DOCSLIB.ORG

The Iron Regulatory Protein/Iron Responsive Element (IRP/IRE) System: Functional Studies of New Target Mrnas and Pathological Implicatio Ns for Novel IRE Mutations

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

The Iron Regulatory Protein/Iron Responsive Element (IRP/IRE) system: functional studies of new target mRNAs and pathological implicatio ns for novel IRE mutations Sara Luscieti ADVERTIMENT . La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX ( www.tdx.cat ) i a través del Dipòsit Digital de la UB ( diposit.ub.edu ) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposici ó des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB . No s’autori tza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA . La consulta de esta tesis queda condicionada a la aceptación de las siguientes condiciones de uso: La difusión de esta tesis por medio del servicio TDR ( www.tdx.cat ) y a través del Repositorio Di gital de la UB ( diposit.ub.edu ) ha sido autorizada por los titulares de los derechos de propiedad intelectual únicamente para usos privados enmarcados en actividades de investigación y docencia. No se autoriza su reproducción con finalidades de lucro ni su difusión y puesta a disposición desde un sitio ajeno al servicio TDR o al Repositorio Digital de la UB . No se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR o al Repositorio Digital de la UB (framing). Esta reserva de derechos afecta tanto al resumen de presentación de la tesis como a sus contenidos. En la utilización o cita de partes de la tesis es obligado indicar el nombre de la persona autora. WARNING . On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX ( www.tdx.cat ) service and by the UB Digital Repository ( diposit.ub.edu ) has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized n or its spreading and availability from a site foreign to the TDX service or to the UB Digital Repository . Introducing its content in a window or frame foreign to th e TDX service or to the UB Digital Repository is not authorized (framing). Tho s e rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the au thor. UNIVERSITAT DE BARCELONA FACULTAT DE FARMÀCIA I CIÈNCIES DE L’ALIMENTACIÓ PROGRAMA DE DOCTORAT EN BIOMEDICINA The Iron Regulatory Protein/Iron Responsive Element (IRP/IRE) system: functional studies of new target mRNAs and pathological implications for novel IRE mutations. SARA LUSCIETI 2016 UNIVERSITAT DE BARCELONA FACULTAT DE FARMÀCIA I CIÈNCIES DE L’ALIMENTACIÓ PROGRAMA DE DOCTORAT EN BIOMEDICINA The Iron Regulatory Protein/Iron Responsive Element (IRP/IRE) system: functional studies of new target mRNAs and pathological implications for novel IRE mutations. Memòria presentada per Sara Luscieti per optar al títol de doctor per la Universitat de Barcelona Doctoranda: Sara Luscieti Directora: Mayka Sánchez Fernández Tutor: Rafael Oliva Virgili SARA LUSCIETI 2016 SUMMARY SUMMARY Iron is an essential micronutrient required for many cellular reactions and a tight regulation of its metabolism is therefore crucial for health. Cellular iron homeostasis relies on the coordination of iron uptake, storage and mobilization. These processes are controlled post- transcriptionally by the IRP/IRE regulatory system. The Iron Regulatory Proteins (IRP1 and IRP2) can recognize cis-regulatory mRNA motifs termed IREs (Iron Responsive Elements), conserved RNA elements located in the untranslated regions (UTR) of mRNAs that encode for proteins involved in iron metabolism. IRP/IRE interactions regulate the expression of mRNAs encoding for proteins for iron acquisition, storage, utilization and export in response to cellular iron level itself being the interaction of the IRPs with IRE motifs promoted under iron-deficient conditions and abolished in iron-replete conditions. Depending on the location of the IRE, IRPs binding regulates gene expression differentially: IRPs inhibit translation initiation when bound to IREs at the 5’ UTR, while IRPs association with 3’ IREs stabilizes and protects the mRNA from degradation. The lack of control of expression of IRE-containing mRNAs is associated in humans with pathological conditions showing the importance of components of the IRP/IRE regulatory system. In the last decades, significant progress has been made in the iron metabolism field, however, post-transcriptional regulation of gene expression by the IRP/IRE regulatory system has been limited to a small subset of known genes. A genome-wide study carried out by our group to characterize the whole repertoire of mRNAs that can interact with the IRPs, identified 35 novel IRP1 and IRP2 candidate target-genes. This work focused on the validation and functional characterization of one of this candidates: Profilin2 (Pfn2). Pfn2 is an actin-binding protein involved in the control of cytoskeletal dynamics. We identified a conserved IRE in the 3’ UTR of Pfn2 mRNA which is functional in in vitro binding studies with IRP1 and IRP2. Pfn2 mRNA showed preferentially downregulation under iron-excess condition in cell lines and we demonstrated to be regulated by IRPs-mediated stabilization in vivo, since Pfn2 mRNA levels are reduced in a mouse model with Irp1 and Irp2 gene inactivation. Moreover, the reduction of cellular free iron levels by Pfn2 overexpression experiments in cell lines, as well as, the misregulation of iron distribution observed in mice knockout for Pfn2 gene, revealed Pfn2 as a previously unrecognized player in iron metabolism. In addition, we also contributed to the identification of a functional 3’ IRE in human BDH2 mRNA, a protein involved in lipocalin-siderophores iron-trafficking, as well as, in the identification and characterization of two novel L-ferritin IRE mutations (Heidelberg +52G>C and Badalona+36C>U) causative of Hereditary Hyperferritinemia Cataract Syndrome and a novel mutation in ALAS2 IRE demonstrated to be a modifier of clinical severity in a family with Erythropoietic Protoporphyria. RESUMEN RESUMEN El hierro es un micronutriente esencial requerido por múltiples reacciones celulares y una regulación adecuada de su metabolismo es fundamental para la salud. La homeostasis celular del hierro está basada en la coordinación de la absorción, el almacenamiento y la movilización de este elemento. Estos procesos son controlados post-transcripcionalmente por el sistema IRP/IRE. Las proteínas reguladoras del hierro (IRP1 e IRP2) reconocen un motivo estructural presente en el ARNm de algunos genes denominado IRE (Iron Responsive Element). El IRE es un motivo conservado y situado en las regiones no traducidas (UTR) de los ARNm de proteínas implicadas en el metabolismo del hierro. Las interacciones IRP/IRE regulan la expresión de los ARNm que codifican proteínas para la adquisición, el almacenamiento, la utilización y la exportación de hierro en respuesta a los niveles celulares de hierro, porque la interacción de las IRPs con los motivos IRE se ve incrementada a niveles bajos de hierro y disminuida en condiciones altas de hierro. Según la localización de los IRE, las IRPs regulan de manera distinta la expresión de sus dianas: las IRPs inhiben la iniciación de la traducción cuando se unen a IREs situados en el 5' UTR, mientras que su asociación con los IREs del 3' UTR estabiliza y protege dicho ARNm de la degradación. La falta de coordinación de la expresión de genes que contienen IRE está asociada con condiciones patológicas ilustrando la importancia de los componentes del sistema regulador IRP/IRE. En las últimas décadas se han realizado progresos importantes en el campo del metabolismo del hierro. Sin embargo, la regulación post-transcripcional de la expresión génica por el sistema IRP/IRE se ha limitado a un pequeño subconjunto de genes. Un estudio “genome- wide” llevado a cabo en nuestro grupo para la caracterización global del sistema regulador IRP/IRE dio como resultado la identificación de 35 genes diana que se unen a IRP1 e IRP2. La presente tesis tiene como objetivo general validar y caracterizar funcionalmente una de esas dianas: Profilin2 (Pfn2). Pfn2 es una proteína que une actina y que está involucrada en la regulación de la dinámica del citoesqueleto. Hemos identificado un IRE localizado en el 3’ UTR del ARNm de Pfn2; el IRE es funcional en estudios in vitro para la unión a IRP1 e IRP2. El ARNm de Pfn2 viene preferentemente regulado y reducido en condiciones altas de hierro en líneas celulares y hemos demostrado in vivo que es regulado por estabilización mediada de las IRPs, ya que los niveles de ARNm de Pfn2 se encuentran reducidos en ratones a los que los genes de Irp1 y Irp2 han sido inactivados. Así mismo, la sobrexpresión de Pfn2 en líneas celulares asociada a una reducción en los niveles de hierro libre, así como la desregulación de la distribución del hierro encontrada en ratones “knockout” para el gen de Pfn2; convierten a Pfn2 como un actor nuevo en el metabolismo del hierro. Por otro lado hemos contribuido tanto a la identificación de un 3’ IRE funcional en el ARNm humano de BDH2, una proteína involucrada en el tráfico de hierro a través del sistema de lipocalinas-sideróforos, como a la identificación de dos nuevas mutaciones en el IRE de la Ferritina L (Heidelberg +52G>C y Badalona+36C>U) responsables del Síndrome Hereditario de Hiperferritinemia con Cataratas y también a la identificación de una nueva mutación en el IRE de ALAS2 que ha demostrado ser un modificador de la importancia clínica de los síntomas en una familia con Protoporfiria Eritropoyética.
Recommended publications
  • Analysis of the Stability of 70 Housekeeping Genes During Ips Reprogramming Yulia Panina1,2*, Arno Germond1 & Tomonobu M

    Analysis of the Stability of 70 Housekeeping Genes During Ips Reprogramming Yulia Panina1,2*, Arno Germond1 & Tomonobu M

    www.nature.com/scientificreports OPEN Analysis of the stability of 70 housekeeping genes during iPS reprogramming Yulia Panina1,2*, Arno Germond1 & Tomonobu M. Watanabe1 Studies on induced pluripotent stem (iPS) cells highly rely on the investigation of their gene expression which requires normalization by housekeeping genes. Whether the housekeeping genes are stable during the iPS reprogramming, a transition of cell state known to be associated with profound changes, has been overlooked. In this study we analyzed the expression patterns of the most comprehensive list to date of housekeeping genes during iPS reprogramming of a mouse neural stem cell line N31. Our results show that housekeeping genes’ expression fuctuates signifcantly during the iPS reprogramming. Clustering analysis shows that ribosomal genes’ expression is rising, while the expression of cell-specifc genes, such as vimentin (Vim) or elastin (Eln), is decreasing. To ensure the robustness of the obtained data, we performed a correlative analysis of the genes. Overall, all 70 genes analyzed changed the expression more than two-fold during the reprogramming. The scale of this analysis, that takes into account 70 previously known and newly suggested genes, allowed us to choose the most stable of all genes. We highlight the fact of fuctuation of housekeeping genes during iPS reprogramming, and propose that, to ensure robustness of qPCR experiments in iPS cells, housekeeping genes should be used together in combination, and with a prior testing in a specifc line used in each study. We suggest that the longest splice variants of Rpl13a, Rplp1 and Rps18 can be used as a starting point for such initial testing as the most stable candidates.
  • Review Article PTEN Gene: a Model for Genetic Diseases in Dermatology

    Review Article PTEN Gene: a Model for Genetic Diseases in Dermatology

    The Scientific World Journal Volume 2012, Article ID 252457, 8 pages The cientificWorldJOURNAL doi:10.1100/2012/252457 Review Article PTEN Gene: A Model for Genetic Diseases in Dermatology Corrado Romano1 and Carmelo Schepis2 1 Unit of Pediatrics and Medical Genetics, I.R.C.C.S. Associazione Oasi Maria Santissima, 94018 Troina, Italy 2 Unit of Dermatology, I.R.C.C.S. Associazione Oasi Maria Santissima, 94018 Troina, Italy Correspondence should be addressed to Carmelo Schepis, [email protected] Received 19 October 2011; Accepted 4 January 2012 Academic Editors: G. Vecchio and H. Zitzelsberger Copyright © 2012 C. Romano and C. Schepis. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PTEN gene is considered one of the most mutated tumor suppressor genes in human cancer, and it’s likely to become the first one in the near future. Since 1997, its involvement in tumor suppression has smoothly increased, up to the current importance. Germline mutations of PTEN cause the PTEN hamartoma tumor syndrome (PHTS), which include the past-called Cowden, Bannayan- Riley-Ruvalcaba, Proteus, Proteus-like, and Lhermitte-Duclos syndromes. Somatic mutations of PTEN have been observed in glioblastoma, prostate cancer, and brest cancer cell lines, quoting only the first tissues where the involvement has been proven. The negative regulation of cell interactions with the extracellular matrix could be the way PTEN phosphatase acts as a tumor suppressor. PTEN gene plays an essential role in human development. A recent model sees PTEN function as a stepwise gradation, which can be impaired not only by heterozygous mutations and homozygous losses, but also by other molecular mechanisms, such as transcriptional regression, epigenetic silencing, regulation by microRNAs, posttranslational modification, and aberrant localization.
  • Human Phosphatase CDC14A Regulates Actin Organization Through Dephosphorylation of Epithelial Protein Lost in Neoplasm

    Human Phosphatase CDC14A Regulates Actin Organization Through Dephosphorylation of Epithelial Protein Lost in Neoplasm

    Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm Nan-Peng Chena,b, Borhan Uddina,b, Robert Hardta, Wen Dinga, Marko Panica,b, Ilaria Lucibelloa, Patricia Kammererc, Thomas Rupperta, and Elmar Schiebela,1 aZentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ)–ZMBH Allianz, Universität Heidelberg, 69120 Heidelberg, Germany; bHartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), Universität Heidelberg, 69120 Heidelberg, Germany; and cMax Planck Institute of Biochemistry, 82152 Martinsried, Germany Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved April 11, 2017 (received for review November 24, 2016) CDC14 is an essential dual-specificity phosphatase that counteracts Because there have been no systematic attempts to identify CDK1 activity during anaphase to promote mitotic exit in Saccharo- phospho-sites that are regulated by hCDC14A, we know relatively myces cerevisiae. Surprisingly, human CDC14A is not essential for little about the identity of either the proteins that are dephos- cell cycle progression. Instead, it regulates cell migration and cell phorylated by hCDC14A or, indeed, the kinases that counteract adhesion. Little is known about the substrates of hCDC14A and hCDC14A in human cells. The only known substrate of hCDC14A the counteracting kinases. Here, we combine phospho-proteome that acts within the actin cytoskeleton is the protein Kibra (20, 21). profiling and proximity-dependent biotin identification to identify However, considering the broad distribution of hCDC14A hCDC14A substrates. Among these targets were actin regulators, throughout the actin network, it is highly likely that hCDC14A including the tumor suppressor eplin. hCDC14A counteracts EGF- dephosphorylates additional actin-associated proteins.
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation

    Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation

    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
  • Genotype Frequency Distributions of 28 SNP Markers in Two Commercial

    Genotype Frequency Distributions of 28 SNP Markers in Two Commercial

    Li et al. BMC Genetics (2020) 21:12 https://doi.org/10.1186/s12863-020-0815-z RESEARCH ARTICLE Open Access Genotype frequency distributions of 28 SNP markers in two commercial lines and five Chinese native chicken populations Jing-Jing Li1, Long Zhang2, Peng Ren1, Ye Wang1, Ling-Qian Yin1, Jin-Shan Ran1, Xian-Xian Zhang1 and Yi-Ping Liu1* Abstract Background: Modern breeding in the poultry industry mainly aims to produce high-performance poultry lines and breeds in two main directions of productivity, meat and eggs. To understand more about the productive potential of lowly selected Chinese native chicken populations, we selected 14 representative SNP markers strongly associated with growth traits or carcass traits and 14 SNP markers strongly associated with egg laying traits through previous reports. By using the MassArray technology, we detected the genotype frequency distributions of these 28 SNP markers in seven populations including four lowly selected as well as one moderately selected Sichuan native chicken populations, one commercial broiler line and one commercial layer line. Results: Based on the genotype frequency distributions of these 28 SNP markers in 5 native chicken populations and 2 commercial lines, the results suggested that these Chinese indigenous chicken populations have a relatively close relationship with the commercial broiler line but a marked distinction from the commercial layer line. Two native chicken breeds, Shimian Caoke Chicken and Daheng Broilers, share similar genetic structure with the broiler line. Conclusions: Our observations may help us to better select and breed superior domestic chickens and provide new clues for further study of breeding programs in local chicken populations.
  • Induction of Micrornas, Mir-155, Mir-222, Mir-424 and Mir-503, Promotes Monocytic Differentiation Through Combinatorial Regulation

    Induction of Micrornas, Mir-155, Mir-222, Mir-424 and Mir-503, Promotes Monocytic Differentiation Through Combinatorial Regulation

    Leukemia (2010) 24, 460–466 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ‘ ORIGINAL ARTICLE Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation ARR Forrest1,2, M Kanamori-Katayama1, Y Tomaru1, T Lassmann1, N Ninomiya1, Y Takahashi1, MJL de Hoon1, A Kubosaki1, A Kaiho1, M Suzuki1, J Yasuda1, J Kawai1, Y Hayashizaki1, DA Hume3 and H Suzuki1 1LSA Technology Development Unit, Omics Science Center, RIKEN Yokohama Institute, Yokohama, Kanagawa, Japan; 2The Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Queensland, Australia and 3The Roslin Institute, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Roslin, UK Acute myeloid leukemia (AML) involves a block in terminal that might promote differentiation. MicroRNAs are short 21–22 differentiation of the myeloid lineage and uncontrolled prolif- nucleotide RNA molecules that cause translational repression eration of a progenitor state. Using phorbol myristate acetate and degradation of multiple mRNAs by binding target sequences (PMA), it is possible to overcome this block in THP-1 cells 0 5 (an M5-AML containing the MLL-MLLT3 fusion), resulting in in their 3 UTRs. Multiple microRNAs are implicated in differentiation to an adherent monocytic phenotype. As part of differentiation of cell lineages including skeletal muscle, FANTOM4, we used microarrays to identify 23 microRNAs that adipocytes and neurons6–8 and in mammals, the microRNA are regulated by PMA. We identify four PMA-induced micro- subsystem is essential for embryonic development. Dicer RNAs (mir-155, mir-222, mir-424 and mir-503) that when over- is required for differentiation of embryonic stem cells,9 Dicer expressed cause cell-cycle arrest and partial differentiation and (À/À) knockouts are embryonic lethal10 and Ago2 (À/À) when used in combination induce additional changes not seen 11 by any individual microRNA.
  • Human Leucine-Rich Repeat Proteins: a Genome-Wide Bioinformatic Categorization and Functional Analysis in Innate Immunity

    Human Leucine-Rich Repeat Proteins: a Genome-Wide Bioinformatic Categorization and Functional Analysis in Innate Immunity

    Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity Aylwin C. Y. Nga,b,1, Jason M. Eisenberga,b,1, Robert J. W. Heatha, Alan Huetta, Cory M. Robinsonc, Gerard J. Nauc, and Ramnik J. Xaviera,b,2 aCenter for Computational and Integrative Biology, and Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; bThe Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142; and cMicrobiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 Edited by Jeffrey I. Gordon, Washington University School of Medicine, St. Louis, MO, and approved June 11, 2010 (received for review February 17, 2010) In innate immune sensing, the detection of pathogen-associated proteins have been implicated in human diseases to date, notably molecular patterns by recognition receptors typically involve polymorphisms in NOD2 in Crohn disease (8, 9), CIITA in leucine-rich repeats (LRRs). We provide a categorization of 375 rheumatoid arthritis and multiple sclerosis (10), and TLR5 in human LRR-containing proteins, almost half of which lack other Legionnaire disease (11). identifiable functional domains. We clustered human LRR proteins Most LRR domains consist of a chain of between 2 and 45 by first assigning LRRs to LRR classes and then grouping the proteins LRRs (12). Each repeat in turn is typically 20 to 30 residues long based on these class assignments, revealing several of the resulting and can be divided into a highly conserved segment (HCS) fol- protein groups containing a large number of proteins with certain lowed by a variable segment (VS).
  • Human Phosphatase CDC14A Is Recruited to the Cell Leading Edge to Regulate Cell Migration and Adhesion

    Human Phosphatase CDC14A Is Recruited to the Cell Leading Edge to Regulate Cell Migration and Adhesion

    Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion Nan-Peng Chena,b, Borhan Uddina,b, Renate Voitc, and Elmar Schiebela,1 aZentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ)-Zentrum für Molekulare Biologie (ZMBH) Allianz, 69120 Heidelberg, Germany; bHartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, Universität Heidelberg, 69120 Heidelberg, Germany; and cMolecular Biology of the Cell II, Deutsches Krebsforschungszentrum (DKFZ), 69120 Heidelberg, Germany Edited by James E. Cleaver, University of California, San Francisco, CA, and approved December 17, 2015 (received for review August 6, 2015) Cell adhesion and migration are highly dynamic biological pro- With mitotic exit, the proteins that were phosphorylated by cesses that play important roles in organ development and CDK1 are dephosphorylated, so that cells can return to the cancer metastasis. Their tight regulation by small GTPases and nonmitotic, interphase status (13). In Saccharomyces cerevisiae, protein phosphorylation make interrogation of these key pro- cell-division cycle 14 (Cdc14) is the major phosphatase that cesses of great importance. We now show that the conserved dual- counteracts Cdk1 activity after the metaphase–anaphase transi- specificity phosphatase human cell-division cycle 14A (hCDC14A) tion (14). The function of Cdc14 in budding yeast has been well associates with the actin cytoskeleton of human cells. To un- studied and two regulatory networks have been identified that derstand hCDC14A function at this location, we manipulated native promote the release of Cdc14 from the RENT complex in the PD loci to ablate hCDC14A phosphatase activity (hCDC14A )inun- nucleolus: “Cdc Fourteen Early Anaphase Release” (FEAR) and transformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines “Mitotic Exit Network” (MEN) (15–17).
  • Transdifferentiation of Human Mesenchymal Stem Cells

    Transdifferentiation of Human Mesenchymal Stem Cells

    Transdifferentiation of Human Mesenchymal Stem Cells Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Tatjana Schilling aus San Miguel de Tucuman, Argentinien Würzburg, 2007 Eingereicht am: Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Martin J. Müller Gutachter: PD Dr. Norbert Schütze Gutachter: Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbstständig angefertigt und keine anderen als die von mir angegebenen Hilfsmittel und Quellen verwendet habe. Des Weiteren erkläre ich, dass diese Arbeit weder in gleicher noch in ähnlicher Form in einem Prüfungsverfahren vorgelegen hat und ich noch keinen Promotionsversuch unternommen habe. Gerbrunn, 4. Mai 2007 Tatjana Schilling Table of contents i Table of contents 1 Summary ........................................................................................................................ 1 1.1 Summary.................................................................................................................... 1 1.2 Zusammenfassung..................................................................................................... 2 2 Introduction.................................................................................................................... 4 2.1 Osteoporosis and the fatty degeneration of the bone marrow..................................... 4 2.2 Adipose and bone
  • Download (4MB)

    Download (4MB)

    DISSERTATION submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by M.Sc. Daniela Casarrubea born in Palermo (Italy) Oral examination: 1st February 2013 A NEW MOUSE MODEL WITH GAIN OF IRON REGULATORY PROTEIN 1 FUNCTION Referees: 1. Dr. Darren Gilmour 2. Prof. Dr. Klaus Unsicker SUMMARY SUMMARY Disorders of iron metabolism account for some of the most common human diseases, such as anemias and hemochromatosis. To maintain physiological iron balance, homeostatic mechanisms are normally in place both at the systemic and the cellular level. Cellular iron homeostasis is secured by Iron Regulatory Proteins (IRP) −1 and −2 through their binding to cis- regulatory iron-responsive elements (IRE) in target mRNAs encoding proteins with key functions in iron metabolism. In turn, the IRE−binding activity of the two IRPs is feedback regulated by the cellular labile iron pool. Mouse models with IRP deficiency have contributed valuable insights into the in vivo roles of the IRP/IRE system as well as mammalian iron biology. However, the physiological consequences of gain of IRP function have so far remained unexplored. To investigate the importance of adequate IRP expression in vivo, we have generated a mouse model allowing conditional gain of IRP function using Cre/Lox technology. This new line expresses a flag-tagged IRP1 mutant (IRP1*), which escapes iron-mediated regulation, being constitutively active in its IRE binding form. Systemic expression of the IRP1* transgene from the Rosa26 locus yields viable animals with gain of IRE-binding activity in all organs that were analyzed.
  • Live-Cell Imaging Rnai Screen Identifies PP2A–B55α and Importin-Β1 As Key Mitotic Exit Regulators in Human Cells

    Live-Cell Imaging Rnai Screen Identifies PP2A–B55α and Importin-Β1 As Key Mitotic Exit Regulators in Human Cells

    LETTERS Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells Michael H. A. Schmitz1,2,3, Michael Held1,2, Veerle Janssens4, James R. A. Hutchins5, Otto Hudecz6, Elitsa Ivanova4, Jozef Goris4, Laura Trinkle-Mulcahy7, Angus I. Lamond8, Ina Poser9, Anthony A. Hyman9, Karl Mechtler5,6, Jan-Michael Peters5 and Daniel W. Gerlich1,2,10 When vertebrate cells exit mitosis various cellular structures can contribute to Cdk1 substrate dephosphorylation during vertebrate are re-organized to build functional interphase cells1. This mitotic exit, whereas Ca2+-triggered mitotic exit in cytostatic-factor- depends on Cdk1 (cyclin dependent kinase 1) inactivation arrested egg extracts depends on calcineurin12,13. Early genetic studies in and subsequent dephosphorylation of its substrates2–4. Drosophila melanogaster 14,15 and Aspergillus nidulans16 reported defects Members of the protein phosphatase 1 and 2A (PP1 and in late mitosis of PP1 and PP2A mutants. However, the assays used in PP2A) families can dephosphorylate Cdk1 substrates in these studies were not specific for mitotic exit because they scored pro- biochemical extracts during mitotic exit5,6, but how this relates metaphase arrest or anaphase chromosome bridges, which can result to postmitotic reassembly of interphase structures in intact from defects in early mitosis. cells is not known. Here, we use a live-cell imaging assay and Intracellular targeting of Ser/Thr phosphatase complexes to specific RNAi knockdown to screen a genome-wide library of protein substrates is mediated by a diverse range of regulatory and targeting phosphatases for mitotic exit functions in human cells. We subunits that associate with a small group of catalytic subunits3,4,17.
  • The Anaphase Promoting Complex Impacts Repair Choice by Protecting Ubiquitin Signalling at DNA Damage Sites

    The Anaphase Promoting Complex Impacts Repair Choice by Protecting Ubiquitin Signalling at DNA Damage Sites

    ARTICLE Received 13 Oct 2016 | Accepted 25 Apr 2017 | Published 12 Jun 2017 DOI: 10.1038/ncomms15751 OPEN The anaphase promoting complex impacts repair choice by protecting ubiquitin signalling at DNA damage sites Kyungsoo Ha1,2,3,*, Chengxian Ma4,*, Han Lin3, Lichun Tang1,2, Zhusheng Lian5, Fang Zhao6, Ju-Mei Li7, Bei Zhen1,2, Huadong Pei1,2, Suxia Han5, Marcos Malumbres8, Jianping Jin7, Huan Chen1,2, Yongxiang Zhao6, Qing Zhu4 & Pumin Zhang1,2,3 Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APCCdh1 plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1. We show that the removal of USP1 depends on APCCdh1 and requires Chk1 activation known to be catalysed by ssDNA-RPA-ATR sig- nalling at the ends designated for HDR, linking the status of end processing to RIF1 or BRCA1 recruitment. 1 National Center for Protein Sciences Beijing, Life Sciences Park, Beijing 102206, China. 2 State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China.