategies to substantially improve the immune response against HPV16-related cancer. In this study, we aimed to design a novel peptide-based self-assembled nanoparticle HPV16 vaccine by combining the cell-penetrating peptide HIV-1 Tat(49-57) that was fused with the HPV16 E7(49-57) cytotoxic T lymphocyte (CTL) epitope and the granulocyte-macrophage colony stimulating factor (GM-CSF) gene, and to investigate how it improves the immune response and the therapeutic outcome ex vivo and in vivo. Nanoparticles were prepared and identified by transmission electron microscopy (TEM), gel retardation and DNase I protection assays. This type of vaccine formulation formed the 20-80nm nanoparticles, and greatly improved epitope-specific immunity both ex vivo and in vivo. Importantly, this vaccine type was associated with decreased tumor growth and enhanced long-term survival in the prophylactic and therapeutic mouse models. The underlying mechanisms were determined to involve priming of enhanced frequency of CD8(+) memory T subtype cells. These results suggest that the nanoparticle Tat-E7/pGM-CSF represents a promising novel approach to enhance the potency of peptide-based cervical cancer vaccines, and this vaccine design strategy may act as a useful reference for research of virus-associated diseases and specific tumor immunotherapies.
Copyright © 2011 Elsevier Ltd. All rights reserved.
A periplasmic arsenite-binding protein involved in regulating arsenite oxidation.
Source
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaDepartments of Land Resources and Environmental Sciences Immunology and Infectious Disease Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA Department of Soil, Water, and Environmental Science, The University of Arizona, Tucson, AZ 85721, USA.
Abstract
Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund List of Hazardous Substances. Microbial redox transformations are the principal drivers of As chemical speciation, which in turn dictates As mobility and toxicity. Consequently, in order to manage or remediate environmental As, land managers need to understand how and why microorganisms react to As. Studies have demonstrated a two-component signal transduction system comprised of AioS (sensor kinase) and AioR (response regulator) is involved in regulating microbial AsIII oxidation, with the AsIII oxidase structural genes aioB and aioA being upregulated by AsIII. However, it is not known whether AsIII is first detected directly by AioS or by an intermediate. Herein we demonstrate the essential role of a periplasmic AsIII-binding protein encoded by aioX, which is upregulated by AsIII. An ΔaioX mutant is defective for upregulation of the aioBA genes and consequently AsIII oxidation. Purified AioX expressed without its TAT-type signalpeptide behaves as a monomer (MW 32 kDa), and Western blots show AioX to be exclusively associated with the cytoplasmic membrane. AioX binds AsIII with a K(D) of 2.4 µM AsIII; however, mutating a conserved Cys108 to either alanine or serine resulted in lack of AsIII binding, lack of aioBA induction, and correlated with a negative AsIII oxidation phenotype. The discovery and characterization of AioX illustrates a novel AsIII sensing mechanism that appears to be used in a range of bacteria and also provides one of the first examples of a bacterial signal anchor protein.
© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.
Synergistic effects of conjugating cell penetrating peptides and thiomers on non-viral transfection efficiency.
Source
Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck, Innrain 52, Josef Möller Haus, 6020 Innsbruck, Austria.
Abstract
Nanoparticles generated by complex coacervation of plasmid DNA (pDNA) and modified chitosans namely chitosan-thioglycolic acid (TGA) conjugate and chitosan-HIV-1 Tat peptide conjugate were evaluated as gene delivery systems. In order to optimize transfection efficiency, chitosan-HIV-1 Tat peptide conjugate was combined with chitosan-TGA before its complexation with pDNA. Particle size and zeta potential measurements were performed to characterize the generated nanoparticles. The nanoparticles transfection efficiencies were assessed by exploitation of the green fluorescent protein (GFP) reporter gene. HEK293 cells were incubated for 24 h with the nanoparticles and the GFP positive cells were observed by fluorescence microscopy. The nanoparticles in the size range of 200-300 nm could transfect HEK293 cells as a model cell line with different transfection efficiencies. Unlike chitosan-TGA, chitosan-HIV-1Tat peptide led to increased zeta potential of nanoparticles as compared to unmodified chitosan. The transfection efficiency of the nanoparticles generated by combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was comparatively higher than that of the nanoparticles generated by either chitosan-TGA or the combination of chitosan-HIV-1 Tat peptide with unmodified chitosan. After 72 h of incubation, the combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was found to be 7.12- and 67.37 times more efficient than unmodified chitosan and pDNA alone, respectively and showed a synergistic effect in transfection of pDNA into the cells. Moreover, none of the nanoparticles showed any severe cytotoxicity. Accordingly, this strategy might result in a potent carrier for gene delivery.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Enhanced Nuclear Import and Transfection Efficiency of TAT Peptide-Based Gene Delivery Systems Modified by Additional Nuclear Localization Signals.
Source
Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University , Wuhan 430072, P. R. China.
Abstract
Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs toTAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, andTAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.
Enantiomer-Specific Bioactivities of Peptidomimetic Analogues of Mastoparan and Mitoparan: Characterization of Inverso Mastoparan as a Highly Efficient Cell Penetrating Peptide.
Source
Research Institute in Healthcare Science, School of Applied Sciences, University of Wolverhampton , Wulfruna Street, Wolverhampton, WV1 1LY, United Kingdom.
Abstract
Retro-inverso transformation has commonly been employed as a strategy both for the synthesis of proteolytically stablepeptide analogues and for the detailed investigation of structure activity relationships. Herein, we adopted a similar strategy to probe the structure activity relationships of two biologically active tetradecapeptides. Analogues of the α-helical mastoparan, and the highly potent apoptogenic analogue mitoparan, were synthesized using d-amino acids assembled in both endogenous (inverso) and reverse (retro-inverso) orientations. For a more comprehensive comparison, our studies also included the retro l-enantiomer of both peptides. Contrary to expectation, comparative investigations of cytotoxicity, mast cell degranulation, and cellular penetration demonstrated that, while retro-inverso transformation abrogated the associated biological activities of these helical peptides, inverso homologues retained their bioactivities. Moreover, inverso mastoparan demonstrated the highest translocation efficacy of all analogues with much improved uptake kinetics compared to other cell penetrating peptides (CPPs) including the commonly employed inert vectors penetratin and tat. Data presented herein thus propound the utility of inverso mastoparan as a highly efficient peptidevector. Furthermore, correlation analysis of plasma membrane translocation and intracellular uptake efficacy further supports a two-compartment model of CPP import whereby the intracellular accumulation of polycationic peptides is dependent upon both the efficiency of transport into the cell and their subsequent accretion at distinct subcellular loci.
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