Browsing by Author "Huidobro Toro, J. Pablo"
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- ItemA cis-Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES(2013) Valiente Echeverria, Fernando; Vallejos, Maricarmen; Monette, Anne; Pino, Karla; Letelier, Alejandro; Huidobro Toro, J. Pablo; Mouland, Andrew J.; López Lastra, Marcelo Andrés
- ItemP2Y(1) Receptor Activation Elicits Its Partition out of Membrane Rafts and Its Rapid Internalization from Human Blood Vessels: Implications for Receptor Signaling(AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2008) Norambuena, Andres; Poblete, M. Ines; Donoso, M. Veronica; Espinoza, C. Sofia; Gonzalez, Alfonso; Huidobro Toro, J. PabloThe nucleotide P2Y(1) receptor ( P2Y(1) R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y(1) R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y(1) R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1) R and obliterated the P2Y(1) R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0] hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y(1) R agonist, not only displaced within 4 min the P2Y(1) R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N-6-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1) R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1) R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y(1) R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1) R to membrane rafts, highlighting the role of this microdomain in P2Y(1) R signaling.
- ItemReactive Oxygen Species Potentiate the P2X(2) Receptor Activity through Intracellular Cys(430)(SOC NEUROSCIENCE, 2009) Coddou, Claudio; Codocedo, Juan F.; Li, Shuo; Lillo, Juan G.; Acuna Castillo, Claudio; Bull, Paulina; Stojilkovic, Stanko S.; Huidobro Toro, J. PabloP2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X(2)R and inhibits the activity of P2X(4)R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X(2) subunit, termed P2X(2a), and a splice variant lacking a 69 residue segment in the C terminal, termed P2X(2b), as the donors for intracellular and transmembrane segments and the P2X(4) subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X(4/2a) chimera but was absent in oocytes expressing P2X(4/2b) chimera. Site-directed mutagenesis experiments revealed that the Cys(430) residue mediates effects of mercury on the P2X(2a)R activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H2O2) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATP-evoked P2X(2a)R and P2X(4/2a)R currents but not P2X(2b)R and P2X(2a)-C430A and P2X(2a)-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H2O2 potentiation. Alkylation of Cys(430) residue with methylmethane-thiosulfonate also abolished the mercury and H2O2 potentiation. Altogether, these results are consistent with the hypothesis that the Cys(430) residue is an intracellular P2X(2a)R redox sensor.
- ItemThe Elav-like protein HuR exerts translational control of viral internal ribosome entry sites(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009) Rivas Aravena, Andrea; Ramdohr, Pablo; Vallejos, Maricarmen; Valiente Echeverria, Fernando; Dormoy Raclet, Virginie; Rodriguez, Felipe; Pino, Karla; Holzmann, Cristian; Huidobro Toro, J. Pablo; Gallouzi, Imed Eddine; Lopez Lastra, MarceloThe human embryonic-lethal abnormal vision (ELAV)-like protein. HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1]RES activity and acts as an activator of the HCV]RES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require direct binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression. (C) 2009 Elsevier Inc. All rights reserved.