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)

Stem cell technologies hold great potential for the treatment of type 1 diabetes, provided that functional transplantable beta-cells can be selectively generated in an efficient manner. Such a process should recapitulate, at least to a certain extent, the embryonic development of beta-cells in vitro. However, progress at identifying the transcription factors involved in beta-cell development has not been accompanied by a parallel success at unraveling the pattern of their instructive extracellular signals. Here we present proof of principle of a novel approach to circumvent this problem, based on the use of the HIV/TAT protein transduction domain. Neurogenin 3 (ngn3), a factor whose expression is essential for pancreatic endocrine differentiation, was fused to the TAT domain. Administration of TAT/ngn3 to cultured pancreatic explants results in efficient uptake, nuclear translocation, and stimulation of downstream reporter and endogenous genes. Consistent with the predicted activity of the protein, e9.5 and e13.5 mouse pancreatic explants cultured in the presence of TAT/ngn3 show an increased level of endocrine differentiation compared with control samples. Our results raise the possibility of sequentially specifying stem/progenitor cells toward the beta-cell lineage, by using the appropriate sequence and combination of TAT-fused transcription factors.
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PMID:TAT-mediated neurogenin 3 protein transduction stimulates pancreatic endocrine differentiation in vitro. 1573 48

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

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. Deltap85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-Deltap85). Recombinant TAT-Deltap85 inhibited interleukin (IL)-5-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. beta(2)-Integrin-dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-Deltap85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, beta(1)-integrin-mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-Deltap85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-delta inhibitor, also blocked beta(2)-integrin adhesion of eosinophils caused by IL-5, whereas beta(1) adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKCdelta from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKCdelta. Western blot analysis demonstrated that extracellular signal-regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-delta. These data suggest that extracellular signal-regulated kinase-mediated adhesion of beta(2)-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKCdelta pathway.
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PMID:Regulation of interleukin-5-induced beta2-integrin adhesion of human eosinophils by phosphoinositide 3-kinase. 1580 51

The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.
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PMID:Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2. 1584 May 24

Human embryonic stem cells (hESCs) are an in vitro model system for the study of human early development and a potential source for cell-based therapies. An efficient strategy for cellular manipulation of hESCs may be highly valuable for the analysis of gene function involved in human embryogenesis and the development of cell-based therapies via induced differentiation into particular cell types. However, plasmid transfection of hESCs has low efficiency and viral transduction may not be the method of choice for cell-based therapies due to genome integration. To overcome these limitations, we applied protein transduction technology that can transfer proteins into cells via direct penetration across the lipid bilayer. Here, we show that the FITC dye fused to the TAT protein transduction domain (PTD) was efficiently transferred into hESCs. In addition, the PDX1 transcription factor, which plays a central role in pancreatic development, was transferred into hESCs as a fusion form of TAT PTD. The transduced TAT-PDX1 activated its downstream target genes and induced insulin protein production in hESCs. These results demonstrate that protein transduction could be used in the cellular manipulation of hESCs and would provide a significant breakthrough for basic and therapeutic research in hESCs.
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PMID:Cellular manipulation of human embryonic stem cells by TAT-PDX1 protein transduction. 1596 17

Cytotoxic T lymphocytes (CTLs) are the most powerful weapon of the immune system to eliminate cells infected by intracellular parasites or tumors. However, very often, escape mechanisms overcome CTL immune surveillance by impairing the classical HLA class I antigen-processing pathway. Here, we describe a strategy for CTL activation based on the ability of Tat to mediate transcellular delivery of viral proteins encompassing HLA class I-restricted epitopes. In this system, the recombinant protein TAT-NpFlu containing the transduction domain of Tat of human immunodeficiency virus type 1 fused to the amino acid region 301 to 498 of the nucleoprotein of influenza A virus is proven to sensitize different human cells to lysis by HLA-B27-restricted, Flu 383-391-specific CTL lines. The fusion protein is processed very effectively, since a comparable biological effect is obtained with an amount of protein between 1 and 2 orders of magnitude lower than that of the synthetic peptide. Interestingly, while part of TAT-NpFlu undergoes fast and productive cleavage, a large amount of it remains intact for up to 24 h. Confocal microscopy shows that TAT-NpFlu accumulates in the trans-Golgi network (TGN), where it starts to be detectable 1 h after transduction. Using TAT-NpFlu mutants and hybrid constructs, we demonstrate that enrichment in the TGN occurs only when the carboxy-terminal region of NpFlu (amino acids 400 to 498) is present. These data disclose an unconventional route for presentation of epitopes restricted for HLA class I molecules.
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PMID:The C terminus of the nucleoprotein of influenza A virus delivers antigens transduced by Tat to the trans-golgi network and promotes an efficient presentation through HLA class I. 1630 24

The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused beta-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-beta-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material.
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PMID:Cytoplasmic transduction peptide (CTP): new approach for the delivery of biomolecules into cytoplasm in vitro and in vivo. 1646 53

The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit beta1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and beta1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either beta1 integrin or PP2A in a complex at the IGF-IR.
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PMID:Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration. 1670 58

1. Myristoylated pseudosubstrate of PKCzeta (mPS) - a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCzeta pseudosubstrate region -- is considered a selective cell-permeable inhibitor of PKCzeta. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2. mPS at micromolar concentrations (1-10 microM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell-permeable version of PKCzeta pseudosubstrate fused to the HIV-TAT membrane-translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3. In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4. Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5. Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKC should be done with caution, especially in endothelial cells.
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PMID:Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation. 1671 18

Epstein-Barr virus (EBV) infects and transforms resting B lymphocytes in vitro. The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-transplant lymphoproliferative disease causes significant complications in transplant recipients, including loss of the transplanted organ and even death. The limited treatment options include, nonspecific targeting of B cell surface antigens with monoclonal antibodies or withdrawal of immunosuppression. These therapies fail in approximately 50% of patients. Clearly, treatments that specifically target EBV-infected cells are desirable. The EBV antigen EBNA3C regulates cell cycle by targeting critical cellular complexes such as cyclin A/cdk2, SCF(Skp2), and Rb. Here, we use a 20-amino-acid EBNA3C-derived peptide, fused to an HIV TAT tag for efficient delivery, to disrupt cell cycle regulation by EBNA3C. The peptide inhibited hyperproliferation of EBV-infected B cell lines and reduced in vitro immortalization of primary B lymphocytes by EBV. Importantly, the peptide inhibited lymphoblastoid outgrowth from the blood of an EBV-positive transplant patient in vitro.
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PMID:A peptide-based inhibitor for prevention of B cell hyperproliferation induced by Epstein-Barr virus. 1687 48


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