Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Molecular Cell Research (v.1793, #2)
TRPC channels and store-operated Ca2+ entry by Ginés M. Salido; Stewart O. Sage; Juan A. Rosado ⁎ (pp. 223-230).
Store-operated calcium entry (SOCE) is a major mechanism for Ca2+ influx. Since SOCE was first proposed two decades ago many techniques have been used in attempting to identify the nature of store-operated Ca2+ (SOC) channels. The first identified and best-characterised store-operated current is ICRAC, but a number of other currents activated by Ca2+ store depletion have also been described. TRPC proteins have long been proposed as SOC channel candidates; however, whether any of the TRPCs function as SOC channels remains controversial. This review attempts to provide an overview of the arguments in favour and against the role of TRPC proteins in the store-operated mechanisms of agonist-activated Ca2+ entry.
Keywords: TRPC; Store-operated Ca; 2+; entry; Type II IP; 3; receptor; STIM1; Orai1
EphrinB3 is an anti-apoptotic ligand that inhibits the dependence receptor functions of EphA4 receptors during adult neurogenesis by Céline Furne; Jerome Ricard; Jorge Ruben Cabrera; Laurent Pays; John R. Bethea; Patrick Mehlen; Daniel J. Liebl (pp. 231-238).
Eph receptors have been implicated in regulating a diverse array of cellular functions in the developing nervous system. Recently, Eph receptors have been shown to promote cell death in adult germinal zones; however, their mechanisms of action remain ill-defined. In this study, we demonstrate that EphA4 is a new member of the dependence receptors family, which can initiate cell death in the absence of its ligand ephrinB3. Upon removal of its ligand, EphA4 triggers cell death that is dependent on caspase activation as caspase inhibitors prevent cell death. EphA4 itself is cleaved by caspase-3-like caspase in the intracellular domain at position D773/774, which is necessary for cell death initiation as mutation of the cleavage site abolishes apoptosis. In the adult subventricular zone, abolishing ephrinB3 results in increased cell death, while the absence of EphA4 results in excessive numbers of neuroblasts. Furthermore, infusion of soluble ephrinB3 into the lateral ventricle reduced cell death, and together these results support a dependence role for EphA4 in adult neurogenesis.
Keywords: Ephrin; Eph receptor; Dependence receptor; Apoptosis
Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts by Martina Frasson; Maurizio Vitadello; Anna Maria Brunati; Nicoletta La Rocca; Elena Tibaldi; Lorenzo A. Pinna; Luisa Gorza; Arianna Donella-Deana (pp. 239-252).
The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation.
Keywords: HSP; Src tyrosine kinase; Protein translocation; Endoplasmic reticulum; Skeletal muscle; Differentiation
The inhibition of the epidermal growth factor (EGF) pathway enhances TGF-β-induced apoptosis in rat hepatoma cells through inducing oxidative stress coincident with a change in the expression pattern of the NADPH oxidases (NOX) isoforms by Patricia Sancho; Esther Bertran; Laia Caja; Irene Carmona-Cuenca; Miguel M. Murillo; Isabel Fabregat (pp. 253-263).
Transforming growth factor-beta (TGF-β) induces apoptosis in hepatocytes, through a mechanism mediated by reactive oxygen species (ROS) production. Numerous tumoral cells develop mechanisms to escape from the TGF-β-induced tumor suppressor effects. In this work we show that in FaO rat hepatoma cells inhibition of the epidermal growth factor receptor (EGFR) with the tyrphostin AG1478 enhances TGF-β-induced cell death, coincident with an elevated increase in ROS production and GSH depletion. These events correlate with down-regulation of genes involved in the maintenance of redox homeostasis, such as γ-GCS and MnSOD, and elevated mitochondrial ROS. Nonetheless, not all the ROS proceed from the mitochondria. Emerging evidences indicate that ROS production by TGF-β is also mediated by the NADPH oxidase (NOX) system. TGF-β-treated FaO cells induce nox1 expression. However, the treatment with TGF-β and AG1478 greatly enhanced the expression of another family member: nox4. NOX1 and NOX4 targeted knock-down by siRNA experiments suggest that they play opposite roles, because NOX1 knockdown increases caspase-3 activity and cell death, whilst NOX4 knock-down attenuates the apoptotic process. This attenuation correlates with maintenance of GSH and antioxidant enzymes levels. In summary, EGFR inhibition enhances apoptosis induced by TGF-β in FaO rat hepatoma cells through an increased oxidative stress coincident with a change in the expression pattern of NOX enzymes.
Keywords: Abbreviations; EGFR; epidermal growth factor receptor; γ-GCS; gamma-glutamylcysteine synthetase; GEE; glutathione-ethyl-ester; GSH; reduced glutathione; HCC; hepatocellular carcinoma; Mn-SOD; Mn-superoxide dismutase; ROS; reactive oxygen species; TGF-β; transforming growth factor betaTGF-beta; ROS; NADPH oxidase; NOX1; NOX4; Apoptosis
DLG1/SAP97 modulates transforming growth factor α bioavailability by Anne-Laure Surena; Giselle P. de Faria; Jeanne-Marie Studler; Franck Peiretti; Morgane Pidoux; Jacques Camonis; Hervé Chneiweiss; Etienne Formstecher; Marie-Pierre Junier (pp. 264-272).
TGFα and its receptor EGFR participate in the development of a wide range of tumors including gliomas, the main adult primary brain tumors. TGFα soluble form results from the cleavage by the metalloprotease TACE/ADAM17 of the extracellular part of its transmembrane precursor, pro-TGFα. To gain insights into the mechanisms underlying TGFα bioavailability, a yeast two-hybrid screen was performed to identify proteins interacting with pro-TGFα intracellular domain (ICD). DLG1/SAP97 (Discs Large Gene 1 or Synapse Associated Protein 97) was found to interact with both pro-TGFα and TACE ICDs through distinct PDZ domains. An in vivo pro-TGFα–DLG1–TACE complex was detected in U251 glioma cells and in gliomas-derived tumor initiating cells. Interaction between DLG1 and TACE diminished in response to stimulations promoting pro-TGFα shedding. Manipulation of DLG1 levels revealed dual actions of DLG1 on pro-TGFα shedding, favoring approximation of pro-TGFα and TACE, while limiting TACE full shedding activity. These results show that DLG1 participates in the control of TGFα bioavailability through its dynamic interaction with the growth factor precursor and TACE.
Keywords: EGF; MAGUK; erbB; Tumor suppressor gene
Intracellular zinc increase inhibits p53−/− pancreatic adenocarcinoma cell growth by ROS/AIF-mediated apoptosis by M. Donadelli; E. Dalla Pozza; M.T. Scupoli; C. Costanzo; A. Scarpa; M. Palmieri (pp. 273-280).
We show that treatment with non-toxic doses of zinc in association to the ionophore compound pyrrolidine dithiocarbamate (PDTC) inhibits p53−/− pancreatic cancer cell growth much more efficiently than gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Both the metal chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine and the radical scavenger N-acetyl-l-cysteine are able to recover cell growth inhibition by Zn/PDTC, demonstrating that this effect depends on the increased levels of intracellular zinc and of reactive oxygen species (ROS). Zn/PDTC treatment induces a strong apoptotic cell death that is associated to ROS-dependent nuclear translocation of the mitochondrial factor AIF, but not to the regulation of apoptotic genes and caspase activation. Primary fibroblasts are more resistant than pancreatic cancer cells to Zn/PDTC treatment and exhibit a lower basal and Zn/PDTC-induced enhancement of intracellular zinc. We show that Zn/PDTC induces p53 proteasomal degradation and that the proteasome inhibitor MG132 further increases fibroblast growth inhibition by Zn/PDTC, suggesting that p53 degradation plays an important role in fibroblast resistance to Zn/PDTC.
Keywords: Pancreatic adenocarcinoma; Zinc; Pyrrolidine dithiocarbamate; Oxidative stress; p53
Alternatively spliced exon 5 of the FERM domain of protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells by Pil-Soo Seo; Jong-Jin Jeong; Lixiao Zeng; Christos G. Takoudis; Brendan J. Quinn; Anwar A. Khan; Toshihiko Hanada; Athar H. Chishti ⁎ (pp. 281-289).
Direct physical linkage of MAGUKs to the actin cytoskeleton was first established by the interaction of erythrocyte p55 with the FERM domain of protein 4.1R. Subsequently, it was reported that p55 binds to a 51-amino acid peptide, encoded by exon 10, located within the FERM domain of protein 4.1R. In this study, we investigated the nature of the p55–FERM domain binding interface and show that p55 binds to a second 35-amino acid peptide, encoded by an alternatively spliced exon 5, located within the FERM domain of protein 4.1R. Competition and Surface Plasmon Resonance-binding measurements suggest that the peptides encoded by exons 5 and 10 bind to independent sites within the D5 domain of p55. Interestingly, the full length 135 kDa isoform of protein 4.1R containing both exons 5 and 10 was targeted exclusively to the plasma membrane of epithelial cells whereas the same isoform without exon 5 completely lost its membrane localization capacity. Together, these results indicate that p55 binds to two distinct sites within the FERM domain, and the alternatively spliced exon 5 is necessary for the membrane targeting of protein 4.1R in epithelial cells. Since sequences similar to the exon 5-peptide of protein 4.1R and D5 domain of p55 are conserved in many proteins, our findings suggest that a similar mechanism may govern the membrane targeting of other FERM domain containing proteins.
Keywords: Abbreviations; FERM; (; F; for 4.1 protein,; E; for Ezrin,; R; for Radixin,; M; for Moesin); MBP; maltose binding protein; 4.1R; human erythrocyte protein 4.1; MAGUKs; membrane associated guanylate kinase homologues; GST; glutathione S-transferase; PCR; polymerase chain reaction; Dlg; discs large tumor suppressor protein; p55; human erythrocyte p55; PBS; phosphate buffered saline; SPR; Surface Plasmon ResonanceProtein 4.1R; Erythrocyte p55; Membrane associated guanylate kinase homologue; FERM domain; Maltose binding protein; Surface plasmon resonance
Beta-catenin modulates the level and transcriptional activity of Notch1/NICD through its direct interaction by Yun Hye Jin; Hangun Kim; Hyunkyung Ki; Ilhwan Yang; Narae Yang; Kwang Youl Lee; Nacksung Kim; Hee-Sae Park; Kwonseop Kim ⁎ (pp. 290-299).
Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. Although there is evidence that the two main signaling pathways can modulate each other, the precise mechanism is not completely understood. This report shows that β-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that β-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that β-catenin could upregulate the level of Notch1 and NICD, possibly by competing the common ubiquitin-dependent degradation machinery. In addition, β-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by β-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. β-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either β-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and β-catenin are sufficient to induce ALP activation. Furthermore, effects of β-catenin on Notch1 were dramatically diminished by overexpressed LEF1. Overall, our data suggest that β-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway.
Keywords: Beta-catenin; Notch1; NICD; HES1; LEF1; Osteogenesis
WNT signaling promotes Nkx2.5 expression and early cardiomyogenesis via downregulation of Hdac1 by Zhiqiang Liu; Tao Li; Yinan Liu; Zhuqing Jia; Yanming Li; Chenguang Zhang; Ping Chen; Kangtao Ma; Nabeel Affara; Chunyan Zhou (pp. 300-311).
The cardiac transcription factor NKX2.5 plays a crucial role in cardiomyogenesis, but its mechanism of regulation is still unclear. Recently, epigenetic regulation has become increasingly recognized as important in differentiation and development. In this study, we used P19CL6 cells to investigate the regulation of Nkx2.5 expression by methylation and acetylation during cardiomyocyte differentiation. During the early stage of differentiation, Nkx2.5 expression was upregulated, but the methylation status of the Nkx2.5 promoter did not undergo significant change; while the acetylation levels of histones H3 and H4 were increased, accompanied by a significant reduction in Hdac1 expression. Suppression of Hdac1 activity stimulated cardiac differentiation accompanied by increased expression of cardiac-specific genes and cell cycle arrest. Overexpression of Hdac1 inhibited cardiomyocyte formation and downregulated the expressions of Gata4 and Nkx2.5. Mimicking induction of the WNT pathway inhibited Hdac1 expression with upregulated Nkx2.5 expression. WNT3a and WNT3 downregulated the expression of Hdac1, contrary to the effect of SFRP2 and GSK3β. Cotransfection of β -catenin and Lef1 significantly downregulated the expression of Hdac1. Our data suggest that WNT signaling pathway plays important roles in the regulation of Hdac1 during the early stage of cardiomyocyte differentiation and that the downregulation of Hdac1 promotes cardiac differentiation.
Keywords: P19CL6 cell; Cardiomyogenesis; Epigenetic; Nkx2.5; HDAC1; WNT
Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells by F. Houben ⁎; C.H.M.P. Willems; I.L.J. Declercq; K. Hochstenbach; M.A. Kamps; L.H.E.H. Snoeckx; F.C.S. Ramaekers; J.L.V. Broers (pp. 312-324).
The nuclear lamina and the cytoskeleton form an integrated structure that warrants proper mechanical functioning of cells. We have studied the correlation between structural alterations and migrational behaviour in fibroblasts with and without A-type lamins. We show that loss of A-type lamins causes loss of emerin and nesprin-3 from the nuclear envelope, concurring with a disturbance in the connection between the nucleus and the cytoskeleton in A-type lamin-deficient ( lmna −/−) cells. In these cells functional migration assays during in vitro wound healing revealed a delayed reorientation of the nucleus and the microtubule-organizing center during migration, as well as a loss of nuclear oscillatory rotation. These observations in fibroblasts isolated from lmna knockout mice were confirmed in a 3T3 cell line with stable reduction of lmna expression due to RNAi approach. Our results indicate that A-type lamins play a key role in maintaining directional movement governed by the cytoskeleton, and that the loss of these karyoskeletal proteins has important consequences for functioning of the cell as a mechanical entity.
Keywords: Nuclear lamina; Cytoskeleton; Cellular mechanics; MTOC; Wound healing
A membrane network of receptors and enzymes for adenine nucleotides and nucleosides by Klaus Schicker; Simon Hussl; Giri K. Chandaka; Kristina Kosenburger; Jae-Won Yang; Maria Waldhoer; Harald H. Sitte; Stefan Boehm ⁎ (pp. 325-334).
Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y1, P2Y2, P2Y12, P2Y13, P2X2, A1, A2A receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y1, P2Y12, P2Y13, A1, A2A, and P2X2 receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y1/P2Y1, A2A/P2Y1, A2A/P2Y12) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.
Keywords: Adenosine receptor; P2Y receptor; P2X receptor; NTPDase; FRET
A large ribonucleoprotein particle induced by cytoplasmic PrP shares striking similarities with the chromatoid body, an RNA granule predicted to function in posttranscriptional gene regulation by Simon Beaudoin; Benoît Vanderperre; Catherine Grenier; Isabelle Tremblay; Frederic Leduc; Xavier Roucou (pp. 335-345).
The observation that PrP is present in the cytosol of some neurons and non-neuronal cells and that the N-terminal signal peptide is slightly inefficient has brought speculations concerning a possible function of the protein in the cytosol. Here, we show that cells expressing a cytosolic form of PrP termed cyPrP display a large juxtanuclear cytoplasmic RNA organelle. Although cyPrP spontaneously forms aggresomes, we used several mutants to demonstrate that the assembly of this RNA organelle is independent from cyPrP aggregation. Components of the organelle fall into three classes: mRNAs; proteins, including the RNAseIII family polymerase Dicer, the decapping enzyme Dcp1a, the DEAD-box RNA helicase DDX6, and the small nuclear ribonucleoprotein-associated proteins SmB/B′/N; and non-coding RNAs, including rRNA 5S, tRNAs, U1 small nuclear RNA, and microRNAs. This composition is similar to RNA granules or chromatoid bodies from germ cells, or planarian stem cells and neurons, which are large ribonucleoprotein complexes predicted to function in RNA processing and posttranscriptional gene regulation. The domain of PrP encompassing residues 30 to 49 is essential for the formation of the RNA particle. Our findings confirm the intriguing relation between PrP and RNA in cells, and underscore an unexpected function for cytosolic PrP: assembling a large RNA processing center which we have termed PrP-RNP for PrP-induced ribonucleoprotein particle.
Keywords: Abbreviations; CyPrP; EGFP; cytoplasmic prion protein genetically fused to EGFP; DDX6; DEAD-box RNA helicase 6; EGFP; enhanced green fluorescent protein; miRNA; microRNA; PrP; prion protein; PrP-RNP; cyPrP-induced RiboNucleoprotein Particle; SmB/B′/N; Small nuclear ribonucleoprotein-associated proteins B/B′/NPrion protein; Chromatoid body; Aggresome; RNA granule; Cytoplasmic prion
The zinc finger protein A20 targets TRAF2 to the lysosomes for degradation by Lianyun Li; Nia Soetandyo; Qiuyan Wang; Yihong Ye (pp. 346-353).
The zinc finger-containing protein A20 is a negative regulator of TNF-induced JNK (c-Jun-N-terminal kinase) and NFκB (nuclear factor κB) signaling. A20 is an unusual enzyme that contains both ubiquitinating and deubiquitinating activities. Although A20 is mostly localized in the cytosol, our recent studies reveal that a fraction of A20 can associate with a lysosome-interacting compartment in a manner that requires its carboxy terminal zinc fingers, but independent of its ubiquitin modifying activities. Whether the lysosome-associated A20 has a function in cellular signaling is unclear. Here, we demonstrate that A20 is capable of targeting an associated signaling molecule such as TRAF2 to the lysosomes for degradation. This process is dependent on the membrane tethering zinc finger domains of A20, but does not require A20 ubiquitin modifying activity. Our findings suggest a novel mode of A20 action that involves lysosomal targeting of signal molecules bound to A20.
Keywords: Abbreviations; TNF; tumor necrosis factor; JNK; c-Jun-N-terminal kinase; NFκB; nuclear factor κB; TRAF; TNF receptor-associated factor; RIP; receptor interacting protein; TNFR; TNF receptor; OTU; ovarian tumor; MEF; mouse embryonic fibroblastA20; TRAF2; NFκB; Ubiquitin; Lysosomal degradation
Caveolin-1 regulates glioblastoma aggressiveness through the control of α5β1 integrin expression and modulates glioblastoma responsiveness to SJ749, an α5β1 integrin antagonist by Sophie Martin; Erika C. Cosset; Jérôme Terrand; Anne Maglott; Ken Takeda; Monique Dontenwill (pp. 354-367).
Caveolin-1 plays a checkpoint function in the regulation of processes often altered in cancer. Although increased expression of caveolin-1 seems to be the norm in the glioma family of malignancies, populations of caveolin-1 positive and negative cells coexist among glioblastoma specimens. As no data are available to date on the contribution of such cells to the phenotype of glioblastoma, we manipulated caveolin-1 in the glioblastoma cell line U87MG. We showed that caveolin-1 plays a critical role in the aggressiveness of glioblastoma. We identified integrins as the main set of genes affected by caveolin-1. We reported here that the phenotypic changes observed after caveolin-1 modulation were mediated by α5β1 integrins. As a consequence of the regulation of α5β1 levels by caveolin-1, the sensitivity of cells to the specific α5β1 integrin antagonist, SJ749, was affected. Mediator of caveolin-1 effects, α5β1 integrin, is also a marker for glioma aggressiveness and an efficient target for the treatment of glioma especially the ones exerting the highest aggressive phenotype.
Keywords: α5β1 integrin; Caveolin-1; α5β1 integrin antagonist; SJ749; Human glioma
Transcriptional regulation of hypoxia-inducible factor 1α by HIPK2 suggests a novel mechanism to restrain tumor growth by Lavinia Nardinocchi; Rosa Puca; Diego Guidolin; Anna S. Belloni; Gianluca Bossi; Carine Michiels; Ada Sacchi; Maurizio Onisto; Gabriella D'Orazi ⁎ (pp. 368-377).
HIPK2 has been implicated in restraining tumor progression by more than one mechanism, involving both its catalytic and transcriptional co-repressor functions. Starting from the finding that HIPK2 knockdown by RNA-interference (HIPK2i) induced significant up-regulation of HIF-1α mRNA and of its target VEGF in tumor cells, we evaluated the role of HIPK2 in transcriptional regulation of HIF-1α. We found that HIPK2 overexpression downmodulated both HIF-1α reporter activity and mRNA levels and showed that HIPK2 was bound in vivo to the HIF-1α promoter likely in a multiprotein co-repressor complex with histone deacetylase 1 (HDAC1). Thus, the HIF-1α promoter was strongly acetylated following HIPK2 knockdown. The HIF-1α-dependent VEGF transcription was evaluated by co-transfection of a dominant negative (DN) construct of HIF-1α that inhibited VEGF reporter activity induced by HIPK2 knockdown. HIF-1α and VEGF up-regulation in HIPK2i cells correlated with increased vascularity of tumor xenografts in vivo and tube formation in HUVEC in vitro. These findings provide the first evidence of HIPK2-mediated transcriptional regulation of HIF-1α that might play a critical role in VEGF expression.
Keywords: HIF-1α mRNA; HIF-1α promoter; ChIP; HIPK2; Luciferase activity; RNA interference
HIV-1 Tat C-terminus is cleaved by calpain 1: Implication for Tat-mediated neurotoxicity by Giovanni Passiatore; Slava Rom; Davide Eletto; Francesca Peruzzi ⁎ (pp. 378-387).
HIV-Encephalopathy (HIVE) is a common neurological disorder associated with HIV-1 infection and AIDS. The activity of the HIV trans-activating protein Tat is thought to contribute to neuronal pathogenesis. While Tat proteins from primary virus isolates consist of 101 or more amino acids, 72 and 86 amino acids forms of Tat are commonly used for in vitro studies. Although Tat72 contains the minimal domain required for viral replication, other activities of Tat appear to vary according to its length, sub-cellular localization, cell type and the stage of cellular differentiation. In this study, we investigated the stability of intracellular Tat101 during proliferation and differentiation of neuronal cells in culture. We have utilized rat neuronal progenitors as a model of neuronal cell proliferation and differentiation, as well as rat primary cortical neurons as a model of fully differentiated cells. Our results indicate that, upon internalization, Tat101 was degraded more rapidly in proliferating cells than in cells which either underwent neuronal differentiation or were fully differentiated. Intracellular degradation of Tat was prevented by the calpain 1 inhibitor, ALLN, in both proliferating and differentiated cells. Inhibition of calpain 1 by calpastatin peptide also prevented Tat cleavage. In vitro calpain digestion and mass spectrometry analysis further demonstrated that the sequence of Tat sensitive to calpain cleavage was located in the C-terminus of this viral protein, between amino acids 68 and 69. Moreover, cleavage of Tat101 by calpain 1 increased neurotoxic effect of this viral protein and presence of the calpain inhibitor protected neuronal cells from Tat-mediated toxicity.
Keywords: HIV-Tat; Neurotoxicity; Calpain
Tat subunit stoichiometry in Arabidopsis thaliana challenges the proposed function of TatA as the translocation pore by Mario Jakob; Susanne Kaiser; Michael Gutensohn; Peter Hanner; Ralf Bernd Klösgen (pp. 388-394).
The twin arginine translocation (Tat) machinery which is capable of transporting folded proteins across lipid bilayers operates in the thylakoid membrane of plant chloroplasts as well as in the cytoplasmic membrane of bacteria. It is composed of three integral membrane proteins (TatA, TatB, and TatC) which form heteromeric complexes of high molecular weight that accomplish binding and transport of substrates carrying Tat pathway-specific signal peptides. Western analyses using affinity purified antibodies showed in both, juvenile and adult tissue from Arabidopsis thaliana, an approximately equimolar ratio of the TatB and TatC components, whereas TatA was detectable only in minor amounts. Upon Blue Native-PAGE, TatB and TatC were found in four heteromeric TatB/C complexes possessing molecular weights of approximately 310, 370, 560 and 620 kDa, respectively, while TatA was detected only in a molecular weight range below 200 kDa. The implications of these findings on the currently existing models explaining the mechanism of Tat transport are discussed.
Keywords: ΔpH/Tat-pathway; Tat complex; Thylakoid membrane; Blue Native-PAGE
PKCδ-dependent functional switch of rpS3 between translation and DNA repair by Tae-Sung Kim; Hag Dong Kim; Joon Kim ⁎ (pp. 395-405).
Ribosomal protein S3 (rpS3) is critically involved in translation as a component of the 40S ribosomal subunit and participates in the processing of DNA damage, functioning as a damage DNA endonuclease. However, it is not yet known how the function of rpS3 switches between translation and DNA repair. Here we show that PKCδ phosphorylates rpS3 resulting in its mobilization in the nucleus to repair damaged DNA. Phosphorylated rpS3 was only detected in non-ribosomal rpS3 and the repair endonuclease activity of rpS3 was increased by its phosphorylation. In addition, rpS3 knock-down cells showed more sensitivity to genotoxic stress than control cells, and this sensitivity was corrected by overexpressed wild-type rpS3 but not by phosphorylation defective rpS3. In conclusion, we propose that the destiny of rpS3 molecules between translation and DNA repair is regulated by PKCδ-dependent phosphorylation.
Keywords: PKCδ; rpS3; Src; Endonuclease activity
Agonist-dependent phosphorylation of the formyl peptide receptor is regulated by the membrane proximal region of the cytoplasmic tail by Elena S. Suvorova; Jeannie M. Gripentrog; Algirdas J. Jesaitis; Heini M. Miettinen (pp. 406-417).
Formyl peptide receptor (FPR) is a chemoattractant G protein-coupled receptor (GPCR) involved in the innate immune response against bacteria. Receptor activation is terminated by receptor phosphorylation of two serine- and threonine-rich regions located in the distal half of the cytoplasmic tail. In this study we show that introduction of an amino acid with a bulky side chain (leucine or glutamine) adjacent to a single leucine, L320, in the membrane-proximal half of the cytoplasmic tail, significantly enhanced receptor phosphorylation, β-arrestin1/2 translocation, and receptor endocytosis, without affecting Gi-mediated ERK1/2 activation and release of intracellular calcium. In addition, the point mutations resulted in diminished susceptibility to trypsin, suggesting a conformation different from that of wild type FPR. Alignment of the FPR sequence with the rhodopsin sequence showed that L320 resides immediately C-terminal of an amphipathic region that in rhodopsin forms helix 8. Deletion of seven amino acids (Δ309–315) from the predicted helix 8 of FPR (G307–S319) caused reduced cell signaling as well as defects in receptor phosphorylation, β-arrestin1/2 translocation and endocytosis. Thus, the amino acid content in the N-terminal half of the cytoplasmic tail influences the structure and desensitization of FPR.
Keywords: Abbreviations; FPR; formyl peptide receptor; C5aR; anaphylatoxin C5a receptor; GPCR; G protein-coupled receptor; fMLF; f-methionine–leucine–phenylalanine; EndoH; endoglycosidase H; PNGase F; N-glycosidase F; mAb; monoclonal antibody; CHO; Chinese hamster ovary; GRK; G protein-coupled receptor kinase; ERK1/2; extracellular signal-regulated kinase 1/2Chemoattractant receptor; Cell signaling; Desensitization; Protein folding; Cytoplasmic helix 8
Uncoupling between Ig somatic hypermutation and oncogene mutation in mouse lymphoma by Christelle Vincent; Véronique Truffinet; Remi Fiancette; Barbara Petit; Nadine Cogné; Michel Cogné; Yves Denizot ⁎ (pp. 418-426).
Burkitt lymphoma (BL) features translocations linking c- myc to the immunoglobulin heavy chain (IgH) locus. By inserting a c- myc gene under the control of the 3′IgH locus control region (LCR) into the mouse genome, we generated c- myc–3′LCR mice that develop clonal BL or diffuse anaplastic lymphoma. We show in the present study that while BL from c- myc–3′LCR mice would be classified as pre-germinal center (GC) cells due to the absence of both BCL-6 expression and somatic hypermutation (SHM) in VH sequences, they show a high level of SHM focused on the c- myc oncogene itself. This observation suggests that the c- myc–3′IgH LCR tandem association drives development of lymphoma from naïve B cells by specifically recruiting AID activity on c- myc in a process that early becomes independent from antigen selection and where the successive rounds of SHM rather rely on the selection of the most efficient mutations for oncogene deregulation. Similar to the translocated c- myc gene in human BL, mutations were found in first exon and 5′ flanking sequences of transgenic c- myc and specially focused on negative regulatory elements, thus leading to high and constitutive oncogene expression. In conclusion while 3′IgH transcriptional enhancers in c- myc–3′LCR mice first simply act in cis to slightly stimulate c- myc transcription in untransformed B cells, the occurrence of lymphoma appears to result from an additional mechanism necessitating AID-driven mutations within the first exon and 5′ flanking sequences which does not occur in parallel but rather circumvents antigen-driven selection.
Keywords: Abbreviations; BL; Burkitt lymphoma; LCR; locus control region; Ig; immunoglobulin; CSR; class switch recombination; ES; embryonic stem; GC; germinal center; AID; activation-induced cytidine deaminaseB cell; Cell activation; Gene regulation
Attenuation of doxorubicin-induced cardiac injury by mitochondrial glutaredoxin 2 by Nicole M. Diotte; Ye Xiong; Jinping Gao; Balvin H.L. Chua; Ye-Shih Ho (pp. 427-438).
While the cardiotoxicity of doxorubicin (DOX) is known to be partly mediated through the generation of reactive oxygen species (ROS), the biochemical mechanisms by which ROS damage cardiomyocytes remain to be determined. This study investigates whether S-glutathionylation of mitochondrial proteins plays a role in DOX-induced myocardial injury using a line of transgenic mice expressing the human mitochondrial glutaredoxin 2 (Glrx2), a thiotransferase catalyzing the reduction as well as formation of protein–glutathione mixed disulfides, in cardiomyocytes. The total glutaredoxin (Glrx) activity was increased by 76% and 53 fold in homogenates of whole heart and isolated heart mitochondria of Glrx2 transgenic mice, respectively, compared to those of nontransgenic mice. The expression of other antioxidant enzymes, with the exception of glutaredoxin 1, was unaltered. Overexpression of Glrx2 completely prevents DOX-induced decreases in NAD- and FAD-linked state 3 respiration and respiratory control ratio (RCR) in heart mitochondria at days 1 and 5 of treatment. The extent of DOX-induced decline in left ventricular function and release of creatine kinase into circulation at day 5 of treatment was also greatly attenuated in Glrx2 transgenic mice. Further studies revealed that heart mitochondria overexpressing Glrx2 released less cytochrome c than did controls in response to treatment with tBid or a peptide encompassing the BH3 domain of Bid. Development of tolerance to DOX toxicity in transgenic mice is also associated with an increase in protein S-glutathionylation in heart mitochondria. Taken together, these results imply that S-glutathionylation of heart mitochondrial proteins plays a role in preventing DOX-induced cardiac injury.
Keywords: Abbreviations; DOX; doxorubicin; ROS; reactive oxygen species; GSH; glutathione; protein–SSG; protein–glutathione mixed disulfides; Glrx1; glutaredoxin 1; Glrx2; glutaredoxin Trx1, thioredoxin 1, Trx2, thioredoxin 2; RCR; respiratory control ratio; P/O ratio; ADP/O ratio; CuZnSOD; copper–zinc superoxide dismutase; MnSOD; manganese superoxide dismutase; Gpx1; glutathione peroxidase 1; α-MyHC; α-myosin heavy chain; BCA; bicinchoninic acid; HED; 2-hydroxyethyl disulfide; NEM; N; -ethylmaleimideOxidative stress; Protein; S; -glutathionylation; Transgenic mice; Mitochondria; Cytochrome; c