Browsing by Author "Bull, Paulina"

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    Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor
    (2007) Acuna-Castillo, Claudio; Coddou, Claudio; Bull, Paulina; Brito, Jocelyn; Huidobro-Toro, J. Pablo
    The P2X(7) receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X(7) receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 mu mol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X(7) receptor allosteric modulation.
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    Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptor channel -: Critical roles of Cys132 for zinc potentiation and Asp138 for copper inhibition
    (2007) Coddou, Claudio; Acuna-Castillo, Claudio; Bull, Paulina; Huidobro-Toro, J. Pablo
    Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X(4) receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His(140), a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr(123) and Thr(146) for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys(132), but not Cys(126), proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp(138), but not Asp(131) was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp(129), Asp131, and Thr(133) had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability.
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    Functional analysis of the endoxylanase B (xynB) promoter from Penicillium purpurogenum
    (2008) Diaz, Jheimmy; Chavez, Renato; Larrondo, Luis F.; Eyzaguirre, Jaime; Bull, Paulina
    In Penicillium purpurogenum, the gene encoding endoxylanase B (xynB) is highly expressed by xylan and repressed by glucose at the transcriptional level. The promoter of this gene has a modular structure, with eight putative XlnR binding sites in tandem (XlnR module), and upstream from them, four putative CreA binding sites (CreA module). Promoter fragments containing different modules were inserted into a plasmid, pAN49-1, which contains a basal fungal promoter linked to a reporter gene (lacZ) and its expression was studied in vivo in Aspergillus nidulans. The XlnR module is able to trigger high beta-galactosidase activity in the presence of xylan, but the lack of most XlnR sites notoriously reduces this enzymatic activity. No enzyme induction is observed if the orientation of the promoter fragment is inverted. The presence of the CreA module is necessary for glucose repression when beta-galactosidase activity is previously induced by xylan. However, when transformant strains containing the XlnR module but lacking all CreA sites were grown in glucose without pre-induction in xylan, a low beta-galactosidase activity was observed compared with the same transformants grown in xylan. These results agree with a double-lock regulatory mechanism for both direct and indirect repression of xylanolytic genes by glucose.
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    Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module
    (2006) Gordillo, Felipe; Caputo, Valentina; Peirano, Alessandra; Chavez, Renato; Van Beeumen, Jozef; Vandenberghe, Isabel; Claeyssens, Marc; Bull, Paulina; Ravanal, Maria Cristina; Eyzaguirre, Jaime
    At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family I of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates. (c) 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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    Reactive 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. Pablo
    P2X 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.
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    Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation
    (SOC NEUROSCIENCE, 2010) Perez de Arce, Karen; Varela Nallar, Lorena; Farias, Olivia; Cifuentes, Alejandra; Bull, Paulina; Couch, Brian A.; Koleske, Anthony J.; Inestrosa, Nibaldo C.; Alvarez, Alejandra R.
    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95.
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    The copper-dependent ACE1 transcription factor activates the transcription of the mco1 gene from the basidiomycete Phanerochaete chrysosporium
    (2008) Canessa, Paulo; Alvarez, Jose Miguel; Polanco, Ruben; Bull, Paulina; Vicuna, Rafael
    We have previously identified and functionally characterized the transcription factor ACE1 (Pc-ACE1) from Phanerochaete chrysosporium. In Saccharomyces cerevisiae, ACE1 activates the copper-dependent transcription of target genes through a DNA sequence element named ACE. However, the possible target gene(s) of Pc-ACE1 were unknown. An in silico search led to the identification of putative ACE elements in the promoter region of mco1, one of the four clustered genes encoding multicopper oxidases (MCOs) in P. chrysosporium. Since copper exerts an effect at the transcriptional level in MCOs from several organisms, in this work we analysed the effect of copper on transcript levels of the clustered MCO genes from P. chrysosporium, with the exception of the transcriptionally inactive mco3. Copper supplementation of cultures produced an increment in transcripts from genes mco1 and mco2, but not from mco4. Electrophoretic mobility-shift assays revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the promoter of mco1. In addition, using a cell-free transcription system, we showed that in the presence of cuprous ion, Pc-ACE1 induces activation of the promoter of mco1, but not that of mco2.
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    The PcACE1 transcription factor from Phanerochaete chrysosporium contains a Cys- and Ser-rich transactivation domain
    (2011) Barham, Feiruz; Roman, Patricia; Bull, Paulina
    Transcription factor Ace1 from Saccharomyces cerevisiae regulates the expression of target genes when the copper concentration reaches 200 mu M levels. We are studying the ortholog of Ace1 from fungus Phanerochaete chrysosporium PcACE1, isolated by complementation in yeast. In this report we show the localization of the transactivation region of PcACE1. Different PcACE1 fragments were ligated in frame to the GAL4 DNA-binding domain by site-directed mutagenesis in a suitable yeast expression vector. Transformation of an appropriate Saccharomyces cerevisiae strain was used as host. This strain contains the fusion GAL1:lacZ in its genome under the control of promoter sequences recognized by GAL4. Finally, we measured beta-galactosidase activity in each yeast clone. The activation of the reporter gene is proportional to the transactivation capacity of the transcription factor PcACE1.