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)

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.
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PMID:Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras. 1219 40

We have reported previously that HIV-TAT-dominant negative (dn) Ras inhibits eosinophil adhesion to ICAM-1 after activation by IL-5 and eotaxin. In this study, we evaluated the role of Ras in Ag-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of dnRas, which was fused to an HIV-TAT protein transduction domain (TAT-dnRas). Uptake of TAT-dnRas (t(1/2) = 12 h) was demonstrated in leukocytes after i.p. administration. OVA-sensitization significantly increased eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid 24 h after final challenge. Treatment of animals with 3-10 mg/kg TAT-dnRas blocked the migration of eosinophils from 464 +/- 91 x 10(3)/ml to 288 +/- 79 x 10(3)/ml with 3 mg/kg of TAT-dnRas (p < 0.05), and further decreased to 116 +/- 63 x 10(3)/ml after 10 mg/kg TAT-dnRas (p < 0.01). Histological examination demonstrated that inflammatory cell infiltration (largely eosinophils and mononuclear cells) and mucin production around the airways caused by OVA were blocked by TAT-dnRas. OVA challenge also caused airway hyperresponsiveness to methacholine, which was dose dependently blocked by treatment with TAT-dnRas. TAT-dnRas also blocked Ag-induced IL-4 and IL-5, but not IFN-gamma, production in lung tissue. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by pretreatment with TAT-dnRas. By contrast, TAT-green fluorescent protein or dnRas lacking the TAT protein transduction domain did not block airway inflammation, cytokine production, or airway hyperresponsiveness. We conclude that Ras mediates Th2 cytokine production, airway inflammation, and airway hyperresponsiveness in immune-sensitized mice.
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PMID:Blockade of airway inflammation and hyperresponsiveness by HIV-TAT-dominant negative Ras. 1453 Mar 63

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Deltap85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85alpha, which was fused to HIV-TAT (TAT-Deltap85). Intraperitoneal administration of TAT-Deltap85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3-10 mg/kg of TAT-Deltap85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Deltap85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.
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PMID:Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-TAT. 1462 11

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

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is part of a complex signaling system that affects a variety of important cell functions. PTEN antagonizes the action of PI3K by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. In the present study, we used a TAT fusion protein transduction system to elucidate the role of PTEN in eosinophils and airway inflammation. A small region of the HIV TAT protein (YGRKKRRQRRR), a protein transduction domain known to enter mammalian cells efficiently, was fused to the N terminus of PTEN. Flow cytometric analysis of annexin V- and propidium iodide-stained cells was used to assess eosinophil survival. A chemotaxis assay was performed using a Boyden chamber. Cell analysis in bronchoalveolar lavage fluid and histological examinations were performed using OVA-challenged A/J mice. We found that TAT-PTEN was successfully internalized into eosinophils and functioned as a phosphatase in situ. TAT-PTEN, but not a TAT-GFP control protein, blocked the ability of IL-5 to prevent the apoptosis of eosinophils from allergic subjects. The eotaxin-induced eosinophil chemotaxis was inhibited by TAT-PTEN in a dose-dependent manner. Intranasal pretreatment with TAT-PTEN, but not TAT-GFP, significantly inhibited the OVA-induced eosinophil infiltration in bronchoalveolar lavage fluid. Histological examination of the lung, including H&E and Alcian blue/periodic acid-Schiff staining, revealed that TAT-PTEN, but not TAT-GFP, abrogated eosinophilic inflammation and mucus production. Our results suggest that PTEN negatively regulates eosinophil survival, chemotaxis, and allergic inflammation. The pharmacological targeting of PTEN may constitute a new strategy for the treatment of eosinophilic disorders.
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PMID:Transduction of phosphatase and tensin homolog deleted on chromosome 10 into eosinophils attenuates survival, chemotaxis, and airway inflammation. 1805 52

We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after beta(2)-integrin ligation. Western blot analysis showed that Pyk2 was activated by phosphorylation at Y402 after eosinophil adhesion to BSA-coated plates after activation with IL-5, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn(2)(+). To determine the role of Pyk2 in regulating eosinophil migration, we used a transducable dominant-negative inhibitor of Pyk2, TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2. TAT-Pyk2-CT blocked tyrosine phosphorylation of Pyk2 caused by beta(2)-integrin adhesion, but did not block adhesion of eosinophils to plated BSA. TAT-Pyk2-CT also blocked subsequent spreading and migration of eosinophils caused by IL-5, PAF, or fMLP. Spreading eosinophils stained with FITC-conjugated phalloidin showed elongation and formation of multiple fillopodia and lamellipodia, whereas nonspreading eosinophils were smaller and round. Treatment of eosinophils with TAT-Pyk2-CT had no effect on the initial cell polarization, but blocked the formation of fillopodia and lamellipodia in adherent cells. Migration of eosinophils through Transwell plates caused by IL-5, PAF, or fMLP was blocked significantly after inhibition of Pyk2. These data indicate that Pyk2, although not involved in beta(2)-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after beta(2)-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by beta(2)-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration.
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PMID:Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion. 1836 25

The objective of this investigation was to determine the role of Pyk2, an intracellular nonreceptor protein tyrosine kinase for postadhesive inflammatory cell migration, on airway inflammation and hyperresponsiveness in immune-sensitized mice. Blockade of Pyk2 was effected by intraperitoneal administration of dominant-negative C-terminal Pyk2 fused to a TAT protein transduction domain (TAT-Pyk2-CT). Ovalbumin challenge elicited infiltration of both eosinophils and lymphocytes into airways, increased mucus-containing epithelial cells, and caused increased airway hyperresponsiveness to methacholine in immune-sensitized mice. Pretreatment with 10 mg/kg TAT-Pyk2-CT intraperitoneally blocked all of these effects and further decreased secretion of Th2 cytokine IL-4, IL-5, and IL-13 into the bronchoalveolar lavage fluid. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Pyk2-CT. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. We conclude that Pyk2, which is essential for inflammatory cell migration in vitro, regulates airway inflammation, Th2 cytokine secretion, and airway hyperresponsiveness in the ovalbumin-sensitized mice during antigen challenge in vivo.
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PMID:Inhibition of Pyk2 blocks airway inflammation and hyperresponsiveness in a mouse model of asthma. 1952 Sep 18