Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E. coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.
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PMID:[The PTD domain of Tat protein enhance GFP protein delivering into myeloma cell SP2/0]. 1256 Dec 18

Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (TAT PTD) into proteins facilitates rapid, efficient entry into cells. We previously showed that rTAT-PTD-OVA-transduced dendritic cells (DC) stimulated antigen (Ag)-specific CTL and Th cells, and vaccinated against OVA-expressing tumors. Here we studied B16 melanoma in C57BL/6 mice, using murine tyrosinase-related protein 2 (Trp2) as a candidate tumor Ag. We produced a 472-amino acid N-terminal fragment of Trp2 protein (rTrp2Delta) with or without PTD. Although PTD-deficient rTrp2Delta was ineffective, mice given rTAT-PTD-Trp2Delta-transduced DC were efficiently primed for Trp2(180-188) peptide-specific and B16-reactive CTL. In 58% of such mice, growth of melanomas was prevented. Trp2(180-188) peptide-pulsed DC protected 35% of recipients, and irradiated GM-CSF-producing B16 cells protected 75%. rTAT-PTD-Trp2Delta-transduced DC induced a more vigorous memory response to B16 rechallenge than the other regimens, and protected 30% of recipients from progressive tumor development in treatment studies. In this setting, Trp2 peptide-treated DC protected 20% and irradiated GM-CSF-producing cells protected 0%. Both tumor prevention and tumor treatment were CD8(+) T cell dependent. Vaccination with rTAT-PTD-Trp2Delta-transduced DC induced a robust CTL response and durable anti-melanoma immunity. This approach should be clinically applicable, and offers theoretical and practical advantages to those that are in current use.
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PMID:Dendritic cells transduced with TAT protein transduction domain-containing tyrosinase-related protein 2 vaccinate against murine melanoma. 1267 50

Transplantation of islets is becoming an established method for treating type 1 diabetes. However, viability of islets is greatly affected by necrosis/apoptosis induced by oxidative stress and other insults during isolation and subsequent in vitro culture. Expression of cytoprotective proteins, such as heme oxygenase-1 (HO-1), reduces the deleterious effects of oxidative stress in transplantable islets. We have generated a fusion protein composed of HO-1 and TAT protein transduction domain (TAT/PTD), an 11-aa cell penetrating peptide from the human immunodeficiency virus TAT protein. Transduction of TAT/PTD-HO-1 to insulin-producing cells protects against TNF-alpha-mediated cytotoxicity. TAT/PTD-HO-1 transduction to islets does not impair islet physiology, as assessed by reversion of chemically induced diabetes in immunodeficient mice. Finally, we report that transduction of HO-1 fusion protein into islets improves islet viability in culture. This approach might have a positive impact on the availability of islets for transplantation.
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PMID:Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic beta-cells. 1276 12

Four discrete mechanisms for the pathogenesis of PTD have been described but they share a final common pathway. Moreover, although the mechanisms have distinct clinical characteristics, they are not mutually exclusive. As an example, triplet gestations are more likely to be associated with periconceptional intrauterine manipulations predisposing to infection, as well as fetal growth restriction, decidual hemorrhage, and pathologic uterine distention. An improved understanding of these pathologic pathways has led to the development of new tests to predict PTD. Use of multiple markers (eg, serum CRH, salivary E3, cervical IL-6, TAT, and fFN) holds promise for implementing targeted interventions to prevent PTD.
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PMID:Testing for risk of preterm delivery. 1284 48

The protein transduction system has been employed for delivery of bioactive proteins into cells via an endocytotic mechanism. However, trapping of endocytosed proteins in the endosome may significantly attenuate biological actions in cells. The present investigation demonstrated that endosomal release of transduced protein could be artificially accelerated by exposure to fluorescent light. Exposure to light at 480 nm stimulated endosomal release of transduced FITC-11 arginine-protein transduction domain (11R-PTD), TAT-PTD and Antennapedia-PTD. Moreover, FITC-11R-p53 protein was released from endosomes following stimulation with light. These data suggest that photo-acceleration is a more efficient strategy in terms of the protein transduction system.
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PMID:Photo-acceleration of protein release from endosome in the protein transduction system. 1530 52

Viability of isolated islets is one of the main obstacles limiting islet transplantation success. It has been reported that overexpression of Bcl-2/Bcl-XL proteins enhances islet viability. To avoid potential complications associated with long-term expression of anti-apoptotic proteins, we investigated the possibility of delivering Bcl-XL or its anti-apoptotic domain BH4 to islets by protein transduction. Bcl-XL and BH4 molecules were fused to TAT/PTD, the 11-aa cell penetrating peptide from HIV-1 transactivating protein, generating TAT-Bcl-XL and TAT-BH4, respectively. Transduction efficiency was assessed by laser scanning confocal microscopy of live islets. Biological activity was tested as the ability to protect NIT-1 insulinoma cell line from death induced by staurosporine or serum deprivation. Spontaneous caspase activation in human islets and cytotoxicity caused by IL-1beta were significantly reduced in the presence of TAT-Bcl-XL and TAT-BH4. We conclude that both TAT proteins are biologically active after transduction and could be an asset in the improvement of islet viability.
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PMID:Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets. 1536 75

Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPP(TAT(PTD))] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPP(TAT(PTD)) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPP(TAT(PTD)) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPP(TAT(PTD)) uptake and are detected only on fibroblasts showing Fg-CPP(TAT(PTD)) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPP(TAT(PTD)) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.
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PMID:The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence. 1562 54

For a long time, as the most prominent subnuclear structure, nucleolus has been recognized as a main site where rRNA processing and ribosomal subunit assemblies take place. It has not been until recently that additional functions of nucleolus have begun to be proposed. In this study, we for the first time demonstrate that Survivin-deltaEx3, a novel functionally splice variant of Survivin localizes in the nucleoli where it degrades rapidly through ubiquitin-proteosome pathway. Several lines of evidences provided in this report support this finding (i) a novel nucleolar localization sequence (NoLS, MQRKPTIRRKNLRLRRK) and a novel degradation signal (aa92-aa137) within Survivin-deltaEx3 were identified (ii) proteasome inhibitors MG132 or ALLN greatly inhibits degradation of Survivin-deltaEx3 and polyubiquitination of Survivin-deltaEx3 was detected (iii) heterologous proteins such as TAT-PTD or p14ARF, when fused to this putative degradation signal, result in a significant degradation within the nucleolus. In addition, the nucleolar localization and degradation of Survivin-deltaEx3 appear to be required for its antiapoptotic function, since neither NoLS-deleted nor degradation signal-deleted Survivin-deltaEx3 retains protective effect against Doxorubicin-induced apoptosis. Thus, our results have provided evidences to suggest that besides cytosol, nucleus, endoplsmic reticulum (ER) or lysosomes, nucleolus may also operate important protein degradation pathway, which has been overlooked previously.
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PMID:Identification of a novel nucleolar localization signal and a degradation signal in Survivin-deltaEx3: a potential link between nucleolus and protein degradation. 1573 64

Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized protein transduction. Protein transduction is achieved by fusing Ngb to the TAT/PTD transduction domain, a peptide originated from the HIV transcriptional transactivator protein. Our study proved that TAT-Ngb is an efficient fusion protein capable of protecting the human islets in culture from loss of cell mass and function, thus increasing the quality of transplantable islets. If the islets could be cultured for a longer period of time without suffering harmful effects, it would be possible to precondition the recipient and there would be more time to assess their quality and function before transplantation.
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PMID:Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction. 1580 6

Delivering cytoprotective proteins/peptides into pancreata prior to islet isolation through protein transduction (PT) is a novel strategy to enhance the yield of viable transplantable islets. Previous work has shown that the protein transduction domain PTD-5 efficiently transduced islets via the pancreatic duct. TAT/PTD is a well-characterized PTD with the capability to cross even the hemato-encephalic barrier. In this study, we investigated the utilization of the 11-aa TAT protein transduction domain (TAT/PTD) to deliver peptides or proteins of different sizes ranging from 1.2 to 120 kDa, as the TAT/PTD and TAT/PTD-BH4 peptide, or the TAT/PTD-beta-galactosidase fusion protein, into islets through the pancreatic duct. Using flow cytometry analysis we found that TAT/PTD derivatives transduced practically 100% of the islet cell population. Moreover, confocal laser scanning microscopy in live, nonfixed islets confirmed these results assessing transduction of TAT/PTD molecules into intact nondisaggregated islets. TAT-beta-galactosidase peptide conjugated to FITC was not compartment selective, as both cytoplasmic and nucleic cellular compartments were positively stained. Furthermore, TAT-beta-galactosidase peptide delivery was highly effective, as even cells located in the inner core region of the islets were transduced. Finally, transduced TAT-beta-galactosidase fusion protein was biologically active after islet isolation and manipulation, and islet insulin secretion capability was not compromised by peptide transduction. These findings suggest that the transduction of chimeric TAT/PTD proteins can represent an efficient tool of molecular delivery independent of the size, to enhance or modify a specific phenotype at the nuclei or cytoplasmic level.
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PMID:Delivery of TAT/PTD-fused proteins/peptides to islets via pancreatic duct. 1605 6


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