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)

Although tightly regulated programmed cell death (apoptosis) possesses great importance for tissue homeostasis, several pathologic processes are associated with organ failure due to adversely activated cell apoptosis. Transient increase in apoptosis has been shown to cause organ damage during fulminant hepatitis B, autoimmune diseases, ischemia-reperfusion injury, sepsis, or allograft rejection. A defined and temporary inhibition of cell apoptosis may therefore be of high clinical relevance. Activation of death receptors results in caspase-8 recruitment to the death-inducing signaling complex, which initiates the apoptotic process through cleavage of caspase-8 and downstream substrates. This initial step may be inhibited by the caspase-8 inhibitor FLIP (FLICE inhibitory protein). To specifically inhibit the initiation of death receptor-mediated apoptosis we constructed a fusion protein containing FLIP fused N-terminally to the human immunodeficiency virus TAT domain. This TAT domain allows the fusion protein to cross the cell membrane and thus makes the FLIP domain able to interfere with the death-inducing signaling complex inside of the cell. We observed that incubation of lymphocytic Jurkat or BJAB cells with TAT-FLIPS proteins significantly inhibits Fas-induced activation of procaspase-8 and downstream caspases, preventing cells from undergoing apoptosis. Systemic application of TAT-FLIPS prolongs survival and reduces multi-organ failure due to Fas-receptor-mediated lethal apoptosis in mice. Therefore, application of cellular FLIPS in the form of a TAT fusion protein may open a promising, easily applicable new tool for providing protection against transient, pathologically increased apoptosis in various diseases.
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PMID:Transduction of the TAT-FLIP fusion protein results in transient resistance to Fas-induced apoptosis in vivo. 1530 99

Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides.
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PMID:A ubiquitin-based assay for the cytosolic uptake of protein transduction domains. 1566 32

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

Induction of heat shock protein 70 (Hsp70) via sublethal stress protects neurons from subsequent lethal injuries. Here we show that specific and efficient intracellular transduction of Hsp70 can be achieved utilizing an 11 amino acid leading sequence from human immunodeficiency virus (TAT-Hsp70) in primary neuronal cultures. Western blot and immunohistochemistry demonstrated intracellular accumulation of Hsp70 in insoluble protein fractions and mitochondrial compartments. We then examined the effects of Hsp70 overexpression using TAT-Hsp70 in models of nitrosative and excitotoxic neuronal death in vitro. Neurons were pre-incubated with 300 nM TAT-Hsp 70 overnight, then exposed to either peroxynitrite (ONOO-) or glutamate. TAT-Hsp70 maintained cellular respiration, inhibited extracellular lactate dehydrogenase release, and/or reduced cell death assessed by flow cytometry vs. vehicle, wild-type Hsp70, and TAT-beta-galactosidase controls. Hsp70 transduction using a TAT fusion protein is an effective method to selectively increase Hsp70 in neurons and is sufficient to provide neuroprotection from nitrosative stress and excitotoxicity. Further study is needed to confirm whether TAT-Hsp70 is protective in in vivo models of brain injury.
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PMID:Selectively increasing inducible heat shock protein 70 via TAT-protein transduction protects neurons from nitrosative stress and excitotoxicity. 1599 87

Certain viruses can infect neurons and cause persistent infections with restricted expression of viral proteins. To study the consequences of such viral proteins on synaptic functions, the effects of two influenza A virus proteins, the nonstructural protein 1 (NS1) and the nucleoprotein (NP), were analyzed in cultures of rat hippocampal neurons. Transduction of the NS1 and NP proteins into the neurons was performed by applying the 11-amino acid peptide transduction domain (PTD) of human immunodeficiency virus (HIV) TAT coupled to the viral proteins. Neurons exposed to the NS1 and NP fusion proteins (NS1-PTD and NP-PTD, respectively) for 4 h were immunopositive for these proteins as diffuse cytoplasmic and nuclear distribution. After exposure for 48 h to NP-PTD, a punctate pattern of the immunolabel appeared in dendritic spinelike processes. Electrophysiologically, a reduction in both the frequency of spontaneous excitatory synaptic activity and in the amplitude of the miniature excitatory postsynaptic currents were recorded after exposing the hippocampal neurons to NP-PTD between 17 and 22 days in culture. These changes may reflect disturbances in postsynaptic functions. No such alterations in synaptic activities were recorded after exposure to NS1-PTD or to green fluorescent protein-PTD, which was used as a control. Based on these findings the authors hypothesize that the viral NP, by its localization to dendritic spinelike structures, interferes with the expression or anchoring of postsynaptic glutamate receptors and thereby disturbs synaptic functions. Thus a persistent viral infection in the brain may be associated with functional disturbances at the synaptic level.
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PMID:Effects on synaptic activity in cultured hippocampal neurons by influenza A viral proteins. 1616 82

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

Protein transduction domains (PTDs) offer an exciting therapeutic opportunity for the treatment of many diseases. An 11-amino acid fragment of human immunodeficiency type 1 (HIV-1) TAT-protein can transduce large, biologically active proteins into mammalian cells; recent evidence has shown an in vivo PTD for the 116 kDa beta-galactosidase protein. However, there is little information on the in vivo distribution of the TAT fusion protein to define the viability of PTDs for human studies. In this study we examined the tissue kinetics and tissue distribution of the PTD-transduced TAT fusion protein in mice. Low (100 microg) or high (500 microg) doses of TAT-beta-galactosidase fusion protein were administrated to mice through four routes (portal vein, i.v., i.p., and oral). Tissues were harvested 15 min, 1h, 6h, 10h, and 24h after treatment. Distribution of beta-galactosidase in various tissues was analysed by in situ staining, enzymatic activity assay, and Western blot analysis. Beta-galactosidase enzyme activity was observed in all tissues (liver, kidney, spleen, lung, bowel, and brain). Beta-galactosidase activity peaked at 15 min in most tissues after portal vein, i.v., and i.p. administration and at 1h after oral dosing in all tissues. Beta-galactosidase activity in the liver at 15 min after portal vein injection (67 milliunits [mU]/mg) was higher than after i.v. (9.8 mU/mg), i.p. (4.4 mU/mg), and oral (0.3 mU/mg) dosing. In situ staining and Western blot results correlated closely with beta-galactosidase enzyme activity assay. The median initial half-life for activity was 2.2h, ranging from 1.2h to 3.4h (coefficient of variation=28.9%). The bioavailability of beta-galactosidase activity after an orally administered PTD was 24%. This study details the kinetics and tissue distribution of delivering of a model TAT fusion protein into the mouse via PTD. These data allow rational selection of delivery route and schedules for therapeutic PTD and will aid the use of TAT fusion protein transduction in the development of protein therapies.
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PMID:The kinetics and tissue distribution of protein transduction in mice. 1637 28

The direct intracellular delivery of proteins has, until recently, been difficult to achieve, due primarily to the bioavailability barrier of the plasma membrane. During the past 15 years, a variety of peptides called protein transduction domains (PTDs) or cell penetrating peptides (CPPs), have been characterized for their ability to translocate into live cells. The most commonly studied are homeodomain transcription factors such as Antennapedia, the herpes simplex virus (HSV) type 1 protein VP22, and the human immunodeficiency virus (HIV-1) transactivator TAT protein. Recently, polyarginine exhibits even greater efficiency in terms of delivery of several peptides and proteins. Numerous examples of biologically active full-length proteins and peptides have been delivered to cells and tissues, both in vitro and in vivo. These studies offer new avenues for treatment of several diseases. The main mechanism of protein transduction is an electrostatic interaction with the plasma membrane, penetration into cells by macropinocytosis, and a release to cytoplasm and nuclei by retrograde transport. Moreover, the intercellular transfer of endogenous transcription factors, such as TAT and homeoproteins, seems to point to an original and important mode of signal transduction. The protein transduction systems have opened up several possibilities, not only for the development of new peptide/protein drugs but also for consideration of their physiological and developmental implications.
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PMID:Protein transduction technology: a novel therapeutic perspective. 1650 84

Future gene therapy for brainstem variant amyotrophic lateral sclerosis may be technically difficult if gene therapy vectors are injected near vital cardiorespiratory centers or if large portions of the tongue and pharyngeal muscles must be peripherally injected for retrograde transport of vectors to motor neurons. In this study we show that it is possible to deliver recombinant proteins to the hypoglossal nuclei without brainstem or muscle injections, by taking advantage of enhanced uptake of fusion proteins containing the protein transduction domain from the human immunodeficiency virus TAT protein. Adenoviral vectors expressing either TAT-modified or native beta-glucuronidase (beta-gluc) were injected into the lateral cerebral ventricles of mice, transducing ventricular epithelium down to the level of the obex in the brainstem. There was significant uptake into the hypoglossal nuclei of TAT-modified but not native beta-glucuronidase. The TAT-modified beta-gluc appeared to encompass half or more of the hypoglossal nuclei as visualized by retrograde labeling with cholera toxin subunit b in adjacent sections. TAT-modification of gene products may allow a relatively non-invasive approach to brainstem gene therapy via cerebroventricular injection.
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PMID:A TAT-modified fusion protein efficiently penetrates mouse hypoglossal nuclei from transduced ependyma. 1665 May 76

Polyomavirus BK (BKV) is a serious problem for immunocompromised patients, where latent virus can enter into the lytic cycle causing cytolytic destruction of host cells. BKV infects >80% of the population worldwide during childhood and then remains in a latent state in the kidney. In the context of immunosuppression in kidney transplant patients, reactivation of the viral early promoter (BKV(E)) results in production of T antigen, enabling virus replication and transition from latency to the lytic phase, causing polyomavirus-associated nephropathy. Reactivation of BKV can also cause complications such as nephritis, atypical retinitis and haemorrhagic cystitis in AIDS patients. Here, the effects of human immunodeficiency virus type 1 (HIV-1) proteins Tat and Vpr on BKV transcription were investigated and it was demonstrated that Tat dramatically stimulated BKV(E). Site-directed mutagenesis analysis of potential Tat-responsive transcriptional motifs complemented by an electrophoretic mobility shift assay (EMSA) showed that Tat activated BKV(E) by inducing binding of the NF-kappaB p65 subunit to a kappaB motif near the 3' end of BKV(E). In addition, a sequence within the 5' UTR of BKV(E) transcripts (BKV(E)-TAR) was identified that is identical to the HIV-1 transactivation response (TAR) element. The BKV(E)-TAR sequence bound TAT in RNA EMSA assays and deletion of the BKV(E)-TAR sequence eliminated Tat transactivation of BKV(E) transcription. Thus, Tat positively affected BKV(E) transcription by a dual mechanism and this may be important in diseases involving BKV reactivation in AIDS patients.
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PMID:Activation of early gene transcription in polyomavirus BK by human immunodeficiency virus type 1 Tat. 1669 Sep 19


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