Protein Transduction Domain Fusion Research Articles

Transduced Tat-aldose Reductase Protects Hippocampal Neuronal Cells against Oxidative Stress-induced Damage.

Aldose reductase (AR) protein, a member of the NADPH-dependent aldo-keto reductase family, reduces a wide range of aldehydes and enhances cell survival by inhibition of oxidative stress. Oxidative stress is known as one of the major pathological factor in ischemia. Since the precise function of AR protein in ischemic injury is fully unclear, we examined the function of AR protein in hippocampal neuronal (HT-22) cells and in an animal model of ischemia in this study. Cell permeable Tat-AR protein was produced by fusion of protein transduction domain in Tat for delivery into the cells. Tat-AR protein transduced into HT-22 cells and significantly inhibited cell death and regulated the mitogen-activate protein kinases (MAPKs), Bcl-2, Bax, and Caspase-3 under oxidative stress condition. In an ischemic animal model, Tat-AR protein transduced into the brain tissues through the blood-brain barrier (BBB) and drastically decreased neuronal cell death in hippocampal CA1 region. These results indicate that transduced Tat-AR protein has protective effects against oxidative stress-induced neuronal cell death in vitro and in vivo, suggesting that Tat-AR protein could be used as potential therapeutic agent in ischemic injury.

GFP Transduction Into HeLa Cells Using Atmospheric-Pressure Helium Plasma Jet

An atmospheric-pressure helium plasma jet has been widely used for biological applications; due to the nature, this plasma jet is cold and can be generated in the atmosphere. In this paper, we examined whether the atmospheric-pressure helium plasma jet enables the transfer of proteins without protein transduction domain (PTD) into cells. To examine the effect of plasma exposure on the protein transduction to cells, we observed the His-green fluorescent protein (GFP)-added cells after plasma exposure. A large number of GFP-positive cells were observed in the samples after plasma exposure although few GFP-positive cells were observed in control. These data suggested that plasma exposure induced protein transduction. To examine the major causes of increase in the GFP-positive cells after plasma exposure, we observed the His-GFP-added cells after various stimulations. A denatured medium of cell culture by plasma exposure induced protein transduction as well as plasma exposure sample. These data suggested that plasma exposure induced protein transduction by the denatured medium after plasma exposure. These findings suggest that the plasma jet may become a useful tool for protein transduction to cells in that the construction step of PTD fusion protein is not necessary. Furthermore, the plasma jet enables the proteins to transfer into cells without changing the nature of the protein by unnecessary PTD.

Development of a cell permeable competitive antagonist of RhoA and CRMP4 binding, TAT-C4RIP, to promote neurite outgrowth.

Neurons fail to re-extend their processes within the central nervous system environment in vivo, and this is partly because of inhibitory proteins expressed within myelin debris and reactive astrocytes that actively signal to the injured nerve cells to limit their growth. The ability of the trans-acting activator of transcription (TAT) protein transduction domain (PTD) to transport macromolecules across biological membranes raises the possibility of developing it as a therapeutic delivery tool for nerve regeneration. Most studies have produced TAT PTD fusion protein in bacteria, which can result in problems such as protein solubility, the formation of inclusion bodies and the lack of eukaryotic posttranslational modifications. While some groups have investigated the production of TAT PTD fusion protein in mammalian cells, these strategies are focused on generating TAT PTD fusions that are targeted to the secretory pathway, where furin protease as well as other proteases can cleave the TAT PTD. As an alternative to mutating the furin cleavage site in the TAT PTD, we describe a novel method to generate cytosolic TAT PTD fusion proteins and purify them from cell lysates. Here, we use this method to generate TAT-C4RIP, a cell permeable competitive antagonist of binding between the small GTPase RhoA and the cytosolic phosphoprotein Collapsin response mediator protein 4 (CRMP4). We demonstrate that TAT-C4RIP transduces cells in vitro and in vivo and retains its biological activity to attenuate myelin inhibition in an in vitro neurite outgrowth assay.

Imaging and Targeting of Hypoxic Tumor Cells by Use of HIF-1

Tumor hypoxia is a potential therapeutic problem because it is closely associated with resistance to anti-cancer therapies and with the phenomenon of malignant progression. Therefore, although hypoxic tumor cells account for a very limited area in a solid tumor, conquering tumor hypoxia is crucial for treatment of malignant tumors. To image hypoxic tumor cells in vivo, we isolated a transfectant clone HeLa/5HRE-Luc, whose luciferase activity under hypoxic conditions was more than 100-fold of the one under aerobic conditions, and monitored the luciferase activity in HeLa/5HRE-luc xenografts with an in vivo real-time imaging system. To target tumor hypoxia, we recently constructed a fusion protein POP33, which is composed of the protein transduction domain (PTD), a part of HIF-1 α ODD and the dormant form of caspase-3, procaspase-3. PTD fusion proteins are previously demonstrated to be delivered to every cell in the whole body. POP33 did not affect well-oxygenized cells but efficiently increased caspase-3 activity and induced cell death to hypoxic cells in vitro. To investigate if POP33 can target hypoxic tumor cells in vivo, we monitored the luciferase activity in orthotopically transplanted human pancreatic cancer cells during POP33 treatment. The metastasis of the pancreatic cancer was significantly suppressed and their lucif erase activity was reduced during the sequential administration of POP33. These data demonstrate that POP33 specifically targets tumor hypoxia and provide direct evidence that hypoxic tumor cells play a crucial role in t metastasis of pancreatic cancers.

HIV-1 TAT protein transduction domain mediates enhancement of enzyme prodrug cancer gene therapy in vitro: a study with TAT-TK-GFP triple fusion construct

Protein transduction domain (PTD) from HIV-1 TAT protein has been reported to translocate across the mammalian cell membrane, also as a part of fusion proteins. However, the true nature of TAT-mediated intercellular spreading is still under debate because it has been claimed to be a fixation artifact. To study the spreading of TAT fusion proteins and their potency to enhance thymidine kinase/ ganciclovir (HSV-TK/GCV) cancer gene therapy, we constructed a novel triple fusion protein containing TAT PTD, HSV-TK and green fluorescent protein (TAT-TK-GFP). This fusion protein has three functional domains in the same polypeptide, allowing reliable determination of the relationship between transduction rate and cell killing efficiency. TAT-TK-GFP was cloned into a lentivirus vector and used for analyses of TAT-mediated protein translocation and enhancement of HSV-TK/GCV cytotoxicity. The triple fusion protein was expressed correctly in vitro, but cell-to-cell translocation was not observed in rat glioma cells (BT4C). However, TAT-TK-GFP made BT4C and SKOV3.ip1 (human ovarian carcinoma) cells significantly more sensitive to ganciclovir than TK-GFP, whereas the effect in PC-3 human prostate carcinoma cells was more subtle. It was also observed that growth in lower serum concentration (2.5-5%) abolished the enhancement in BT4C cells, suggesting that high proliferation rate is one of the factors that contribute to TAT PTD-mediated enhancement of cytotoxicity. In summary, our results indicate that TAT PTD fusion proteins do not translocate intercellularly at detectable levels, but enhancement of the HSV-TK/GCV cytotoxicity can be detected in rat and human tumor cell lines in vitro.

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein

It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.