Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells.
ABSTRACT Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H(2)O(2), the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzyme.
Article: Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes.[show abstract] [hide abstract]
ABSTRACT: This study was performed to investigate the transduction of a full-length superoxide dismutase (SOD) protein fused to transactivator of transcription (Tat) into human chondrocytes, and to determine the regulatory function of transduced Tat-SOD in the inflammatory cytokine induced catabolic pathway. The pTat-SOD expression vector was constructed to express the basic domain of HIV-1 Tat as a fusion protein with Cu, Zn-SOD. We also purified histidine-tagged SOD without an HIV-1 Tat and Tat-GFP as control proteins. Cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were cultured in both a monolayer and an explant. For the transduction of fusion proteins, cells/explants were treated with a variety of concentrations of fusion proteins. The transduced protein was detected by fluorescein labeling, western blotting and SOD activity assay. Effects of transduced Tat-SOD on the regulation of IL-1 induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression was assessed by the Griess reaction and reverse transcriptase PCR, respectively. Tat-SOD was successfully delivered into both the monolayer and explant cultured chondrocytes, whereas the control SOD was not. The intracellular transduction of Tat-SOD into cultured chondrocytes was detected after 1 hours, and the amount of transduced protein did not change significantly after further incubation. SOD enzyme activity increased in a dose-dependent manner. NO production and iNOS mRNA expression, in response to IL-1 stimulation, was significantly down-regulated by pretreatment with Tat-SOD fusion proteins. This study shows that protein delivery employing the Tat-protein transduction domain is feasible as a therapeutic modality to regulate catabolic processes in cartilage. Construction of additional Tat-fusion proteins that can regulate cartilage metabolism favorably and application of this technology in in vivo models of arthritis are the subjects of future studies.Arthritis research & therapy 02/2006; 8(4):R96. · 4.27 Impact Factor
Article: The importance of valency in enhancing the import and cell routing potential of protein transduction domain-containing molecules.[show abstract] [hide abstract]
ABSTRACT: Protein transduction domains (PTDs) are peptides that afford the internalization of cargo macromolecules (including plasmid DNA, proteins, liposomes, and nanoparticles). In the case of polycationic peptides, the efficiency of PTDs to promote cellular uptake is directly related to their molecular mass or their polyvalent presentation. Similarly, the efficiency of routing to the nucleus increases with the number of nuclear localization signals (NLS) associated with a cargo. The quantitative enhancement, however, depends on the identity of the PTD sequence as well as the targeted cell type. Thus the choice and multivalent presentation of PTD and NLS sequences are important criteria guiding the design of macromolecules intended for specific intracellular localization. This review outlines synthetic and recombinant strategies whereby PTDs and signal sequences can be assembled into multivalent peptide dendrimers and promote the uptake and routing of their cargoes. In particular, the tetramerization domain of the tumour suppressor p53 (p53tet) is emerging as a useful scaffold to present multiple routing and targeting moieties. Short cationic peptides fused to the 31-residue long p53tet sequence resulted in tetramers displaying a significant enhancement (up to 1000 fold) in terms of their ability to be imported into cells and delivered to the cell nucleus in relation to their monomeric analogues. The design of future polycationic peptide dendrimers as effective delivering vehicles will need to incorporate selective cell targeting functions and provide solutions to the issue of endosomal entrapment.Biochimica et Biophysica Acta 04/2006; 1758(3):355-63. · 4.66 Impact Factor
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ABSTRACT: A secreted peptide Pep27 initiates the cell death program in S. pneumoniae through signal transduction. This study was undertaken to evaluate the relation between the structure and cytotoxic activity of Pep27 and its analogues on cancer cells. Pep27anal2 characterized substituting (2R-->W), (4E-->W), (11S-->W) and (13Q-->W) in native Pep27, exhibited greater hydrophobicity and anticancer activity than Pep27 and other analogues. The IC50 values of Pep27anal2 were approximately 10 - 30 microM in a number of cell lines (AML-2, HL-60, Jurkat, MCF-7 and SNU-601). Confocal microscopy showed that Pep27anal2-FITC was localized in the plasma membrane, and then moving from the membrane to subcellular compartments with the initiation of membrane blebbing. Flow cytometric analysis using propidium iodide and Annexin V also revealed that Pep27anal2 induced apoptosis with minor membrane damage. Electron microscopy revealed that Pep27 induced apoptosis in Jurkat cells. The anticancer activity of Pep27anal2 was neither abrogated by pan-caspase inhibitor (Z-VAD-fmk) nor related to cytochrome c release from mitochondria. The 3D solution structures of these two Pep27 peptides revealed that both form a random coil conformation in water; however, they adopted stable alpha-helical conformations in solutions. The results indicate that Pep27anal2 can penetrate the plasma membrane, and then induce apoptosis in both caspase-and cytochrome c-independent manner. The hydrophobicity of Pep27anal2 appears to play an important role in membrane permeabilization and/or anticancer properties. The structure-functional relationships of these peptides are also discussed. It is proposed that Pep27anal2 is a potential candidate for anticancer therapeutic agents.Cancer Cell International 08/2005; 5:21. · 1.97 Impact Factor