DOCSLIB.ORG

Cloning of Receptor Tyrosine Kinases from Anvsia

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cloning of Receptor Tyrosine Kinases from Anvsia Cloning of Receptor Tyrosine Kinases From ANvsia Jonathan Hislop Neurology and Neurosurgery, McGill University, Montreal. August, 1998 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Master of Science @Jonathan Hislop, 1998 National Library Biblioth$ue nationale du Cana a Acquisitions and Acquisitions et Bibbgraphic Semices m~icesbibliographiques 395 Wdlngton Street 395, rue Weilinglori OItawaON K1AW OnawaON K1AON4 CariPds Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in rnicrofonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substaatial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othenirise de celle-ci ne doivent être imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract I have cloned two putative receptor tyrosine kinases (RTKs) fiom Aplysia californica, designated Aror and Anif. Aror is homologous to RTKs fiom the Ror subfarnily. Furthemore, Aror contains an extracellular immunoglobulin-like domain, cysteine-rich region, and kringie domain. These domains are d characteristic of Ror recepton. Amf is homologous to members of the Ret and FGF receptor subfamilies of RTKs. However, Aruf does not have the extracellular domains typical of those receptors. Furthemore the degree of identity between Anif and either the Ret or FG F recepton is closer to that between members of different RTK subfamilies, han between membea of the sarne subfamily. Therefore, Amf encodes an RTK belonging to a novel subfamily related to the Ret/FGFR subfamily. J'ai cloné deux molécules qui sont de presum6s récepteurs tyrosine kinase (RTK) provenant de Aplysia cal@rrnica, et désignées Aror et Ad. Aror est homologue aw membres de la sous-famille Ror des RTK. De plus, Aror comprend un domaine extra- cellulaire apparenté aux immunoglobulines, une région riche en cystéine et un domaine kringle. Ces domaines sont tous caractéristiques des récepteurs Ror. Anif est homologue aux membres des sous-familles Ret et FGFR des RTK. Cependant, Anif n'a pas le domaine extra-cellulaire qui est typique de ces récepteurs. De plus, le degré d'identité entre Anif et l'un ou l'autre de Ret ou FGFR est plus prb du degré d'identité partagé entre les membres des diffdrentes sous-familles RTK que de celui partagé entre les membres d'une même sous-famille. Donc, Adencode un RTK appartenant à une nouvelle sous-famille reliée à celles de Ret et de FGFR, Acknowledgements 1 would like to thank my supervisor Wayne Sossin for his time and patience as a teacher. I'd also like to thank the other members of my advisory cornmittee, Phi1 Barker and David Kaplan, for their helpful comments and direction throughout this project. Thanks also go to David Ragsdale, and Peter MacPhearson for their encouragement and support. My biggest surprise about doing research has ken how much i've enjoyed workine- with everyone in our lab. John Dyer showed me how to perfom many of the procedures for the project, and was a great teacher. 1 enjoyed working with him tremendously, and listening to Monty Python with him. A number of other people €rom the lab also helped me learn various procedures, including Tony Pepio, Xiao-Tang Fan, and Stefanie Yanow. 1 had a great tirne working with al1 of them as well as others from the lab, such as Hedy Chung, Tina Vouloumanos, Darcy Scott, and Arash Nakhost. Poking fun at Arash was particularly rewarding. My family and fnends were also always supportive of me during the project. But my cat Checkers objected to this entire flair and wants lots of attention to make up for it. He also wants to try one of the Aplysia. III Statement of Originalitv The cloning of each of the receptoa discussed in this study (designated Aror, and Anif) constitute original scholmhip. Neither of these receptors have been previously cloned. 1 pexformed aii the procedures discussed in the study with the exception that most of the DNA sequencing was perfomed by our laboratory technician Xiao-Tang Fan, and by Bio S&T Inc., and The W.M. Keck Biotechnology Resource Laboratory. The rationale for the project was devised by Dr. Wayne Sossin. Table of Contents Abstract ................................................................................................................................. 1 Résumé ................................................................................................................................ II Acknow ledgements............................................................................................................. 111 Staternent of Originality ......................................................................................................iV Table of Contents ................ ... ............................................................................................. V List of Tables and Figures .......................................................................................... VI1 List of Abbreviations ......................................................................................................VI11 introduction .......................................................................................................................... 1 Receptor Tyrosine Kinases: Structure and Function ........................................................ 1 Oved Structure............................................................................................................ 1 The Extracellular Domain ..............................................................................................-3 The Kinase Domain ....................................................................................................... 3 RTK Activation and Signal Transduction .....................................................................4 RTKs Have Diverse Roles in Many Organisms ...........................................................7 RTKs in Invertebrates .................................................................................................1 O RTKs and Memory Formation in Aplysia californica .......................................... 1 O Serotonin Induces Translation and Intemediate Memory in Aplysia ........................ I 1 RTKs and Intermediate Memory in Aplysia .............................................................. 12 Rationale and Objectives ............................................................................................. 1 2 Cloning of RTKs fiom Aplysia ................................................................................. 1 3 Materials and Methods ..................................................................................................... 1 8 Construction of cDNA Library ...................................................................................... 18 PCR Based Screen and Design of Oligodegenerate bers............................................ 18 Screening for Full Length Clows ....................................................................................20 Cloning of Aplysia RTKs ................................................................................................25 Amplification of PCR Eragments ................................................................................. 25 Aror Hy bridization Screen .......................................................................................... 26 Aror encodes a putative RTK related to the Ror subfamily ....................................... 27 Anif Hybridization Screen ........................................ .....................,..........*..*,...*,.*.*....30 Anif encodes a putative RTK related to the RetIFGF receptor subfamilies ...............31 Discussion .......................................................................................................................... 45 Summary .........................................................................................................................45 Additional RTK Fragments Amplified ..........................................................................A5 Featwes of Aror ..............................................................................................................46 Extracellular Domain.................................................................................................... 46 Intracellular Domain ................................ .. ..............................................................47 Features of Amf ............................................................................................................. -49 Extraceilular Domain ....................................................................................................49 intracellular Domain ...................................................................................................-50 Future Directions ............................................................................................................51 References .........................................................................................................................
Load more

Recommended publications
  • Download the Abstract Book
    1 Exploring the male-induced female reproduction of Schistosoma mansoni in a novel medium Jipeng Wang1, Rui Chen1, James Collins1 1) UT Southwestern Medical Center. Schistosomiasis is a neglected tropical disease caused by schistosome parasites that infect over 200 million people. The prodigious egg output of these parasites is the sole driver of pathology due to infection. Female schistosomes rely on continuous pairing with male worms to fuel the maturation of their reproductive organs, yet our understanding of their sexual reproduction is limited because egg production is not sustained for more than a few days in vitro. Here, we explore the process of male-stimulated female maturation in our newly developed ABC169 medium and demonstrate that physical contact with a male worm, and not insemination, is sufficient to induce female development and the production of viable parthenogenetic haploid embryos. By performing an RNAi screen for genes whose expression was enriched in the female reproductive organs, we identify a single nuclear hormone receptor that is required for differentiation and maturation of germ line stem cells in female gonad. Furthermore, we screen genes in non-reproductive tissues that maybe involved in mediating cell signaling during the male-female interplay and identify a transcription factor gli1 whose knockdown prevents male worms from inducing the female sexual maturation while having no effect on male:female pairing. Using RNA-seq, we characterize the gene expression changes of male worms after gli1 knockdown as well as the female transcriptomic changes after pairing with gli1-knockdown males. We are currently exploring the downstream genes of this transcription factor that may mediate the male stimulus associated with pairing.
    [Show full text]
  • Dissecting Mechanisms Controlling Neural Network Formation in Drosophila Melanogaster
    Dissecting mechanisms controlling neural network formation in Drosophila melanogaster Hong Long Department of Neurology and Neurosurgery McGill University Montreal, Quebec, Canada August 2009 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the Ph.D. degree in Neuroscience © Hong Long, 2009 1 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-66458-2 Our file Notre référence ISBN: 978-0-494-66458-2 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L’auteur conserve la propriété du droit d’auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,034,913 B2 Hunt Et Al
    USOO8034913B2 (12) United States Patent (10) Patent No.: US 8,034,913 B2 Hunt et al. (45) Date of Patent: Oct. 11, 2011 (54) RECOMBINANTLY MODIFIED PLASMIN FOREIGN PATENT DOCUMENTS CA 2045869 12/1991 (75) Inventors: Jennifer A. Hunt, Raleigh, NC (US); WO WO 97.27331 A2 7/1997 Valery Novokhatny, Raleigh, NC (US) WO WO99,05322 A1 2/1999 WO WOO1,9436.6 A1 12/2001 WO WOO2,5O290 A1 6, 2002 (73) Assignee: Grifols Therapeutics Inc., Research WO WOO3/O54232 A2 7, 2003 Triangle Park, NC (US) WO WO 2004/052228 A2 6, 2004 WO WO 2005,105990 11, 2005 WO WO 2007/047874 4/2007 (*) Notice: Subject to any disclaimer, the term of this WO WO 2009,0734.71 6, 2009 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 854 days. Hunt et al. Simplified recombinant plasmin: Production and func tional comparison of a novel thrombolytic molecule with plasma (21) Appl. No.: 11/568,023 derived plasmin. Thromb. Haemost 100:413-419, 2008.* Andrianov, S.I., et al., “Peculiarities of Hydrolytic Action of Plasmin, (22) PCT Filed: Apr. 21, 2005 Miniplasmin, Microplasmin and Trypsin on Polymeric Fibrin.” Ukr. Biokhim. Zh., 64(3): 14-20 (1992). Anonick, P. et al., “Regulation of Plasmin, Miniplasmin and (86). PCT No.: PCT/US2OOS/O13562 Streptokinase-Plasmin Complex by-O.--Antiplasmin, O.-- S371 (c)(1) Macroglobulin, and Antithrombin III in the Presence of Heparin.” . Thrombosis Res., 59: 449-462 (1990). (2), (4) Date: Feb. 1, 2008 Bennett, D., et al., “Kinetic Characterization of the Interaction of Biotinylated Human Interleukin 5 with an Fc Chimera of its Receptor (87) PCT Pub.
    [Show full text]
  • The Molecular Basis of Blood Coagulation Review
    Cell, Vol. 53, 505-518, May 20, 1988, Copyright 0 1988 by Cell Press The Molecular Basis Review of Blood Coagulation Bruce Furie and Barbara C. Furie into the fibrin polymer. The clot, formed after tissue injury, Center for Hemostasis and Thrombosis Research is composed of activated platelets and fibrin. The clot Division of Hematology/Oncology mechanically impedes the flow of blood from the injured Departments of Medicine and Biochemistry vessel and minimizes blood loss from the wound. Once a New England Medical Center stable clot has formed, wound healing ensues. The clot is and Tufts University School of Medicine gradually dissolved by enzymes of the fibrinolytic system. Boston, Massachusetts 02111 Blood coagulation may be initiated through either the in- trinsic pathway, where all of the protein components are present in blood, or the extrinsic pathway, where the cell- Overview membrane protein tissue factor plays a critical role. Initia- tion of the intrinsic pathway of blood coagulation involves Blood coagulation is a host defense system that assists in the activation of factor XII to factor Xlla (see Figure lA), maintaining the integrity of the closed, high-pressure a reaction that is promoted by certain surfaces such as mammalian circulatory system after blood vessel injury. glass or collagen. Although kallikrein is capable of factor After initiation of clotting, the sequential activation of cer- XII activation, the particular protease involved in factor XII tain plasma proenzymes to their enzyme forms proceeds activation physiologically is unknown. The collagen that through either the intrinsic or extrinsic pathway of blood becomes exposed in the subendothelium after vessel coagulation (Figure 1A) (Davie and Fiatnoff, 1964; Mac- damage may provide the negatively charged surface re- Farlane, 1964).
    [Show full text]
  • Differential Binding of Plasminogen, Plasmin, and Angiostatin4.5 to Cell Surface B-Actin: Implications for Cancer-Mediated Angiogenesis
    Research Article Differential Binding of Plasminogen, Plasmin, and Angiostatin4.5 to Cell Surface B-Actin: Implications for Cancer-Mediated Angiogenesis Hao Wang,1 Jennifer A. Doll,1 Keyi Jiang,1 Deborah L. Cundiff,1 Jarema S. Czarnecki,1 Mindy Wilson,2 Karen M. Ridge,2 and Gerald A. Soff1,3 1Division of Hematology/Oncology and 2Division of Pulmonary and Critical Care Medicine, Department of Medicine and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois and 3Division of Hematology/ Oncology, State University of New York Downstate Medical Center, Brooklyn, New York Abstract Several angiostatin isoforms have been reported, differing in Angiostatin4.5 (AS4.5) is the product of plasmin autoproteol- kringle content. Angiostatins consisting of plasminogen kringles 1 f to 3, kringles 1 to 4, or angiostatin4.5 (AS4.5), which includes ysis and consists of kringles 1 to 4 and 85% of kringle 5. In 78 529 culture, cancer cell surface globular B-actin mediates plasmin kringles 1 to 4 plus 85% of kringle 5 (Lys -Arg ), have been autoproteolysis to AS4.5. We now show that plasminogen generated in vitro by a variety of proteinases. However, it is still not binds to prostate cancer cells and that the binding colocalizes well understood which angiostatin isoforms are present naturally with surface B-actin, but AS4.5 does not bind to the cell in the human body (5, 8–13). We have shown previously that surface. Plasminogen and plasmin bind to immobilized AS4.5 is a naturally occurring human form (14). AS4.5 is generated B-actin similarly, with a K of f140 nmol/L.
    [Show full text]
  • Supplementary Materials
    Supplementary Materials 0.3 0.274 0.28 0.2735 0.26 0.273 0.24 0.2725 0.22 0.2 0.272 ccTM−score 0.18 0.2715 0.16 0.271 0.14 0.2705 0.12 0.1 0.27 1 2 3 4 5 6 7 8 9 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 d gap penalty cut Figure S1. Selection of the optimal values for dcut and gap penalty. The experiments were done on 50% randomly selected groups from the SABmark_twi dataset. The gap penalty on the left panel is 0 and the dcut on the right panel is 4. 1 0.9 0.8 PCC=0.94 0.7 0.6 0.5 0.4 ACC of the MSTA 0.3 0.2 0.1 0 0 0.2 0.4 0.6 0.8 1 ACC of the PSA Figure S2. The correlation between the accuracy of the PSA and the MSTA. The data used in this figure are available in Table S5. Table S1. The influence of TM-score normalization. TM-score_s/TM-score_b is the mean TM-score normalized by the size of the smaller/bigger protein. ccTM-score_s and ccTM-score_b are the mean common core TM-scores of the corresponding MSTA. Dataset HOMSTRAD SABmark_sup SABmark_twi SISY-multiple Number of groups 398 425 209 86 Mean length difference 17.5 34.1 43.4 65.2 Pairwise TM-score_s 0.810 0.683 0.575 0.692 Pairwise TM-score_b 0.756 0.597 0.476 0.571 No.
    [Show full text]
  • Evolution of Enzyme Cascades from Embryonic Development to Blood
    Opinion TRENDS in Biochemical Sciences Vol.27 No.2 February 2002 67 as the upstream, middle and downstream proteases) Evolution of enzyme that undergo sequential zymogen activation, followed by cleavage of a terminal substrate by the downstream protease (Fig. 1). Activation of the cascades from upstream protease might occur by contact with a non-enzymatic ligand or by cleavage by another protease. Furthermore, there are alternate routes of embryonic activating the middle and downstream proteases for some of the cascades, especially for vertebrate complement and clotting. However, for the sake of development to simplicity, the focus of this discussion is confined to the functional cores and terminal substrates depicted in Fig. 1. blood coagulation In addition to its position within an individual cascade, each enzyme can be classified according to highly conserved evolutionary markers that divide Maxwell M. Krem and Enrico Di Cera serine proteases into discrete lineages and indicate the relative ages of those lineages [4] (Box 1). When the above classification system is applied to proteases Recent delineation of the serine protease cascade controlling dorsal–ventral within the cascades (Table 1), a clear pattern patterning during Drosophila embryogenesis allows this cascade to be emerges: the upstream protease is from a more compared with those controlling clotting and complement in vertebrates and recently evolved category than the downstream invertebrates. The identification of discrete markers of serine protease protease. The middle protease belongs to the same evolution has made it possible to reconstruct the probable chronology of category as either the upstream or the downstream enzyme evolution and to gain new insights into functional linkages among the protease, depending on the particular cascade.
    [Show full text]
  • Structural Features of the Kringle Domain Determine the Intracellular Degradation of Under-␥-Carboxylated Prothrombin: Studies of Chimeric Rat͞human Prothrombin
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13654–13660, December 1997 Cell Biology This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 30, 1996. Structural features of the kringle domain determine the intracellular degradation of under-g-carboxylated prothrombin: Studies of chimeric ratyhuman prothrombin WEI WU*, JOHN D. BANCROFT†, AND J. W. SUTTIE*‡ *Department of Biochemistry, College of Agricultural and Life Sciences, and †Department of Pediatrics, The Medical School, University of Wisconsin–Madison, Madison, WI 53706 Contributed by J. W. Suttie, October 3, 1997 ABSTRACT Vitamin K antagonists such as warfarin in- of prothrombin secretion, and an intracellular accumulation of hibit the vitamin K-dependent g-glutamyl carboxylation dur- under-g-carboxylated prothrombin that is degraded in a pre- ing protein processing and block the secretion of under-g- Golgi compartment (5–7). In patients treated with oral anti- carboxylated prothrombin (FII) in the rat but not in the coagulants (4) and in cultures of a human hepatoma (HepG2) human or bovine. Under-g-carboxylated prothrombin is also cell line incubated with warfarin (8), under-g-carboxylated secreted from warfarin-treated human (HepG2) cell cultures prothrombin is secreted. Rat prothrombin (rFII) stably ex- but is degraded in the endoplasmic reticulum in warfarin- pressed in HepG2 cells treated with warfarin is not secreted treated rat (H-35) cell cultures. This differential response to but is degraded intracellularly (8), whereas secretion of en- warfarin has been shown to be determined by the structural dogenous human prothrombin (hFII) is not altered.
    [Show full text]
  • Ep 2655624 B1
    (19) TZZ _T (11) EP 2 655 624 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/62 (2006.01) C07K 16/46 (2006.01) 29.11.2017 Bulletin 2017/48 (86) International application number: (21) Application number: 11820905.5 PCT/US2011/066947 (22) Date of filing: 22.12.2011 (87) International publication number: WO 2012/088461 (28.06.2012 Gazette 2012/26) (54) LINKER PEPTIDES AND POLYPEPTIDES COMPRISING SAME LINKERPEPTIDE UND DIESE ENTHALTENDE POLYPEPTIDE PEPTIDES COUPLEURS ET POLYPEPTIDES LES COMPORTANT (84) Designated Contracting States: US-A1- 2002 103 353 AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • SHERIDAN DOUGLAS L ET AL: "A new way to PL PT RO RS SE SI SK SM TR rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro (30) Priority: 23.12.2010 US 201061426993 P transposition reaction", BMC NEUROSCIENCE, BIOMED CENTRAL, LONDON, GB, vol. 3, no. 1, (43) Date of publication of application: 19 June 2002 (2002-06-19), page 7, XP021015026, 30.10.2013 Bulletin 2013/44 ISSN: 1471-2202, DOI: 10.1186/1471-2202-3-7 • DATABASE UniProt [Online] 23 October 2007 (73) Proprietor: Biogen MA Inc. (2007-10-23), "SubName: Cambridge, MA 02142 (US) Full=AGAP006375-PA;", XP002676589, retrieved from EBI accession no. UNIPROT:A7UU88 (72) Inventors: Database accession no. A7UU88 -& HOLT R A ET • MILLER, Brian, Robert AL: "The Genome Sequence of the Malaria San Diego, CA 92122 (US) Mosquito Anopheles gambiae", SCIENCE, • GLASER, Scott AMERICAN ASSOCIATION FOR THE San Diego, CA 92107 (US) ADVANCEMENT OF SCIENCE, WASHINGTON, • CARAVELLA, Justin DC; US, vol.
    [Show full text]
  • Download Author Version (PDF)
    RSC Advances This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. This Accepted Manuscript will be replaced by the edited, formatted and paginated article as soon as this is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/advances Page 1 of 36 RSC Advances 1 Typical and atypical domain combinations in human protein kinases: Functions, disease causing mutations and conservation in other primates Ramaswamy Rakshambikai a# , Malini Manoharan a# , Mutharasu Gnanavel a,b and Narayanaswamy Srinivasan a* DOI: 10.1039/b000000x Ser/Thr and Tyr protein kinases orchestrate many signalling pathways and hence loss in this balance leads to many disease phenotypes. Due to their high abundance, diversity and importance, efforts have been made in the past to classify kinases and annotate their functions at both gross and fine levels. These kinases are conventionally classified into subfamilies based on the sequences of catalytic domains.
    [Show full text]
  • Disruption of the Kringle 1 Domain of Prothrombin Leads to Late Onset Mortality in Zebrafsh Steven J
    www.nature.com/scientificreports OPEN Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafsh Steven J. Grzegorski1,9, Zhilian Hu1,6, Yang Liu1,7, Xinge Yu1, Allison C. Ferguson1, Hasam Madarati2,3, Alexander P. Friedmann2,3, Deepak Reyon4,5,8, Paul Y. Kim2,3, Colin A. Kretz2,3, J. Keith Joung4,5 & Jordan A. Shavit1* The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete defciency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafsh are able to tolerate severe hemostatic defciencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafsh prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fbrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafsh and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafsh are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.
    [Show full text]
  • Functional Domains and Evolutionary History of the PMEL and GPNMB Family Proteins
    molecules Article Functional Domains and Evolutionary History of the PMEL and GPNMB Family Proteins Paul W. Chrystal 1,2,† , Tim Footz 1,† , Elizabeth D. Hodges 2, Justin A. Jensen 2, Michael A. Walter 1,* and W. Ted Allison 1,2,3,* 1 Department of Medical Genetics, University of Alberta, Edmonton, AB T6G 2R3, Canada; [email protected] (P.W.C.); [email protected] (T.F.) 2 Department of Biological Sciences, University of Alberta, Edmonton, AB T7Y 1C4, Canada; [email protected] (E.D.H.); [email protected] (J.A.J.) 3 Centre for Prions & Protein Folding Disease, University of Alberta, Edmonton, AB T6G 2M8, Canada * Correspondence: [email protected] (M.A.W.); [email protected] (W.T.A.) † These authors contributed equally to this work. Abstract: The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurode- generative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolution- ary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most Citation: Chrystal, P.W.; Footz, T.; ancient paralog of the family. By curating the functional domains in each paralog, we identified Hodges, E.D.; Jensen, J.A.; Walter, PMEL M.A.; Allison, W.T.
    [Show full text]