DOCSLIB.ORG

University of California Riverside

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
University of California Riverside UNIVERSITY OF CALIFORNIA RIVERSIDE Characterization of Tumor Derived B56gamma Mutations and Their Effect on Tumor Suppressor Function of B56gamma PP2A A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology by Yumiko Nobumori June 2013 Dissertation Committee: Dr. Xuan Liu, Chairperson Dr. Frances M. Sladek Dr. Jolinda A. Traugh Copyright by Yumiko Nobumori 2013 The Dissertation of Yumiko Nobumori is approved: _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Committee Chairperson University of California, Riverside ACKNOWLEDGEMENTS First and foremost, I would like to express my deep gratitude to Dr. Xuan Liu for her continued guidance, support, and encouragement to work in this project. I would like to thank the members of my committee Dr. Frances M. Sladek and Dr. Jolinda A. Traugh for valuable advice and encouragement. I would also like to thank past and present members of my laboratory for providing suggestions and assistance. I am grateful to Dr. Ernest Martinez, Dr. Frank U. Sauer, and members of their laboratories, and members of Dr. Frances M. Sladek’s laboratory who have provided research suggestions and experimental advice during laboratory meeting. Last but not least, I would like to thank my husband, Jonathan, for his love, kindness, and constant support throughout entire process. The materials in Chapter 2 were originally published in Oncogene. 2010; 29:3933-3941. G.P. Shouse, Y. Nobumori, and X. Liu. A B56γ mutation in lung cancer disrupts the p53- dependent tumor-suppressor function of protein phosphatase 2A. Copyright © 2010 Nature Publishing Group, a division of Macmillan Publishers Limited. The materials in Chapter 3 were originally published in The journal of biological chemistry. 2012; 287:11030-11036. Y. Nobumori, G.P. Shouse and X. Liu. HEAT repeat 1 motif is required for B56γ-containing protein phosphatase 2A (B56γ-PP2A) holoenzyme assembly and tumor-suppressive function. Copyright © 2012 the American Society for Biochemistry and Molecular Biology. iv ABSTRACT OF THE DISSERTATION Characterization of Tumor Derived B56gamma Mutations and Their Effect on Tumor Suppressor Function of B56gamma PP2A by Yumiko Nobumori Doctor of Philosophy, Graduate Program in Biochemistry and Molecular Biology University of California, Riverside, June 2013 Dr. Xuan Liu, Chairperson The heterotrimeric PP2A holoenzyme consists of a scaffolding A, a catalytic C, and a variable regulatory B subunit. Previous study has reported that B56γ subunit mediates the tumor suppressive function of PP2A. B56γ-PP2A inhibits cell transformation by dephosphorylating and activating p53. In addition, B56γ-PP2A possesses the p53-independent tumor suppressive function although its mechanism is unclear. In the present study, we demonstrate the mechanism of B56γ tumor suppressive function inactivation through characterizing B56γ mutations identified in human cancer. v First, we demonstrate that the A383G and F395C mutations disrupt B56γ from interacting and activating p53. The mutants are unable to dephosphorylate p53 and thus lack the p53-dependent tumor suppressive function. Moreover, we identify the region adjacent to these mutations as the p53 binding domain. This finding implies that the bridging interaction between B56γ and p53 is required for p53 dephosphorylation by PP2A. Second, we show that the C39R mutation within the HEAT repeat 1 disrupts B56γ binding with the AC core and thus abolishes the B56γ tumor suppressive function completely. We further demonstrate that the intact HEAT repeat 1 is required for B56γ to bind the AC core, providing structural insights into the B56γ-PP2A holoenzyme assembly. Further characterization of additional B56γ mutations reveals two classes of mutations with different mechanisms of tumor suppressive function inactivation. Mutations in the first class disrupt the interaction between B56γ and the AC core and thus fail to regulate all substrates and completely lose the tumor suppressive function. Mutations in the second class prevent specific substrates from binding B56γ and partially reduce the tumor suppressive function. Although it remains to be investigated how frequently these mutations occur, our results underline the importance of B56γ tumor suppressive function in human cancer. Finally, we identified the S220N mutation that specifically disrupts the p53-independent tumor suppressive function of B56γ, suggesting that it may disrupt the binding of unknown substrates. Identification of these proteins would contribute to the further understanding of the tumor suppressor role of B56γ in cells. vi TABLE OF CONTENTS Chapter 1: Introduction ................................................................................................................... 1 References ................................................................................................................................ 18 Figure legend ........................................................................................................................... 31 Figures ...................................................................................................................................... 32 Tables ....................................................................................................................................... 33 Chapter 2: A B56γ mutation in lung cancer disrupts the p53-dependent tumor suppressor function of protein phosphatase 2A ........................................................................................................ 35 Abstract .................................................................................................................................... 36 Introduction .............................................................................................................................. 37 Materials and Methods ............................................................................................................. 39 Results ...................................................................................................................................... 41 Discussion ................................................................................................................................ 48 References ................................................................................................................................ 51 Acknowledgements ................................................................................................................... 54 Figure legend ............................................................................................................................ 55 vii Figures ...................................................................................................................................... 59 Chapter 3: HEAT repeat 1 is required for B56γ-PP2A holoenzyme assembly and tumor suppressive function ................................................................................................................. 66 Abstract .................................................................................................................................... 67 Introduction .............................................................................................................................. 68 Materials and Methods ............................................................................................................. 71 Results ....................................................................................................................................... 74 Discussion ................................................................................................................................ 79 References ................................................................................................................................ 82 Acknowledgements ................................................................................................................... 84 Figure legend ............................................................................................................................ 85 Figures....................................................................................................................................... 88 Chapter 4: Characterization of B56γ tumor-associated mutations reveals mechanisms for inactivation of B56γ-PP2A ...................................................................................................... 94 Abstract .................................................................................................................................... 95 Introduction ............................................................................................................................... 96 viii Materials and Methods .............................................................................................................. 98 Results ..................................................................................................................................... 100 Discussion .............................................................................................................................. 108 References .............................................................................................................................
Load more

Recommended publications
  • Genome-Wide Analysis of 5-Hmc in the Peripheral Blood of Systemic Lupus Erythematosus Patients Using an Hmedip-Chip
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1467-1479, 2015 Genome-wide analysis of 5-hmC in the peripheral blood of systemic lupus erythematosus patients using an hMeDIP-chip WEIGUO SUI1*, QIUPEI TAN1*, MING YANG1, QIANG YAN1, HUA LIN1, MINGLIN OU1, WEN XUE1, JIEJING CHEN1, TONGXIANG ZOU1, HUANYUN JING1, LI GUO1, CUIHUI CAO1, YUFENG SUN1, ZHENZHEN CUI1 and YONG DAI2 1Guangxi Key Laboratory of Metabolic Diseases Research, Central Laboratory of Guilin 181st Hospital, Guilin, Guangxi 541002; 2Clinical Medical Research Center, the Second Clinical Medical College of Jinan University (Shenzhen People's Hospital), Shenzhen, Guangdong 518020, P.R. China Received July 9, 2014; Accepted February 27, 2015 DOI: 10.3892/ijmm.2015.2149 Abstract. Systemic lupus erythematosus (SLE) is a chronic, Introduction potentially fatal systemic autoimmune disease characterized by the production of autoantibodies against a wide range Systemic lupus erythematosus (SLE) is a typical systemic auto- of self-antigens. To investigate the role of the 5-hmC DNA immune disease, involving diffuse connective tissues (1) and modification with regard to the onset of SLE, we compared is characterized by immune inflammation. SLE has a complex the levels 5-hmC between SLE patients and normal controls. pathogenesis (2), involving genetic, immunologic and envi- Whole blood was obtained from patients, and genomic DNA ronmental factors. Thus, it may result in damage to multiple was extracted. Using the hMeDIP-chip analysis and valida- tissues and organs, especially the kidneys (3). SLE arises from tion by quantitative RT-PCR (RT-qPCR), we identified the a combination of heritable and environmental influences. differentially hydroxymethylated regions that are associated Epigenetics, the study of changes in gene expression with SLE.
    [Show full text]
  • PPP2R3C Gene Variants Cause Syndromic 46,XY Gonadal
    5 180 T Guran and others PPP2R3C in testis developmentQ1 180:5 291–309 Clinical Study and spermatogenesis PPP2R3C gene variants cause syndromic 46,XY gonadal dysgenesis and impaired spermatogenesis in humans Tulay Guran1, Gozde Yesil2, Serap Turan1, Zeynep Atay3, Emine Bozkurtlar4, AghaRza Aghayev5, Sinem Gul6, Ilker Tinay7, Basak Aru8, Sema Arslan9, M Kutay Koroglu10, Feriha Ercan10, Gulderen Y Demirel8, Funda S Eren4, Betul Karademir9 and Abdullah Bereket1 1Department of Paediatric Endocrinology and Diabetes, Marmara University, 2Department of Genetics, Bezm-i Alem University, 3Department of Paediatric Endocrinology and Diabetes, Medipol University, 4Department of Pathology, Marmara University, School of Medicine, Istanbul, Turkey, 5Department of Medical Genetics, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey, 6Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey, 7Department of Urology, Marmara University, School of Medicine, Istanbul, Turkey, 8Department of Immunology, Yeditepe Correspondence University, Faculty of Medicine, Istanbul, Turkey, 9Department of Biochemistry, Genetic and Metabolic Diseases should be addressed Research and Investigation Center, and 10Department of Histology and Embryology, Marmara University, School of to T Guran Medicine, Istanbul, Turkey Email [email protected] Abstract Context: Most of the knowledge on the factors involved in human sexual development stems from studies of rare cases with disorders of sex development. Here, we have described a novel 46, XY complete gonadal dysgenesis syndrome caused by homozygous variants in PPP2R3C gene. This gene encodes B″gamma regulatory subunit of the protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase involved in the phospho-regulation processes of most mammalian cell types. PPP2R3C gene is most abundantly expressed in testis in humans, while its function was hitherto unknown.
    [Show full text]
  • Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins
    International Journal of Molecular Sciences Article Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins M. Quadir Siddiqui 1,† , Maulik D. Badmalia 1,† and Trushar R. Patel 1,2,3,* 1 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; [email protected] (M.Q.S.); [email protected] (M.D.B.) 2 Department of Microbiology, Immunology and Infectious Disease, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive, Calgary, AB T2N 4N1, Canada 3 Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada * Correspondence: [email protected] † These authors contributed equally to the work. Abstract: Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and func- tions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share Citation: Siddiqui, M.Q.; Badmalia, similarities with transcriptional regulators and have positively charged electrostatic patches, which M.D.; Patel, T.R.
    [Show full text]
  • Inhibition of Polyomavirus DNA Replication by Nucleotide Analogs
    Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. Title Inhibition of polyomavirus DNA replication by nucleotide analogs Author(s) Onwubiko, Nichodemus Okechukwu Publication Date 2017-01-31 Item record http://hdl.handle.net/10379/6277 Downloaded 2021-09-27T20:27:15Z Some rights reserved. For more information, please see the item record link above. Inhibition of Polyomavirus DNA Replication by Nucleotide Analogs Nichodemus Okechukwu Onwubiko Supervised by Professor Heinz-Peter Nasheuer School of Natural Sciences, Biochemistry National University of Ireland, Galway A thesis submitted to the National University of Ireland, Galway for a degree of Doctor of Philosophy January, 2017 Table of Contents Table of contents List of Abbreviation…………………………………………………………….…….…....iv Acknowledgment…………………………………………………………………………..vii Abstract...….………………………………………………………………………………viii 1.0 Introduction.........……………………………………………………………………….1 1.1 Polyomaviruses.............…………………………………………………….………....2 1.1.1 Discovery, Classification and Disease Association of Human Polyomaviruses....2 1.1.1.1 JC Virus..….............……………………………………………………………3 1.1.1.1.1 Reactivation of JCV in Humans.......………..........…….....……………...4 1.1.1.2 BK Virus...……….............…………………………………...……………..….7 1.1.1.2.1 Disease Association of BKV...…………....……............………………...8 1.1.1.2.1.1 Polyomavirus-Associated Nephropathy...…………….................……..8 1.1.1.2.1.2 Polyomavirus-Assoiated Hemorrhagic Cystitis...……………..............10
    [Show full text]
  • Potent Inhibition of Hemangioma Formation in Rats by the Acyclic Nucleoside Phosphonate Analogue Cidofovir1
    (CANCER RESEARCH 58, 2562-2567. June 15. 1998| Potent Inhibition of Hemangioma Formation in Rats by the Acyclic Nucleoside Phosphonate Analogue Cidofovir1 Sandra Liekens,2 Gracida Andrei, Michel Vandeputte, Erik De Clercq, and Johan Neyts Rega Institute for Medical Research, Katholieke L/niversiteit Leuven, B-3000 Leuven, Belgium ABSTRACT genie therapy could improve the life span of hemangioma-bearing children. At present, IFN-a is being used with relative success for the The acyclic nucleoside phosphonate analogue cidofovir elicited a treatment of hemangiomas (7, 8). The mechanism underlying this marked protection against hemangioma growth in newborn rats that had antitumor action is, however, not completely understood. been infected i.p. with a high titer of murine polyomavirus. Untreated, We have developed a novel animal model for the study of strategies infected rats developed cutaneous, ¡.in.,and cerebral hemangiomas asso for the treatment of hemangiomas. Infection of newborn rats with a ciated with severe hemorrhage and anemia leading to death within 3 high titer of the Marseille strain of mPyV3 was found to induce weeks postinfection (p.i.). s.c. treatment with cidofovir at 25 mg/kg, once a week, resulted in a complete suppression of hemangioma development cutaneous, i.m., and intracerebral hemangiomas with a short latency and associated mortality when treatment was initiated at 3 days p.i. (100% period. The cerebral hemangiomas were associated with hemorrhage, survival compared with 0% for the untreated animals). Cidofovir still resulting in severe anemia and subsequent death of the animals within afforded 40% survival and a significant delay in tumor-associated mor 3 weeks postinfection (p.i.).
    [Show full text]
  • Journal of Virology
    JOURNAL OF VIROLOGY Volume 61 October 1987 No. 10 ANIMAL VIRUSES Analysis of Pseudorabies Virus Glycoprotein glll Localization and Modification by Using Novel Infectious Viral Mutants Carrying Unique EcoRI Sites. J. Patrick Ryan, Mary E. Whealy, Alan K. Robbins, and Lynn W. Enquist 2962-2972 Effects of Position and Orientation of the 72-Base-Pair-Repeat Transcriptional Enhancer on Replication from the Simian Virus 40 Core Origin. Settara C. Chandrasekharappa and Kiranur N. Subramanian .................... 2973-2980 Mutants of the Rous Sarcoma Virus Envelope Glycoprotein That Lack the Transmembrane Anchor and Cytoplasmic Domains: Analysis of Intracellular Transport and Assembly into Virions. Lautaro G. Perez, Gary L. Davis, and Eric Hunter....................................... 2981-2988 Sequences of Herpes Simplex Virus Type 1 That Inhibit Formation of Stable TK+ Transformants. Daniel H. Farkas, Timothy M. Block, Paul B. Hart, and Robert G. Hughes, Jr................................................ 2989-2996 Primer-Dependent Synthesis of Covalently Linked Dimeric RNA Molecules by Poliovirus Replicase. John M. Lubinski, Lynn J. Ransone, and Asim Dasgupta ........................................................... 2997-3003 Adaptor Plasmids Simplify the Insertion of Foreign DNA into Helper-Independent Retroviral Vectors. Stephen H. Hughes, Jack J. Greenhouse, Christos J. Petropoulos, and Pramod Sutrave ..................................... 3004-3012 Point Mutation in the S Gene of Hepatitis B Virus for a d/y or w/r Subtypic Change in Two Blood Donors Carrying a Surface Antigen of Compound Subtype adyr or adwr. Hiroaki Okamoto, Mitsunobu Imai, Fumio Tsuda, Takeshi Tanaka, Yuzo Miyakawa, and Makoto Mayumi ................. 3030-3034 Hepatitis A Virus cDNA and Its RNA Transcripts Are Infectious in Cell Culture. Jeffrey 1. Cohen, John R. Ticehurst, Stephen M.
    [Show full text]
  • JOURNAL of VIROLOGY Volume 52 January 1985 No
    JOURNAL OF VIROLOGY Volume 52 January 1985 No. 1 ANIMAL VIRUSES Differential Stability of Host mRNAs in Friend Erythroleukemia Cells Infected with Herpes Simplex Virus Type 1. Barbara A. Mayman and Yutaka Nishioka ............................................................ 1-6 Myristic Acid, a Rare Fatty Acid, Is the Lipid Attached to the Transforming Pro- tein of Rous Sarcoma Virus and Its Cellular Homolog. Janice E. Buss and Bartholomew M. Sefton ............. ................................. 7-12 Genome Organization of Herpesvirus Aotus Type 2. Pawel G. Fuchs, Rudiger Ruger, Herbert Pfister, and Bernhard Fleckenstein ...... ................ 13-18 Isolation and Structural Mapping of a Human c-src Gene Homologous to the Transforming Gene (v-src) of Rous Sarcoma Virus. Carol P. Gibbs, Akio Tanaka, Stephen K. Anderson, Janet Radul, Joseph Baar, Anthony Ridgway, Hsing-Jien Kung, and Donald J. Fujita ........................ 19-24 Molecular Basis of Host Range Variation in Avian Retroviruses. Andrew J. Dorner, Jonathan P. Stoye, and John M. Coffin ......................... 32-39 Assignment of the Temperature-Sensitive Lesion in the Replication Mutant Al of Vesicular Stomatitis Virus to the N Gene. M. David Marks, Jennifer Kennedy-Morrow, and Judith A. Lesnaw .............................. 44-51 Mapping of the Structural Gene of Pseudorabies Virus Glycoprotein A and Identi- fication of Two Non-Glycosylated Precursor Polypeptides. Thomas C. Mettenleiter, Noemi Lukacs, and Hanns-Joachim Rziha ..... ............ 52-57 Preliminary Characterization of an Epitope Involved in Neutralization and Cell Attachment That Is Located on the Major Bovine Rotavirus Glycoprotein. Marta Sabara, James E. Gilchrist, G. R. Hudson, and L. A. Babiuk ...... 58-66 Use of a Bacterial Expression Vector to Map the Varicella-Zoster Virus Major Glycoprotein Gene, gC.
    [Show full text]
  • A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures
    viruses Communication A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures Ryan P. Bennett 1,† , Courtney L. Finch 2,† , Elena N. Postnikova 2 , Ryan A. Stewart 1, Yingyun Cai 2 , Shuiqing Yu 2 , Janie Liang 2, Julie Dyall 2 , Jason D. Salter 1 , Harold C. Smith 1,* and Jens H. Kuhn 2,* 1 OyaGen, Inc., 77 Ridgeland Road, Rochester, NY 14623, USA; [email protected] (R.P.B.); [email protected] (R.A.S.); [email protected] (J.D.S.) 2 NIH/NIAID/DCR/Integrated Research Facility at Fort Detrick (IRF-Frederick), Frederick, MD 21702, USA; courtney.fi[email protected] (C.L.F.); [email protected] (E.N.P.); [email protected] (Y.C.); [email protected] (S.Y.); [email protected] (J.L.); [email protected] (J.D.) * Correspondence: [email protected] (H.C.S.); [email protected] (J.H.K.); Tel.: +1-585-697-4351 (H.C.S.); +1-301-631-7245 (J.H.K.) † These authors contributed equally to this work. Abstract: Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody- based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane.
    [Show full text]
  • PPP2R5D-Related Intellectual Disability and Neurodevelopmental Delay: a Review of the Current Understanding of the Genetics and Biochemical Basis of the Disorder
    International Journal of Molecular Sciences Review PPP2R5D-Related Intellectual Disability and Neurodevelopmental Delay: A Review of the Current Understanding of the Genetics and Biochemical Basis of the Disorder Dayita Biswas 1, Whitney Cary 2,3,* and Jan A. Nolta 1,2,3,* 1 SPARK Program Scholar, Institute for Regenerative Cures, University of California, Sacramento, CA 95817, USA; [email protected] 2 Stem Cell Program, UC Davis School of Medicine, The University of California, Sacramento, CA 95817, USA 3 UC Davis Gene Therapy Program, University of California, Sacramento, CA 95817, USA * Correspondence: [email protected] (W.C.); [email protected] (J.A.N.) Received: 11 December 2019; Accepted: 10 February 2020; Published: 14 February 2020 Abstract: Protein Phosphatase 2 Regulatory Subunit B0 Delta (PPP2R5D)-related intellectual disability (ID) and neurodevelopmental delay results from germline de novo mutations in the PPP2R5D gene. This gene encodes the protein PPP2R5D (also known as the B56 delta subunit), which is an isoform of the subunit family B56 of the enzyme serine/threonine-protein phosphatase 2A (PP2A). Clinical signs include intellectual disability (ID); autism spectrum disorder (ASD); epilepsy; speech problems; behavioral challenges; and ophthalmologic, skeletal, endocrine, cardiac, and genital malformations. The association of defective PP2A activity in the brain with a wide range of severity of ID, along with its role in ASD, Alzheimer’s disease, and Parkinson’s-like symptoms, have recently generated the impetus for further research into mutations within this gene. PP2A, together with protein phosphatase 1 (PP1), accounts for more than 90% of all phospho-serine/threonine dephosphorylations in different tissues.
    [Show full text]
  • 13 Effect of Hypoxia on Expression of the PP2A System in Primary Human Cardiovascular Cells: Post-Translation Modification and Involvement of Hif1α
    Abstracts 13 EFFECT OF HYPOXIA ON EXPRESSION OF THE PP2A activity was decreased (p<0.05) in both HASMC and HCF-av SYSTEM IN PRIMARY HUMAN CARDIOVASCULAR CELLS: following hypoxia (24 hour) while no effect was observed in POST-TRANSLATION MODIFICATION AND HAEC. Protein expression of the catalytic subunit of PP2A INVOLVEMENT OF HIF1a only decreased (p<0.05) after exposure to hypoxia for 72 hour in all cell lines. In HASMC, 24 hour hypoxia I Elgenaidi, JP Spiers. Department of Pharmacology and Therapeutics, Trinity College, increased demethylated PP2CA and phosphorylated PP2CA Dublin, Ireland abundance (p<0.05) and increased PME abundance (p<0.05). DMOG (100 mM) decreased both mRNA and protein Activity 10.1136/heartjnl-2017-311433.13 of PPP2CA. In conclusion, hypoxia suppresses the expression of PP2A Dysregulation of protein phosphorylation has been linked to in cardiac endothelial, smooth muscle and fibroblast with mechanical dysfunction and arrhythmias in a host of highly kinetics that are both cell and time dependent. In HASMC, 1 prevalent cardiovascular disorders. Little is known about the hypoxia cause post-translational modification of PP2A attenuat- effect of hypoxia on PP2A expression and regulation in the ing its activity. Furthermore, prolyl-hydroxylase inhibition cardiovascular system. The aim of this work was to: (i) deter- decreased PP2CA expression suggesting that HIF-1a may play mine the expression profile of PP2A in different cardiovascu- a role in modulating the PP2A system during hypoxia in the lar cells (HAEC, HASMC and HCF-av), investigate how this cardiovascular system. changes under hypoxia and following hydroxylase inhibition, (ii) study post-translational regulation of PP2A in HASMC REFERENCE under hypoxic conditions.
    [Show full text]
  • Temporal Proteomic Analysis of HIV Infection Reveals Remodelling of The
    1 1 Temporal proteomic analysis of HIV infection reveals 2 remodelling of the host phosphoproteome 3 by lentiviral Vif variants 4 5 Edward JD Greenwood 1,2,*, Nicholas J Matheson1,2,*, Kim Wals1, Dick JH van den Boomen1, 6 Robin Antrobus1, James C Williamson1, Paul J Lehner1,* 7 1. Cambridge Institute for Medical Research, Department of Medicine, University of 8 Cambridge, Cambridge, CB2 0XY, UK. 9 2. These authors contributed equally to this work. 10 *Correspondence: [email protected]; [email protected]; [email protected] 11 12 Abstract 13 Viruses manipulate host factors to enhance their replication and evade cellular restriction. 14 We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a 15 comprehensive time course analysis of >6,500 viral and cellular proteins during HIV 16 infection. To enable specific functional predictions, we categorized cellular proteins regulated 17 by HIV according to their patterns of temporal expression. We focussed on proteins depleted 18 with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be 19 necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the 20 B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). 21 Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent 22 hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. 23 The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral 2 24 lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and 25 conserved Vif function.
    [Show full text]
  • Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications
    Stem Cell Rev and Rep DOI 10.1007/s12015-016-9662-8 Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications Behnam Ahmadian Baghbaderani 1 & Adhikarla Syama2 & Renuka Sivapatham3 & Ying Pei4 & Odity Mukherjee2 & Thomas Fellner1 & Xianmin Zeng3,4 & Mahendra S. Rao5,6 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract We have recently described manufacturing of hu- help determine which set of tests will be most useful in mon- man induced pluripotent stem cells (iPSC) master cell banks itoring the cells and establishing criteria for discarding a line. (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Keywords Induced pluripotent stem cells . Embryonic stem Reports, 5(4), 647–659, 2015). In this manuscript, we de- cells . Manufacturing . cGMP . Consent . Markers scribe the detailed characterization of the two iPSC clones generated using this process, including whole genome se- quencing (WGS), microarray, and comparative genomic hy- Introduction bridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibra- Induced pluripotent stem cells (iPSCs) are akin to embryonic tion material and with a reporter subclone and lines made by a stem cells (ESC) [2] in their developmental potential, but dif- similar process from different donors. We believe that iPSCs fer from ESC in the starting cell used and the requirement of a are likely to be used to make multiple clinical products. We set of proteins to induce pluripotency [3]. Although function- further believe that the lines used as input material will be used ally identical, iPSCs may differ from ESC in subtle ways, at different sites and, given their immortal status, will be used including in their epigenetic profile, exposure to the environ- for many years or even decades.
    [Show full text]