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)

To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-beta-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.
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PMID:Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy. 1467 8

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.
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PMID:The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria. 1717 93

The objectives of this study were to develop an innovative investigative model using doxorubicin as a fluorophore to evaluate the skin permeation of nanocarriers and the impact of size and surface characteristics on their permeability. Different doxorubicin-loaded liposomes with mean particle size <130 nm and different surface chemistry were prepared by ammonium acetate gradient method using DPPC, DOPE, Cholesterol, DSPE-PEG 2000 and 1,1-Di-((Z)-octadec-9-en-1-yl) pyrrolidin-1-ium chloride (CY5)/DOTAP/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) as the charge modifier. There was minimal release of doxorubicin from the liposomes up to 8h; indicating that fluorescence observed within the skin layers was due to the intact liposomes. Liposomes with particle sizes >600 nm were restricted within the stratum corneum. DOTAP (p<0.01) and CY5 (p<0.05) liposomes demonstrated significant permeation into the skin than DOPA and PEG liposomes. Tape stripping significantly (p<0.01) enhanced the skin permeation of doxorubicin liposomes but TAT-decorated doxorubicin liposomes permeated better (p<0.005). Blockage of the hair follicles resulted in significant reduction in the extent and intensity of fluorescence observed within the skin layers. Overall, doxorubicin liposomes proved to be an ideal fluorophore-based model. The hair follicles were the major route utilized by the liposomes to permeate skin. Surface charge and particle size played vital roles in the extent of permeation.
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PMID:Doxorubicin liposomes as an investigative model to study the skin permeation of nanocarriers. 2591 Apr 14

Efficient delivery of pharmaceutically active molecules across cellular membranes using cell penetrating peptides (CPPs), such as the cationic human immunodeficiency virus-1 trans-acting activator of transcription peptide (HIV-1 TAT), continues to attract scientific attention in drug design and disease treatment. Experimental results show that the TAT peptide is not only capable of directly penetrating the biological membrane in a passive manner, but also forming physical, membrane-spanning pores that may facilitate transport. Experiments further show that anionic lipids accelerate peptide permeation within a range of mole percentage composition. In this work, we explored the structures and translocation thermodynamics of the cationic TAT peptide across a series of DPPC/DPPS model membranes with the presence of 0-30 mol% cholesterol. We computed the potentials of the mean force by using umbrella sampling molecular dynamics simulations coupled to the Martini coarse-grained force field. We systematically investigated the roles of cholesterol and anionic lipids (membrane surface charge) in TAT peptide translocation. In qualitative agreement with experimental findings, the barrier heights were significantly reduced in the presence of anionic lipids. A toroidal hydrophilic pore was strongly suggested by membrane structure analysis. Cholesterol stabilizes the liquid-ordered (Lo) phase of membranes and increases the elastic stiffness of bilayers. Consequently, it hinders transmembrane pore formation and thus modulates solute permeability, since the liquid-ordered phase suppresses reorientation of the lipid molecules on simulation time scales. Though cholesterol contributes marginally to the total free energy associated with peptide permeation, the coordination of cholesterol to the peptide weakens more favorable peptide-lipid interactions. The addition of the anionic lipid DPPS to the neutral DPPC bilayer leads to the emergence and further enhancement of an interfacially stable state of the peptide due to the favorable peptide-anionic lipid interactions. Translocation free energy barriers decrease in lockstep with increasing DPPS composition in the model bilayers simulated. Finally, we investigated the size of hydrophilic pores emerging in our simulations, as well as the qualitative mobility of the peptide on the membrane surface.
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PMID:Thermodynamics of cell-penetrating HIV1 TAT peptide insertion into PC/PS/CHOL model bilayers through transmembrane pores: the roles of cholesterol and anionic lipids. 2743 87