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Silvia N. J. Moreno - One of the best experts on this subject based on the ideXlab platform.

  • Ca2+ Regulation of Trypanosoma bruCei Phosphoinositide Phospholipase C
    Eukaryotic cell, 2015
    Co-Authors: Sharon King-keller, Christina A. Moore, Roberto Docampo, Silvia N. J. Moreno
    Abstract:

    We CharaCterized a Phosphoinositide Phospholipase C (PI-PLC) from the proCyCliC form (PCF) of Trypanosoma bruCei. The protein Contains a domain organization CharaCteristiC of typiCal PI-PLCs, suCh as X and Y CatalytiC domains, an EF-hand CalCium-binding motif, and a C2 domain, but it laCks a pleCkstrin homology (PH) domain. In addition, the T. bruCei PI-PLC (TbPI-PLC) Contains an N-terminal myristoylation Consensus sequenCe found only in trypanosomatid PI-PLCs. A peptide Containing this N-terminal domain fused to green fluoresCent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatiC aCtivity was stimulated by Ca(2+) ConCentrations below the CytosoliC levels in the parasite, suggesting that the enzyme is Constitutively aCtive. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2), with a higher affinity for PIP2. We found that modifiCation of a single amino aCid in the EF-hand motif greatly affeCted the protein's Ca(2+) sensitivity and substrate preferenCe, demonstrating the role of this motif in Ca(2+) regulation of TbPI-PLC. Endogenous TbPI-PLC loCalizes to intraCellular vesiCles and might be using an intraCellular sourCe of PIP2. KnoCkdown of TbPI-PLC expression by RNA interferenCe (RNAi) did not result in growth inhibition, although enzymatiC aCtivity was still present in parasites, resulting in hydrolysis of PIP2 and a Contribution to the inositol 1,4,5-trisphosphate (IP3)/diaCylglyCerol (DAG) pathway.

  • aCylation dependent export of trypanosoma Cruzi Phosphoinositide speCifiC Phospholipase C to the outer surfaCe of amastigotes
    Journal of Biological Chemistry, 2010
    Co-Authors: Vicente De Paulo Martins, Roberto Docampo, Silvia N. J. Moreno, Michael Okura, Danijela Maric, David M Engman, Mauricio Vieira
    Abstract:

    Phosphoinositide Phospholipase C (PI-PLC) plays an essential role in Cell signaling. A unique Trypanosoma Cruzi PI-PLC (TCPI-PLC) is lipid-modified in its N terminus and loCalizes to the plasma membrane of amastigotes. Here, we show that TCPI-PLC is loCated onto the extraCellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino aCids are neCessary and suffiCient to target the fused GFP to the outer surfaCe of the parasite. Mutagenesis of the prediCted aCylated residues Confirmed that myristoylation of a glyCine residue in the 2nd position and aCyl modifiCation of a Cysteine in the 4th but not in the 8th or 15th position of the Coding sequenCe are required for CorreCt plasma membrane loCalization in T. Cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the Cysteine at the 8th position inCreased its flagellar loCalization. When expressed as fusion ConstruCts with GFP, the N-terminal 6 and 10 amino aCids fused to GFP are predominantly loCated in the Cytosol and ConCentrated in a Compartment that Co-loCalizes with a Golgi Complex marker. The N-terminal 20 amino aCids of TCPI-PLC assoCiate with lipid rafts when dually aCylated. Taken together, these results indiCate that N-terminal aCyl modifiCations serve as a moleCular addressing system for sending TCPI-PLC to the outer surfaCe of the Cell.

  • Developmental Expression of a Trypanosoma Cruzi Phosphoinositide-SpeCifiC Phospholipase C in Amastigotes and Stimulation of Host Phosphoinositide Hydrolysis
    Infection and immunity, 2010
    Co-Authors: Vicente De Paulo Martins, Roberto Docampo, Melina Galizzi, Maria Laura Salto, Silvia N. J. Moreno
    Abstract:

    Phosphoinositide Phospholipase C (PI-PLC) plays an essential role in Cell signaling. A unique Trypanosoma Cruzi PI-PLC (TCPI-PLC) is lipid modified in its N terminus and loCalizes to the outer surfaCe of the plasma membrane of amastigotes. We show here that TCPI-PLC is developmentally regulated in amastigotes and shows two peaks of surfaCe expression during the developmental CyCle of T. Cruzi, the first immediately after differentiation of trypomastigotes into amastigotes and the seCond before differentiation of amastigotes into trypomastigotes. SurfaCe expression of TCPI-PLC CoinCides with phosphatidylinositol 4,5-bisphosphate (PIP2) depletion in the host Cell membrane and with an inCrease in the levels of its produCt, inositol 1,4,5-trisphosphate. During extraCellular differentiation, PI-PLC is seCreted into the inCubation medium. Maximal early expression of TCPI-PLC on the surfaCe of amastigotes and PIP2 depletion CoinCide with host Cytoskeletal Changes, Ca2+ signaling, and transCriptional responses desCribed previously. The presenCe of TCPI-PLC on the outer surfaCe of the plasma membrane of the parasite and the CapaCity to be seCreted and to alter host phospholipids are novel meChanisms of the host-parasite interaCtion.

  • A Toxoplasma gondii Phosphoinositide Phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol.
    The Biochemical journal, 2006
    Co-Authors: Jianmin Fang, Norma Marchesini, Silvia N. J. Moreno
    Abstract:

    The Toxoplasma gondii Phosphoinositide-speCifiC Phospholipase C gene (TgPI-PLC) was Cloned, sequenCed and expressed in EsCheriChia Coli and its enzymatiC CharaCteristiCs were investigated. TgPI-PLC is present in the genome as a single-Copy gene Consisting of 22 exons interrupted by 21 introns, and enCodes a polypeptide of 1097 amino aCids with a prediCted moleCular mass of 121 kDa. In addition to the Conserved CatalytiC X and Y domains, TgPI-PLC Contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When Compared with mammalian delta-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31-35% identity over the whole sequenCe. ReCombinant TgPI-PLC, as well as the native enzyme obtained from Crude membrane extraCts of the parasite, was more aCtive with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. IndireCt immunofluoresCenCe analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme loCalizes in the plasma membrane of the parasites.

  • A Toxoplasma gondii Phosphoinositide Phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol
    Biochemical Journal, 2006
    Co-Authors: Jianmin Fang, Norma Marchesini, Silvia N. J. Moreno
    Abstract:

    The Toxoplasma gondii Phosphoinositide-speCifiC Phospholipase C gene (TgPI-PLC) was Cloned, sequenCed and expressed in EsCheriChia Coli and its enzymatiC CharaCteristiCs were investigated. TgPI-PLC is present in the genome as a single-Copy gene Consisting of 22 exons interrupted by 21 introns, and enCodes a polypeptide of 1097 amino aCids with a prediCted moleCular mass of 121 kDa. In addition to the Conserved CatalytiC X and Y domains, TgPI-PLC Contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When Compared with mammalian δ-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31–35% identity over the whole sequenCe. ReCombinant TgPI-PLC, as well as the native enzyme obtained from Crude membrane extraCts of the parasite, was more aCtive with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. IndireCt immunofluoresCenCe analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme loCalizes in the plasma membrane of the parasites.

John H. Exton - One of the best experts on this subject based on the ideXlab platform.

  • Ciba Foundation Symposium 164 - InteraCtions Among Cell Signalling Systems - Regulation of Phosphoinositide and phosphatidylCholine Phospholipases by G proteins.
    Ciba Foundation symposium, 2007
    Co-Authors: John H. Exton, Stephen J. Taylor, Jonathan S. Blank, Stephen B. Bocckino
    Abstract:

    Two G proteins that regulate Phosphoinositide Phospholipase C in liver plasma membranes have been purified to homogeneity in both the heterotrimeriC and dissoCiated forms. The heterotrimers Contain a 42 kDa or 43 kDa alpha subunit and a 35 kDa beta subunit. The alpha subunits are not ADP-ribosylated by pertussis toxin and are Closely related immunologiCally to members of the reCently identified Gq Class of G proteins. The speCifiC Phosphoinositide Phospholipase C isozyme that responds to the G proteins has been determined to the beta 1 isozyme. GTP analogues stimulate phosphatidylCholine hydrolysis in rat liver plasma membranes. The nuCleotide speCifiCity and Mg2+ dependenCy of the response indiCate that it is mediated by a G protein. PhosphatidiC aCid, diaCylglyCerol, Choline and phosphorylCholine are the produCts, indiCating that both Phospholipase D and C aCtivities are involved. ACtivation of Phospholipase D is also indiCated by the enhanCed produCtion of phosphatidyl-ethanol in the presenCe of ethanol.

  • Phospholipase C-γ, protein kinase C and Ca2+/Calmodulin-dependent protein kinase II are involved in platelet-derived growth faCtor-induCed phosphorylation of Tiam1
    FEBS letters, 1998
    Co-Authors: Ian N. Fleming, Cassondra M. Elliott, John H. Exton
    Abstract:

    In Swiss 3T3 fibroblasts, the RaC1-speCifiC guanine nuCleotide exChange faCtor Tiam1 is phosphorylated by several different agonists. We show here that PDGF induCes threonine phosphorylation of Tiam1 in a time- and dose-dependent manner. Tiam1 phosphorylation was signifiCantly reduCed by the seleCtive protein kinase C inhibitor Ro-31-8220 and by KN93, an inhibitor of Ca2+/Calmodulin-dependent protein kinase II. The Ca2+ Chelator BAPTA/AM totally abrogated Tiam1 phosphorylation, indiCating that Ca2+ is essential for this phosphorylation. Moreover, PDGF-stimulated Tiam1 phosphorylation was markedly reduCed by 72±10% in PLC-γ1 defiCient mouse fibroblasts, Compared to wild-type Cells, indiCating that Phosphoinositide Phospholipase C is involved.

  • DissoCiation of tyrosine phosphorylation and aCtivation of Phosphoinositide Phospholipase C induCed by the protein kinase C inhibitor Ro-31-8220 in Swiss 3T3 Cells treated with platelet-derived growth faCtor
    Biochimica et biophysica acta, 1997
    Co-Authors: Eui-ju Yeo, Joseph J Provost, John H. Exton
    Abstract:

    AbstraCt Platelet-derived growth faCtor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP 2 ) via Phospholipase C- γ 1 (PLC- γ 1) in Swiss 3T3 Cells. Treatment of Cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly deCreased PDGF-induCed tyrosine phosphorylation of PLC- γ 1, but paradoxiCally enhanCed the produCtion of inositol phosphates (InsPs). The inhibitor also Caused an inCrease of PDGF reCeptor tyrosine phosphorylation at later times. The Changes in phosphorylation of the reCeptor were Correlated with alterations in PLC- γ 1 transloCation to the partiCulate fraCtion. Thus, although aCtivation of PLC- γ 1 was assoCiated with phosphorylation of the reCeptor and transloCation of the enzyme to the partiCulate fraCtion, it was dissoCiated from its tyrosine phosphorylation. A non-reCeptor-assoCiated, CytosoliC tyrosine kinase also was found to phosphorylate PLC- γ 1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment deCreased the tyrosine phosphorylation of PLC- γ 1 induCed by PDGF and slowed the transloCation of PLC- γ 1, but Ro-31-8220 produCed further effeCts. The effeCt of Ro-31-8220 to enhanCe the produCtion of InsPs Could not be attributed to inhibition of PKC sinCe InsPs produCtion with PDGF was deCreased in PKC-depleted Cells and a stimulatory effeCt of the inhibitor was still evident. Interestingly, Ro-31-8220 deCreased the radioaCtivity in phosphatidylinositol and inCreased that in phosphatidylinositol 4-phosphate and PtdInsP 2 in Cells labeled with myo[ 3 H]inositol. The inCreased synthesis of PtdInsP 2 Could Contribute to the inCreased produCtion of InsPs induCed by Ro-31-8220. In summary, these results support the ConClusion that the aCtivation of PLC- γ 1 in response to PDGF requires autophosphorylation of the reCeptor and membrane assoCiation of PLC- γ 1, but not phosphorylation of the enzyme. Furthermore, the effeCts of Ro-31-8220 on the tyrosine phosphorylation and aCtivity of PLC- γ 1, and on PtdInsP 2 synthesis Cannot be attributed to inhibition of PKC. © 1997 Elsevier SCienCe B.V. All rights reserved.

  • Role of Rho Family Proteins in Phospholipase D ACtivation by Growth FaCtors
    The Journal of biological chemistry, 1997
    Co-Authors: Jean A. Hess, Annette H. Ross, Rong-guo Qiu, Marc Symons, John H. Exton
    Abstract:

    Treatment of fibroblasts with growth faCtors results in aCtivation of Phospholipase D (PLD). In order to determine the role of the Rho family of small GTPases in growth faCtor-mediated PLD aCtivation, we used Cells transfeCted with wild type and mutant RaC1. In response to epidermal growth faCtor (EGF), PLD aCtivity was greatly inCreased in Rat1 fibroblasts expressing wild type RaC1 (wtRaC1), and Completely abrogated in Cells expressing dominant negative N17RaC1, Consistent with RaC1 mediating the aCtion of this growth faCtor. In Contrast, in Cells treated with platelet-derived growth faCtor (PDGF) or phorbol ester, the wtRaC1 Cells showed little or no enhanCement of PLD aCtivity, and the response was not affeCted in the N17RaC1 Cells, implying that RaC1 played a minimal role in the aCtivation of PLD by PDGF or protein kinase C. Both growth faCtors produCed an attenuated PLD response in Cells expressing Constitutively aCtive V12RaC1, but these Cells showed other Changes, inCluding altered morphology, inCreased basal PLD, and deCreased growth faCtor reCeptor autophosphorylation. The effeCts of EGF and PDGF on Phosphoinositide Phospholipase C aCtivity were not enhanCed in Cells expressing wtRaC1 or inhibited in those expressing N17RaC1. In Cells expressing Constitutively aCtive V12RaC1, basal Phosphoinositide Phospholipase C was elevated, but there were no signifiCant effeCts of EGF or PDGF. We used C3 transferase of Clostridium botulinum, whiCh ADP-ribosylates and inaCtivates RhoA, to investigate the involvement of RhoA in the aCtivation of PLD by PDGF. Cells expressing wtRaC1 and N17RaC1 showed a deCreased PLD in response to PDGF when treated with C3 transferase, indiCating a role for RhoA. In summary, these data indiCate a major role for RaC1 in the aCtivation of PLD by EGF, but not PDGF or protein kinase C.

  • EnhanCed Phospholipase D ACtivity and Altered Morphology inRhoA-Overexpressing RAT1 Fibroblasts
    Biochemical and biophysical research communications, 1996
    Co-Authors: Kenneth C. Malcolm, Carol L. Sable, Cassondra M. Elliott, John H. Exton
    Abstract:

    AbstraCt Small GTP-binding proteins of the Rho family are impliCated in the regulation of Phospholipase D (PLD). However, few studies have addressed their role in agonist-stimulated PLD aCtivity in vivo. Stable lines of Rat1 fibroblasts overexpressing RhoA were shown to have altered morphology. Moreover, they demonstrated inCreased PLD aCtivity when stimulated with lysophosphatidiC aCid, platelet-derived growth faCtor, and phorbol ester, Compared with veCtor-transfeCted Cells. However, Phosphoinositide Phospholipase C aCtivity was unaltered by overexpression of RhoA. These data indiCate a CritiCal downstream role for RhoA in agonist-stimulated PLD aCtivity in intaCt Cells.

Rory A. Fisher - One of the best experts on this subject based on the ideXlab platform.

  • A trunCated form of RGS3 negatively regulates G protein-Coupled reCeptor stimulation of adenylyl CyClase and Phosphoinositide Phospholipase C.
    The Journal of biological chemistry, 1997
    Co-Authors: Tapan K. Chatterjee, Alex K. Eapen, Rory A. Fisher
    Abstract:

    AbstraCt IdentifiCation of a new family of proteins (RGS proteins) that funCtion as negative regulators ofG protein signaling has sparked new understanding of desensitization of this signaling proCess. ReCent studies with several mammalian RGS proteins has delineated their ability to interaCt with and funCtion as GTPase-aCtivating proteins speCifiCally for G proteins in the Gi family. Here, we investigated the funCtional aCtivity of RGS3 and a trunCated form of RGS3 on G protein-Coupled reCeptor-mediated aCtivation of adenylyl CyClase, Phosphoinositide Phospholipase C, and mitogen-aCtivated protein kinase in intaCt Cells. Polymerase Chain reaCtion and 5′-rapid amplifiCation of CDNA ends analyses revealed the tissue-speCifiC expression of a short form of the RGS3 transCript that enCodes the approximate Carboxyl-terminal half of RGS3. This trunCated form of RGS3 (RGS3T) was shown reCently to funCtion as a negative regulator of pheromone signaling in yeast (Druey, K. M., Blumer, K. J., Kang, V. R., and Kehrl, J. H. (1996) Nature 379, 742–746). Baby hamster kidney Cells transiently transfeCted with RGS3T CDNA exhibited a pronounCed impairment in platelet-aCtivating faCtor reCeptor-stimulated inositol phosphate produCtion, a pertussis toxin-insensitive response. Similarly, CalCitonin gene-related peptide reCeptor-stimulated inCreases in intraCellular CAMP and pituitary adenylate-CyClase aCtivating polypeptide reCeptor-stimulated inCreases in both CAMP and inositol phosphates were reduCed signifiCantly in RGS3T transfeCtants Compared with veCtor-transfeCted Control Cells. In Contrast, baby hamster kidney Cells transfeCted with the full-length RGS3 CDNA showed no impairment in CAMP and inositol phosphate produCtion mediated by these G protein-Coupled reCeptors. However, lysophosphatidiC aCid reCeptor-stimulated phosphorylation of endogenous ERK1 and ERK2 was impaired markedly in both RGS3 and RGS3T transfeCtants, demonstrating the funCtional ability of both RGS forms to modulate Gi-mediated signaling. These results provide the first evidenCe for regulatory effeCts of an RGS protein on Gs- and Gq-mediated signaling in intaCt Cells and doCument that the Carboxyl-terminal region of RGS3 Comprises the struCtural domain for this aCtivity.

  • MoleCular Cloning of a Novel Variant of the Pituitary Adenylate CyClase-aCtivating Polypeptide (PACAP) ReCeptor That Stimulates CalCium Influx by ACtivation of L-type CalCium Channels
    Journal of Biological Chemistry, 1996
    Co-Authors: Tapan Kumar Chatterjee, Ram V. Sharma, Rory A. Fisher
    Abstract:

    AbstraCt Pituitary adenylate CyClase-aCtivating polypeptide (PACAP) is a novel neuropeptide that produCes its biologiCal effeCts by interaCting with G protein-Coupled reCeptors. MoleCular Cloning of the PACAP reCeptor revealed the existenCe of five spliCe variant reCeptor forms differing in the third intraCellular loop region, with four variants aCtivating both adenylyl CyClase and Phosphoinositide Phospholipase C and one variant aCtivating only adenylyl CyClase (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., BoCkaert, J., Seeburg, P. H., and Journot, L. (1993) Nature365, 170-175). Here, we report Cloning of a novel PACAP reCeptor variant, designated PACAPR TM4 (transmembrane domain IV), that differs from the previously Cloned short form of the PACAP reCeptor (PACAPR) primarily by disCrete sequenCes loCated in transmembrane domains II and IV. Reverse transCriptase-polymerase Chain reaCtion and primer extension analyses demonstrated tissue-speCifiC differential expression of mRNAs enCoding PACAPR TM4 and spliCe variant forms of the PACAP reCeptor. PACAPR TM4 and PACAPR possess identiCal intraCellular domains, impliCated as primary determinants of G protein reCognition by rhodopsin-like reCeptors. However, unlike the PACAPR, PACAPR TM4 does not aCtivate either adenylyl CyClase or Phosphoinositide Phospholipase C in response to PACAP in either transient or stable expression systems. However, PACAP stimulates inCreases in [Ca2+]i in Cells expressing PACAPR TM4 by aCtivating L-type Ca2+ Channels, a response not eliCited by stimulation with vasoaCtive intestinal polypeptide. The signaling phenotype of PACAPR TM4 is CharaCteristiC of the PACAP reCeptor involved in regulation of insulin seCretion from panCreatiC β islets, a tissue expressing transCripts for PACAPR TM4 but not for PACAPR or its longer spliCe variant forms. These findings are Consistent with a role of PACAPR TM4 in the physiologiCal Control of insulin release by PACAP in β-islet Cells. The finding that PACAPR TM4 has a unique signaling phenotype, although it possesses intraCellular domains identiCal to those of the PACAPR, suggests that reCeptor-G protein reCognition by rhodopsin-like reCeptors Can be determined by sequenCes other than those loCated in intraCellular reCeptor domains.

  • MoleCular Cloning of a Novel Variant of the Pituitary Adenylate CyClase-aCtivating Polypeptide (PACAP) ReCeptor That Stimulates CalCium Influx by ACtivation of L-type CalCium Channels
    The Journal of biological chemistry, 1996
    Co-Authors: Tapan K. Chatterjee, Ram V. Sharma, Rory A. Fisher
    Abstract:

    Pituitary adenylate CyClase-aCtivating polypeptide (PACAP) is a novel neuropeptide that produCes its biologiCal effeCts by interaCting with G protein-Coupled reCeptors. MoleCular Cloning of the PACAP reCeptor revealed the existenCe of five spliCe variant reCeptor forms differing in the third intraCellular loop region, with four variants aCtivating both adenylyl CyClase and Phosphoinositide Phospholipase C and one variant aCtivating only adenylyl CyClase (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., BoCkaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). Here, we report Cloning of a novel PACAP reCeptor variant, designated PACAPR TM4 (transmembrane domain IV), that differs from the previously Cloned short form of the PACAP reCeptor (PACAPR) primarily by disCrete sequenCes loCated in transmembrane domains II and IV. Reverse transCriptase-polymerase Chain reaCtion and primer extension analyses demonstrated tissue-speCifiC differential expression of mRNAs enCoding PACAPR TM4 and spliCe variant forms of the PACAP reCeptor. PACAPR TM4 and PACAPR possess identiCal intraCellular domains, impliCated as primary determinants of G protein reCognition by rhodopsin-like reCeptors. However, unlike the PACAPR, PACAPR TM4 does not aCtivate either adenylyl CyClase or Phosphoinositide Phospholipase C in response to PACAP in either transient or stable expression systems. However, PACAP stimulates inCreases in [Ca2+]i in Cells expressing PACAPR TM4 by aCtivating L-type Ca2+ Channels, a response not eliCited by stimulation with vasoaCtive intestinal polypeptide. The signaling phenotype of PACAPR TM4 is CharaCteristiC of the PACAP reCeptor involved in regulation of insulin seCretion from panCreatiC beta islets, a tissue expressing transCripts for PACAPR TM4 but not for PACAPR or its longer spliCe variant forms. These findings are Consistent with a role of PACAPR TM4 in the physiologiCal Control of insulin release by PACAP in beta-islet Cells. The finding that PACAPR TM4 has a unique signaling phenotype, although it possesses intraCellular domains identiCal to those of the PACAPR, suggests that reCeptor-G protein reCognition by rhodopsin-like reCeptors Can be determined by sequenCes other than those loCated in intraCellular reCeptor domains.

  • The Third IntraCellular Domain of the Platelet-aCtivating FaCtor ReCeptor Is a CritiCal Determinant in ReCeptor Coupling to Phosphoinositide Phospholipase C-aCtivating G Proteins STUDIES USING INTRACELLULAR DOMAIN MINIGENES AND RECEPTOR CHIMERAS
    The Journal of biological chemistry, 1996
    Co-Authors: Steve A. Carlson, Tapan K. Chatterjee, Rory A. Fisher
    Abstract:

    AbstraCt Platelet aCtivating faCtor (PAF) is a potent phospholipid mediator whiCh eliCits a diverse array of biologiCal aCtions by interaCting with G protein-Coupled PAF reCeptors (PAFR). Binding of PAF to PAFRs leads to aCtivation of G protein(s) that stimulate Phosphoinositide Phospholipase C and subsequent intraCellular signaling responses. To identify the potential role of intraCellular domains of the rat PAFR (rPAFR) in signaling, we examined effeCts of transfeCting minigenes enCompassing rPAFR intraCellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol phosphate (IP) produCtion mediated by the Co-transfeCted rPAFR CDNA. Although transfeCtion of the rPAFR1i and rPAFR2i minigenes had no effeCts on PAF-stimulated signaling, transfeCtion of the rPAFR3i minigene inhibited PAF-stimulated IP produCtion by approximately 50% Compared to Controls. The rPAFR3i domain did not inhibit IP produCtion mediated by the multifunCtional rat pituitary adenylate CyClase-aCtivating polypeptide reCeptor (rPACAPR), demonstrating the speCifiCity of the Competition by the rPAFR3i domain. In further experiments, the rPAFR3i domain was engineered onto the homologous domain of a monofunCtional transmembrane variant of the rPACAPR (rPACAPR2) that aCtivates only adenylyl CyClase. The rPACAPR2/rPAFR3i Chimera responded to PACAP with inCreases in IP produCtion whiCh were attenuated nearly Completely in Cells CotransfeCted with the rPAFR3i domain. In Contrast, PACAP had no effeCts on IP produCtion in a reCeptor Chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut). These results demonstrate the ability of the rPAFR3i domain to Confer a Phospholipase C-signaling phenotype to a reCeptor defiCient in this aCtivity and show that this aCtivity is speCifiC for the engineered rPAFR3i domain. These results suggest that the third intraCellular loop of the rPAFR is a primary determinant in its Coupling to Phosphoinositide Phospholipase C-aCtivating G proteins, providing the first insight into the moleCular basis of interaCtion of PAFRs with signal-transduCing G proteins.

Jianmin Fang - One of the best experts on this subject based on the ideXlab platform.

  • A Toxoplasma gondii Phosphoinositide Phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol.
    The Biochemical journal, 2006
    Co-Authors: Jianmin Fang, Norma Marchesini, Silvia N. J. Moreno
    Abstract:

    The Toxoplasma gondii Phosphoinositide-speCifiC Phospholipase C gene (TgPI-PLC) was Cloned, sequenCed and expressed in EsCheriChia Coli and its enzymatiC CharaCteristiCs were investigated. TgPI-PLC is present in the genome as a single-Copy gene Consisting of 22 exons interrupted by 21 introns, and enCodes a polypeptide of 1097 amino aCids with a prediCted moleCular mass of 121 kDa. In addition to the Conserved CatalytiC X and Y domains, TgPI-PLC Contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When Compared with mammalian delta-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31-35% identity over the whole sequenCe. ReCombinant TgPI-PLC, as well as the native enzyme obtained from Crude membrane extraCts of the parasite, was more aCtive with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. IndireCt immunofluoresCenCe analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme loCalizes in the plasma membrane of the parasites.

  • A Toxoplasma gondii Phosphoinositide Phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol
    Biochemical Journal, 2006
    Co-Authors: Jianmin Fang, Norma Marchesini, Silvia N. J. Moreno
    Abstract:

    The Toxoplasma gondii Phosphoinositide-speCifiC Phospholipase C gene (TgPI-PLC) was Cloned, sequenCed and expressed in EsCheriChia Coli and its enzymatiC CharaCteristiCs were investigated. TgPI-PLC is present in the genome as a single-Copy gene Consisting of 22 exons interrupted by 21 introns, and enCodes a polypeptide of 1097 amino aCids with a prediCted moleCular mass of 121 kDa. In addition to the Conserved CatalytiC X and Y domains, TgPI-PLC Contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When Compared with mammalian δ-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31–35% identity over the whole sequenCe. ReCombinant TgPI-PLC, as well as the native enzyme obtained from Crude membrane extraCts of the parasite, was more aCtive with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. IndireCt immunofluoresCenCe analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme loCalizes in the plasma membrane of the parasites.

M Y Follo - One of the best experts on this subject based on the ideXlab platform.

  • SeleCtive ACtivation of NuClear PI-PLCbeta1 During Normal and Therapy-Related Differentiation.
    Current pharmaceutical design, 2016
    Co-Authors: S Mongiorgi, M Y Follo, L Manzoli, Yong Ryoul Yang, Stefano Ratti, James A. Mccubrey, Anna Maria Billi, Pann-ghill Suh, Lucio Cocco
    Abstract:

    NuClear Phosphoinositide-Phospholipase C (PI-PLC) beta1 plays a CruCial role in the moleCular steps that regulate Cell proliferation and differentiation in several experimental models, suCh as myoblasts and hematopoietiC Cells, via interaCtion with other important moleCular players. Indeed, PI-PLCbeta1 and its related moleCules are definitely involved in hematopoiesis, and partiCularly in drug-induCed myeloid or erythroid differentiation. Here, we review the role of nuClear PI-PLCbeta1 signalling in normal hematopoiesis, in pathogenesis and in drug-related induCtion of hematopoietiC differentiation, with partiCular referenCe to the Current therapy of MyelodysplastiC Syndromes (MDS).

  • ACtivation of nuClear inositide signalling pathways during erythropoietin therapy in low-risk MDS patients
    Leukemia, 2012
    Co-Authors: M Y Follo, S Mongiorgi, C Clissa, S Paolini, G Martinelli, A M Martelli, G Fioravanti, L Manzoli, C Finelli, L Cocco
    Abstract:

    Inositide signaling pathways Can have a role in the MyelodysplastiC Syndromes (MDS) progression to aCute myeloid leukemia. Erythropoietin (EPO) is Currently used in low-risk MDS, where it suCCessfully CorreCts anemia in 50–70% of patients. However, some MDS patients are refraCtory to this treatment and little is known about the exaCt moleCular meChanisms underlying the effeCt of EPO in these subjeCts. Here, we investigated the role of inositide pathways in low-risk MDS treated with EPO, mainly foCusing on the Akt/PI-PLC (Phosphoinositide-Phospholipase C) gamma1 axis, whiCh is aCtivated by the EPO reCeptor, and PI-PLCbeta1/CyClin D3 signaling, as CyClin D3 is assoCiated with hematopoietiC proliferation and differentiation. Interestingly, EPO responder patients showed a speCifiC aCtivation of both the Akt/PI-PLCgamma1 pathway and beta-Globin gene expression, while nonresponders displayed an inCrease in PI-PLCbeta1 signaling. Moreover, in normal CD34+ Cells induCed to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induCed Akt phosphorylation and beta-Globin expression. Overall, these findings suggest that PI-PLCbeta1 Can aCt as a negative regulator of erythroid differentiation and Confirm the involvement of Akt/PI-PLCgamma1 pathway in EPO signaling, therefore Contributing to the Comprehension of the effeCt of EPO in low-risk MDS and possibly paving the way to the identifiCation of MDS patients at higher risk of refraCtoriness to EPO treatment.

  • EpigenetiC regulation of nuClear PI-PLCbeta1 signaling pathway in low-risk MDS patients during azaCitidine treatment
    Leukemia, 2012
    Co-Authors: M Y Follo, S Mongiorgi, C Clissa, L Manzoli, C Finelli, D Russo, C Filì, C Colombi, M Gobbi, M Piazzi
    Abstract:

    Phosphoinositide-Phospholipase C (PI-PLC) beta1 Can be Considered a speCifiC target for demethylating therapy in high-risk myelodysplastiC syndrome (MDS) patients, as azaCitidine treatment has been assoCiated with a PI-PLCbeta1-speCifiC promoter demethylation, and induCtion of PI-PLCbeta1 gene and protein expression. However, little is known about the moleCular effeCt of azaCitidine in low-risk MDS or the funCtional meChanisms linked with azaCitidine effeCt on PI-PLCbeta1 promoter. In the present study, we further investigated the role of epigenetiC regulation of PI-PLCbeta1, mainly foCusing on the struCture of the PI-PLCbeta1 promoter. We first examined the effeCt of azaCitidine on PI-PLCbeta1 promoter methylation and gene expression in low-risk MDS. Moreover, we studied the expression of key moleCules assoCiated with the nuClear inositide signaling pathways, suCh as CyClin D3. By applying a Chromatin immunopreCipitation method, we also studied the Correlation between the demethylating effeCt of azaCitidine and the degree of reCruitment to PI-PLCbeta1 promoter of some transCription faCtors impliCated in hematopoietiC stem Cell proliferation and differentiation, as well as of the methyl-CpG-binding domain proteins, whiCh speCifiCally interaCt with methylated DNA. Taken together, our results hint at a speCifiC involvement of PI-PLCbeta1 in epigenetiC meChanisms, and are partiCularly Consistent with the hypothesis of a role for PI-PLCbeta1 in azaCitidine-induCed myeloid differentiation.

  • SynergistiC induCtion of PI-PLCβ1 signaling by azaCitidine and valproiC aCid in high-risk myelodysplastiC syndromes
    Leukemia, 2011
    Co-Authors: M Y Follo, S Mongiorgi, C Clissa, S Paolini, G Martinelli, A M Martelli, C Finelli, F Chiarini, G Ramazzotti, L Cocco
    Abstract:

    The assoCiation between azaCitidine (AZA) and valproiC aCid (VPA) has shown high response rates in high-risk myelodysplastiC syndromes (MDS) Cases with unfavorable prognosis. However, little is known about the moleCular meChanisms underlying this therapy, and moleCular markers useful to monitor the disease and the effeCt of the treatment are needed. Phosphoinositide-Phospholipase C (PI-PLC) β1 is involved in both genetiC and epigenetiC meChanisms of MDS progression to aCute myeloid leukemia. Indeed, AZA as a single agent was able to induCe PI-PLCβ1 expression, therefore providing a promising new tool in the evaluation of response to demethylating therapies. In this study, we assessed the effiCaCy of the Combination of AZA and VPA on induCing PI-PLCβ1 expression in high-risk MDS patients. Furthermore, we observed an inCrease in CyClin D3 expression, a downstream target of PI-PLCβ1 signaling, therefore suggesting a potential Combined aCtivity of AZA and VPA in high-risk MDS in aCtivating PI-PLCβ1 signaling, thus affeCting Cell proliferation and differentiation. Taken together, our findings might open up new lines of investigations aiming at evaluating the role of the aCtivation of PI-PLCβ1 signaling in the epigenetiC therapy, whiCh may also lead to the identifiCation of innovative targets for the epigenetiC therapy of high-risk MDS.

  • ReduCtion of Phosphoinositide-Phospholipase C beta1 methylation prediCts the responsiveness to azaCitidine in high-risk MDS.
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: M Y Follo, S Mongiorgi, C Clissa, Carlo Finelli, Costanza Bosi, Michele Baccarani, Giulia Ramazzotti, Nicoletta Testoni, Francesca Chiarini, Alberto M. Martelli
    Abstract:

    Lipid signaling pathways are involved in Cell growth, differentiation, and apoptosis, and Could have a role in the progression of myelodysplastiC syndromes (MDS) into aCute myeloid leukemia (AML). Indeed, reCent studies showed that Phosphoinositide-Phospholipase (PI-PL)Cbeta1 mono-alleliC deletion Correlates with a higher risk of AML evolution. Also, a single patient treated with azaCitidine, a DNA methyltransferase inhibitor Currently used in MDS, displayed a direCt Correlation between PI-PLCbeta1 gene expression and drug responsiveness. Consequently, we hypothesized that PI-PLCbeta1 Could be a target for demethylating therapy. First, we analyzed the struCture of PI-PLCbeta1 gene promoter, then quantified the degree of PI-PLCbeta1 promoter methylation and gene expression in MDS patients at baseline and during azaCitidine administration. Indeed, PI-PLCbeta1 mRNA inCreased in responder patients, along with a reduCtion of PI-PLCbeta1 promoter methylation. Also, the moleCular response Correlated to and antiCipated the CliniCal outCome, thus suggesting that PI-PLCbeta1 gene reaCtivation Could prediCt azaCitidine responsiveness. Our results demonstrate not only that PI-PLCbeta1 promoter is hypermethylated in high-risk MDS patients, but also that the amount of PI-PLCbeta1 mRNA Could prediCt the CliniCal response to azaCitidine, therefore indiCating a promising new therapeutiC approaCh.