DOCSLIB.ORG

Human Prion-Like Proteins and Their Relevance in Disease

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Human Prion-Like Proteins and Their Relevance in Disease ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Universitat Autònoma de Barcelona Departament de Bioquímica i Biologia Molecular Institut de Biotecnologia i Biomedicina HUMAN PRION-LIKE PROTEINS AND THEIR RELEVANCE IN DISEASE Doctoral thesis presented by Cristina Batlle Carreras for the degree of PhD in Biochemistry, Molecular Biology and Biomedicine from the Universitat Autònoma de Barcelona. The work described herein has been performed in the Department of Biochemistry and Molecular Biology and in the Institute of Biotechnology and Biomedicine, supervised by Prof. Salvador Ventura i Zamora. Cristina Batlle Carreras Prof. Salvador Ventura i Zamora Bellaterra, 2020 Protein Folding and Conformational Diseases Lab. This work was financed with the fellowship “Formación de Profesorado Universitario” by “Ministerio de Ciencia, Innovación y Universidades”. This work is licensed under a Creative Commons Attributions-NonCommercial-ShareAlike 4.0 (CC BY-NC- SA 4.0) International License. The extent of this license does not apply to the copyrighted publications and images reproduced with permission. (CC BY-NC-SA 4.0) Batlle, Cristina: Human prion-like proteins and their relevance in disease. Doctoral Thesis, Universitat Autònoma de Barcelona (2020) English summary ENGLISH SUMMARY Prion-like proteins have attracted significant attention in the last years. The interest in these polypeptides owes to: i) their implication in degenerative diseases; ii) the presence of disease- causing mutations that increase the risk to develop the disease; iii) their ability to establish protein-protein interactions; and iv) their participation in membraneless organelles formation. Yeast prions are proteins with the ability to switch between a soluble an amyloid state as an adaptive mechanism to confront environmental changes. Most of the yeast prions have a disordered prion forming domain (PFD) of low complexity, enriched in Gln and Asn, and depleted of hydrophobic and charged residues. In order to discover novel yeast prions, computational tools were developed and further used to uncover proteins with domains similar to yeast PFDs in other proteomes, including human. These proteins were named prion-like, and their identified regions prion-like domains (PrLDs). PrLDs are similar in composition to yeast PFDs, and they share the ability to aggregate, replicate, and propagate. Cytoplasmic deposits of prion-like proteins have been observed in many degenerative diseases, and genetic mutations in their PrLD have been described. Most of the prion-like proteins are nuclear, but they translocate to the cytoplasm in disease conditions. This phenomenon has been associated with prion-like proteins aggregation into amyloid fibrils. Therefore, in this thesis we identified potential new prion-like proteins’ candidates and experimentally validated the region responsible for aggregation of seven of them. We also improve predictions for disease-causing mutations impact in prion-like proteins aggregation process. It was observed that prion-like proteins have the ability to form dynamic and reversible membraneless organelles, depending on the cellular conditions, for example, under stress. When these changes are too persistent, membraneless formation is not reversible anymore and they result in the appearance of the solid deposits recurrently observed in patients. We are still far from deciphering the exact mechanism of membraneless organelles formation. For this reason, in this thesis, we have studied the human prion-like protein hnRNPDL to try to expand the knowledge of this phenomenon and its connection to disease. We characterized hnRNPDL isoforms self-assembly properties demonstrating the importance of alternative splicing in controlling protein phase separation events, and show that D378N/H disease mutations accelerates hnRNPDL aggregation dramatically reducing its solubility in the muscle of Drosophila. Prion-like proteins are hubs of large protein-protein interaction networks through their PrLDs. Interestingly, a significant number of yeast PFDs have coiled-coil regions (CC) that participate in their aggregation process. We demonstrate here that this is also the case for human PrLDs and characterize the MED15 protein as an example, showing that its PrLD forms amyloid fibrils in a process mediated by its CC, which can account for its role in disease. In general, this thesis is an extensive study of prion-like proteins and their properties. 1 2 Resum en català RESUM EN CATALÀ Les “prion-like proteins” han anat adquirint una atenció cada cop major en els últims anys. El seu interès recau en: i) la seva implicació en malalties degeneratives; ii) la quantitat d’elles que presenten mutacions genètiques que augmenten el risc a patir la malaltia; iii) la seva habilitat d’establir interaccions proteiques; i iv) la seva participació en la formació dels orgànuls sense membrana. Els prions de llevat són proteïnes amb la capacitat de canviar d’un estat soluble a un amiloide com a sistema adaptatiu per fer front a canvis ambientals. La majoria dels prions de llevat tenen un “prion forming domain” (PFD) desordenat i de baixa complexitat, enriquit en Gln i Asn, i empobrit en residus hidrofòbics o amb càrrega. Per tal de descobrir nous prions de llevat, es van desenvolupar eines computacionals que després es van usar per buscar proteïnes amb dominis similars als PFDs de llevat en altres proteomes, incloent l’humà. Aquestes proteïnes es van anomenar “prion-like”, i les seves regions identificades “prion-like domains” (PrLDs). Els PrLDs tenen una composició similar als PFDs de llevat, i comparteixen la capacitat d’agregar, replicar-se i propagar-se. En moltes malalties degeneratives s’han trobat dipòsits citoplasmàtics compostos de “prion-like proteins”, de les quals s’han descrit mutacions genètiques en el seu PrLD. La gran majoria de “prion-like proteins” són nuclears, però es transloquen al citoplasma quan provoquen la malaltia. Aquest fenomen s’ha associat a la seva agregació en forma amiloide. En aquesta tesis hem identificat nous candidats “prion-like” potencials, i hem validat experimentalment per set d’ells la regió responsable de la seva agregació. També hem millorat les prediccions de l’impacte de les mutacions genètiques en el procés d’agregació de les “prion-like proteins”. S’ha vist que les “prion-like proteins” tenen la capacitat de formar orgànuls sense membrana dinàmics i reversibles en funció de les condicions cel·lulars, com per exemple en situacions d’estrès. Quan aquests canvis cel·lulars són massa persistents, aleshores els orgànuls sense membrana deixen de ser reversibles donant lloc als dipòsits sòlids trobats en pacients. Encara estem lluny de conèixer el mecanisme exacte de la formació dels orgànuls sense membrana. Per aquesta raó, en aquesta tesis hem estudiat la “prion-like protein” hnRNPDL humana a fons per intentar expandir el coneixement sobre aquests fenòmens i la seva connexió amb la malaltia. Hem caracteritzat les propietats d’autoassemblatge de les isoformes de hnRNPDL demostrant la importància del splicing alternatiu en controlar els esdeveniments de separació de fase, i ensenyem que les mutacions D378N/H involucrades en malaltia acceleren dramàticament l’agregació de hnRNPDL, reduint la seva solubilitat en el múscul de Drosophila. Les “prion-like proteins” són nuclis de grans xarxes d’interaccions proteiques a través del seu PrLD. S’ha vist que un nombre significatiu de PFDs de llevat tenen regions “coiled-coil” que participen en el seu procés d’agregació. En aquesta tesis demostrem que això també passa pels PrLDs humans i hem caracteritzat la proteïna MED15 com a exemple, mostrant que 3 Resum en català el seu PrLD forma fibres amiloides en un procés mediat pel seu “coiled-coil”, fet que pot explicar el seu rol en malaltia. En general, aquesta tesis és un estudi exhaustiu de les “prion-like proteins” i les seves propietats. 4 Contents CONTENTS 1. LIST OF PUBLICATIONS: ........................................................................................................ 6 2. ABBREVIATIONS ..................................................................................................................... 7 3. INTRODUCTION ....................................................................................................................... 8 4. OBJECTIVES .......................................................................................................................... 13 5. PUBLICATIONS 5.1. PUBLICATION I ............................................................................................................... 15 5.2. PUBLICATION II .............................................................................................................. 33 5.3. PUBLICATION III ............................................................................................................. 41 5.4. PUBLICATION IV ............................................................................................................
Load more

Recommended publications
  • Protein Interaction Network of Alternatively Spliced Isoforms from Brain Links Genetic Risk Factors for Autism
    ARTICLE Received 24 Aug 2013 | Accepted 14 Mar 2014 | Published 11 Apr 2014 DOI: 10.1038/ncomms4650 OPEN Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism Roser Corominas1,*, Xinping Yang2,3,*, Guan Ning Lin1,*, Shuli Kang1,*, Yun Shen2,3, Lila Ghamsari2,3,w, Martin Broly2,3, Maria Rodriguez2,3, Stanley Tam2,3, Shelly A. Trigg2,3,w, Changyu Fan2,3, Song Yi2,3, Murat Tasan4, Irma Lemmens5, Xingyan Kuang6, Nan Zhao6, Dheeraj Malhotra7, Jacob J. Michaelson7,w, Vladimir Vacic8, Michael A. Calderwood2,3, Frederick P. Roth2,3,4, Jan Tavernier5, Steve Horvath9, Kourosh Salehi-Ashtiani2,3,w, Dmitry Korkin6, Jonathan Sebat7, David E. Hill2,3, Tong Hao2,3, Marc Vidal2,3 & Lilia M. Iakoucheva1 Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • Proteogenomic Analysis of Inhibitor of Differentiation 4 (ID4) in Basal-Like Breast Cancer Laura A
    Baker et al. Breast Cancer Research (2020) 22:63 https://doi.org/10.1186/s13058-020-01306-6 RESEARCH ARTICLE Open Access Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer Laura A. Baker1,2, Holly Holliday1,2†, Daniel Roden1,2†, Christoph Krisp3,4†, Sunny Z. Wu1,2, Simon Junankar1,2, Aurelien A. Serandour5, Hisham Mohammed5, Radhika Nair6, Geetha Sankaranarayanan5, Andrew M. K. Law1,2, Andrea McFarland1, Peter T. Simpson7, Sunil Lakhani7,8, Eoin Dodson1,2, Christina Selinger9, Lyndal Anderson9,10, Goli Samimi11, Neville F. Hacker12, Elgene Lim1,2, Christopher J. Ormandy1,2, Matthew J. Naylor13, Kaylene Simpson14,15, Iva Nikolic14, Sandra O’Toole1,2,9,10, Warren Kaplan1, Mark J. Cowley1,2, Jason S. Carroll5, Mark Molloy3 and Alexander Swarbrick1,2* Abstract Background: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Methods: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq.
    [Show full text]
  • Tyr1 Phosphorylation Promotes Phosphorylation of Ser2 on the C-Terminal Domain
    1 Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain 2 of eukaryotic RNA polymerase II by P-TEFb 3 Joshua E. Mayfield1† *, Seema Irani2*, Edwin E. Escobar3, Zhao Zhang4, Nathanial T. 4 Burkholder1, Michelle R. Robinson3, M. Rachel Mehaffey3, Sarah N. Sipe3,Wanjie Yang1, 5 Nicholas A. Prescott1, Karan R. Kathuria1, Zhijie Liu4, Jennifer S. Brodbelt3, Yan Zhang1,5 6 1 Department of Molecular Biosciences, 2Department of Chemical Engineering, 7 3 Department of Chemistry and 5 Institute for Cellular and Molecular Biology, University of 8 Texas, Austin 9 4 Department of Molecular Medicine, Institute of Biotechnology, University of Texas 10 Health Science Center at San Antonio 11 †Current Address: Department of Pharmacology, University of California, San Diego, La 12 Jolla 13 * These authors contributed equally to this paper. 14 Correspondence: Yan Zhang ([email protected]) 15 16 1 17 Summary 18 The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates 19 Ser2 residues of C-terminal domain (CTD) of the largest subunit (RPB1) of RNA 20 polymerase II and is essential for the transition from transcription initiation to 21 elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in 22 vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and 23 hinders understanding of the transition from transcription initiation to elongation. 24 Through in vitro reconstruction of CTD phosphorylation, mass spectrometry 25 analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we 26 uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of 27 P-TEFb and alters its specificity from Ser5 to Ser2.
    [Show full text]
  • Whole Exome Sequencing in Families at High Risk for Hodgkin Lymphoma: Identification of a Predisposing Mutation in the KDR Gene
    Hodgkin Lymphoma SUPPLEMENTARY APPENDIX Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene Melissa Rotunno, 1 Mary L. McMaster, 1 Joseph Boland, 2 Sara Bass, 2 Xijun Zhang, 2 Laurie Burdett, 2 Belynda Hicks, 2 Sarangan Ravichandran, 3 Brian T. Luke, 3 Meredith Yeager, 2 Laura Fontaine, 4 Paula L. Hyland, 1 Alisa M. Goldstein, 1 NCI DCEG Cancer Sequencing Working Group, NCI DCEG Cancer Genomics Research Laboratory, Stephen J. Chanock, 5 Neil E. Caporaso, 1 Margaret A. Tucker, 6 and Lynn R. Goldin 1 1Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 2Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 3Ad - vanced Biomedical Computing Center, Leidos Biomedical Research Inc.; Frederick National Laboratory for Cancer Research, Frederick, MD; 4Westat, Inc., Rockville MD; 5Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; and 6Human Genetics Program, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD, USA ©2016 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2015.135475 Received: August 19, 2015. Accepted: January 7, 2016. Pre-published: June 13, 2016. Correspondence: [email protected] Supplemental Author Information: NCI DCEG Cancer Sequencing Working Group: Mark H. Greene, Allan Hildesheim, Nan Hu, Maria Theresa Landi, Jennifer Loud, Phuong Mai, Lisa Mirabello, Lindsay Morton, Dilys Parry, Anand Pathak, Douglas R. Stewart, Philip R. Taylor, Geoffrey S. Tobias, Xiaohong R. Yang, Guoqin Yu NCI DCEG Cancer Genomics Research Laboratory: Salma Chowdhury, Michael Cullen, Casey Dagnall, Herbert Higson, Amy A.
    [Show full text]
  • Potential Human Transmission of Amyloid Β Pathology: Surveillance and Risks
    This is a repository copy of Potential human transmission of amyloid β pathology: surveillance and risks. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/161306/ Version: Accepted Version Article: Lauwers, E, Lalli, G, Brandner, S et al. (34 more authors) (2020) Potential human transmission of amyloid β pathology: surveillance and risks. Lancet Neurology, 19 (10). pp. 872-878. ISSN 1474-4422 https://doi.org/10.1016/S1474-4422(20)30238-6 © 2020, Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/. Reuse This article is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) licence. This licence only allows you to download this work and share it with others as long as you credit the authors, but you can’t change the article in any way or use it commercially. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Potential human transmission of amyloid-β pathology: surveillance and risks Elsa Lauwers, PhD1,2,#, Giovanna Lalli, PhD3,#, Prof. Sebastian Brandner, MD4,5, Prof. John Collinge, MD6, Prof. Veerle Compernolle, MD7,8, Prof. Charles Duyckaerts, MD9, Prof. Gustaf Edgren, MD10,11, Stéphane Haïk, MD9,12, Prof.
    [Show full text]
  • Emidio Capriotti Phd CURRICULUM VITÆ
    Emidio Capriotti PhD CURRICULUM VITÆ Name: Emidio Capriotti Nationality: Italian Date of birth: February, 1973 Place of birth: Roma, Italy Languages: Italian, English, Spanish Positions Oct 2019 Associate Professor: Department of Pharmacy and Biotechnology (FaBiT). University of Bologna, Bologna, Italy 2016-2019 Senior Assistant Professor (RTD type B): Department of Pharmacy and Biotechnology (FaBiT) and Department of Biological, Geological, and Environmental Sciences (BiGeA). University of Bologna, Bologna, Italy. 2015-2016 Junior Group Leader: Institute of Mathematical Modeling of Biological Systems, University of Düsseldorf, Düsseldorf, Germany 2012-2015 Assistant Professor: Division of Informatics, Department of Pathology, University of Alabama at Birmingham (UAB), Birmingham (AL), USA. 2011-2012 Marie-Curie IOF: Contracted Researcher at the Department of Mathematics and Computer Science, University of Balearic Islands (UIB), Palma de Mallorca, Spain. 2009-2011 Marie-Curie IOF: Postdoctoral Researcher at the Helix Group, Department of Bioengineering, Stanford University, Stanford (CA), USA. 2006-2009 Postdoctoral Researcher in the Structural Genomics Group at Department of Bioinformatics and Genetics, Prince Felipe Research Center (CIPF) Valencia, Spain. 2004-2006 Contract researcher at Department of Biology, University of Bologna, Bologna, Italy. 2001-2003 Ph.D student in Physical Sciences at University of Bologna, Bologna, Italy. Education Sep 2004 Master in Bioinformatics (first level) University of Bologna, Bologna (Italy). Jun 2004 Ph.D. in Physical Sciences University of Bologna, Bologna (Italy). Jul 1999 Laurea (B.S.) Degree in Physical Sciences, score 106/110 University of Bologna, Bologna (Italy). Visiting Jun 2012 – Jul 2012 Prof. Frederic Rousseau and Prof. Joost Schymkowitz, VIB Switch Laboratory, KU Leuven, Leuven (Belgium) May 2009 Prof.
    [Show full text]
  • Termin Translat Trna Utr Mutat Protein Signal
    Drugs & Chemicals 1: Tumor Suppressor Protein p53 2: Heterogeneous-Nuclear Ribonucleo- (1029) proteins (14) activ apoptosi arf cell express function inactiv induc altern assai associ bind mdm2 mutat p53 p73 pathwai protein regul complex detect exon famili genom respons suppress suppressor tumor wild-typ interact intron isoform nuclear protein sensit site specif splice suggest variant 3: RNA, Transfer (110) 4: DNA Primers (1987) codon contain differ eukaryot gene initi amplifi analysi chain clone detect dna express mrna protein region ribosom rna fragment gene genotyp mutat pcr sequenc site speci suggest synthesi polymorph popul primer reaction region restrict sequenc speci termin translat trna utr 5: Saccharomyces cerevisiae Proteins 6: Apoptosis Regulatory Proteins (291) (733) activ apoptosi apoptosis-induc albican bud candida cerevisia complex encod apoptot bcl-2 caspas caspase-8 cell eukaryot fission function growth interact involv death fasl induc induct ligand methyl necrosi pathwai program sensit surviv trail mutant pomb protein requir saccharomyc strain suggest yeast 7: Plant Proteins (414) 8: Membrane Proteins (1608) access arabidopsi cultivar flower hybrid leaf leav apoptosi cell conserv domain express function gene human identifi inhibitor line maiz plant pollen rice root seed mammalian membran mice mous mutant seedl speci thaliana tomato transgen wheat mutat protein signal suggest transport 1 9: Tumor Suppressor Proteins (815) 10: 1-Phosphatidylinositol 3-Kinase activ arrest cell cycl cyclin damag delet dna (441) 3-kinas activ
    [Show full text]
  • Bicyclomycin Sensitivity and Resistance Affect Rho Factor-Mediated Transcription Termination in the Tna Operon of Escherichia Coli
    JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4451–4456 Vol. 177, No. 15 0021-9193/95/$04.0010 Copyright 1995, American Society for Microbiology Bicyclomycin Sensitivity and Resistance Affect Rho Factor-Mediated Transcription Termination in the tna Operon of Escherichia coli CHARLES YANOFSKY* AND VIRGINIA HORN Department of Biological Sciences, Stanford University, Stanford, California 94305-5020 Received 13 March 1995/Accepted 27 May 1995 The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be acquired by mutations at any one of three loci, two of which appear to be rho and rpoB. Zwiefka et al. (24) found that the antibiotic bicyclomycin segment and interacts with the transcribing RNA polymerase (bicozamycin), an inhibitor of the growth of several gram- molecule, causing it to terminate transcription (7, 9).
    [Show full text]
  • Structure of the Rho Transcription Terminator: Mechanism of Mrna Recognition and Helicase Loading
    Cell, Vol. 114, 135–146, July 11, 2003, Copyright 2003 by Cell Press Structure of the Rho Transcription Terminator: Mechanism of mRNA Recognition and Helicase Loading Emmanuel Skordalakes and James M. Berger* this process by a number of factors, such as NusG, Department of Molecular and Cell Biology which serve as antiterminators to ameliorate the termi- University of California, Berkeley nation properties of the enzyme (Sullivan and Gottes- 239 Hildebrand Hall, #3206 man, 1992). Berkeley, California 94720 Tethering of Rho to a rut site is independent of ATP binding and/or hydrolysis (Galluppi and Richardson, 1980; McSwiggen et al., 1988) and is mediated by an Summary N-terminal domain in each protomer of the Rho hexamer (Martinez et al., 1996a, 1996b; Allison et al., 1998; Bri- In bacteria, one of the major transcriptional termina- ercheck et al., 1998). Previous high-resolution NMR and tion mechanisms requires a RNA/DNA helicase known X-ray crystal structures of the N-terminal mRNA binding as the Rho factor. We have determined two structures domain of Rho have provided a structural view of this of Rho complexed with nucleic acid recognition site fold and its interactions with target substrates (Allison mimics in both free and nucleotide bound states to et al., 1998; Briercheck et al., 1998; Bogden et al., 1999). 3.0 A˚ resolution. Both structures show that Rho forms The domain consists of a three-helix bundle that rests a hexameric ring in which two RNA binding sites—a on top of a five-stranded ␤ barrel. The barrel belongs primary one responsible for target mRNA recognition to the oligonucleotide/oligossacharide binding (OB) pro- and a secondary one required for mRNA translocation tein superfamily and is primarily responsible for Rho’s and unwinding—point toward the center of the ring.
    [Show full text]
  • Supplemental Digital Content (Sdc) Sdc, Materials
    SUPPLEMENTAL DIGITAL CONTENT (SDC) SDC, MATERIALS AND METHODS Animals This study used 9-12 week old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). This study conformed to the National Institutes of Health guidelines and was conducted under animal protocols approved by the University of Virginia’s Institutional Animal Care and Use Committee. Murine DCD Lung Procedure Mice were anesthetized by isoflurane inhalation and euthanized by cervical dislocation followed by a 60-minute period of “no-touch” warm ischemia. Mice then underwent extended median sternotomy and midline cervical exposure followed by intubation for the initiation of mechanical ventilation at 120 strokes/minute with room air. The left atrium was vented via an atriotomy followed by infusion of the lungs with 3 mL 4°C Perfadex® solution (Vitrolife Inc., Denver, CO) supplemented with THAM Solution (Vitrolife, Kungsbacka, Sweden), estimating weight-based volume recommendations for pulmonary artery perfusion (140mL/kg) (1). The chest was then packed with ice and the trachea occluded by silk-suture tie at tidal volume (7µL/g body weight) prior to cold static preservation (CSP) for 60 minutes at 4°C. Mice were then randomized into three experimental groups: 1) CSP alone with no EVLP, 2) EVLP with Steen solution and 3) EVLP with Steen solution supplemented with the highly selective A2AR agonist, ATL1223 (30nM, Lewis and Clark Pharmaceuticals, Charlottesville, VA). Mice treated with ATL1223 during EVLP also received ATL1223 treatment (30nM) during the Perfadex flush prior to CSP whereas the EVLP group received vehicle (DMSO) during the flush. CSP lungs, which did not undergo EVLP, underwent immediate functional assessment after re-intubation as described below.
    [Show full text]