DOCSLIB.ORG

Contains Introns Shared by Src Homology 2 Domains in Many Unrelated Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Contains Introns Shared by Src Homology 2 Domains in Many Unrelated Proteins Copyright 2003 by the Genetics Society of America Phospholipase C-␥ Contains Introns Shared by src Homology 2 Domains in Many Unrelated Proteins Charlene M. Manning,1 Wendy R. Mathews,2 Leah P. Fico and Justin R. Thackeray3 Biology Department, Clark University, Worcester, Massachusetts 01610 Manuscript received September 10, 2002 Accepted for publication March 13, 2003 ABSTRACT Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-␥ (PLC-␥) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-␥ evolution and structure-function relationships within the Drosophila PLC-␥ encoded by small wing (sl), we cloned and sequenced the PLC-␥ homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-␥ homologs in other animals. PLC-␥ has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demon- strates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-␥ SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-␥ are derived from an ancestral domain that was shuffled not only into PLC-␥, but also into many other unrelated genes during animal evolution. OUR distinct types of phospholipase C (PLC) pro- X and Y catalytic domains that is unique among PLC F tein, ␤, ␥, ␦, and ε, are produced in mammals by a subtypes. This central region contains one src homology rather diverse family of more than 10 genes. All PLCs 3 (SH3) and two SH2 domains within a split PH domain, catalyze the hydrolysis of the membrane phospholipid implying a series of shufflings and duplications to pro- phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P2] into duce the modern PLC-␥ structure from an ancestral inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. PLC form. These events were apparently completed be- The former is bound by specific receptors embedded fore the parazoan-eumetazoan split, because the sponge in the endoplasmic reticular membrane, leading to a Ephydatia fluviatilis has a PLC-␥ homolog with identical transient increase in intracellular calcium, while the structure to all other animal forms (Koyanagi et al. latter is a direct activator of protein kinase C. The pat- 1998). The SH2 and SH3 domains of PLC-␥ are typical tern of expression, mechanism of activation, and cellu- examples of the many widely distributed modules that lar function vary considerably among the four types are found in many different proteins involved in signal (reviewed by Rebecchi and Pentyala 2000; Lopez et transduction; each domain is presumed to have arisen al. 2001; Song et al. 2001). All PLC proteins have three once and then been spread both by gene duplication domains in common: a C2 domain and catalytic domains and by being co-opted into existing genes by exon shuf- ␤ ␥ ␦ X and Y; three of the PLC types, , , and , also share fling via retrotransposition, illegitimate recombination, an N-terminal pleckstrin homology (PH) domain and or long interspersed nuclear element-mediated 3Ј trans- ␦ EF hands. PLC- may be the ancestral form, because duction (Long 2001). In the case of PLC-␥ these ac- ␤ ␥ ε , , and types are absent from plants and simple quired domains permit specific protein:protein interac- eukaryotes such as yeast. ␥ ␥ tions: the SH2 domains are thought to allow PLC- to PLC- is particularly interesting from an evolutionary bind to specific phosphorylated tyrosine residues on the standpoint, because it has a central region between the cytoplasmic face of activated receptor tyrosine kinases, whereas the SH3 domain is essential in allowing PLC-␥ to activate the phosphatidylinositol-3-OH kinase [PI(3)K] Sequence data from this article have been deposited with the enhancer (Ye et al. 2002). EMBL/GenBank Data libraries under the accession nos. AF543827 and AF543828. PLC-␥ is involved in regulating many aspects of cell 1Present address: Department of Genetics, HHMI, Harvard Medical physiology, including proliferation, differentiation, and School, 200 Longwood Ave., Boston, MA 02115. motility (reviewed by Rebecchi and Pentyala 2000). 2 Present address: Department of Biology, Johns Hopkins University, PLC-␥ activation is triggered by the binding of a wide 3400 N. Charles St., Baltimore, MD 21218. 3Corresponding author: Biology Department, Clark University, 950 variety of growth factors, cytokines, and immunoglobu- Main St., Worcester, MA 01610. E-mail: [email protected] lins to their membrane-bound receptor. Two distinct Genetics 164: 433–442 ( June 2003) 434 C. M. Manning et al. PLC-␥ homologs encoded by different genes—PLC-␥1 further rounds of plating and hybridization. Purified plaques ␭ and PLC-␥2—are present in mammals, whereas all inver- were obtained and -genomic DNA was extracted by the plate lysate method (Sambrook et al. 1989). tebrates described to date have only a single homolog. DNA sequencing: Restriction fragments containing sl exons In Drosophila, the PLC-␥ homolog is encoded by small were identified by Southern blot hybridization using a variety wing (sl; Emori et al. 1994; Thackeray et al. 1998), a of probes corresponding to the sl open reading frame (ORF). gene first identified by Bridges in 1915 (Lindsley and For the D. virilis gene, several of these fragments were sub- ϩ Zimm 1992). Flies lacking sl function are viable, with cloned into pBluescript KS (Stratagene) and sequenced on both strands by primer walking. Most of the D. pseudoobscura mild defects in eye and wing development due to over- gene was sequenced by transposon tagging of a 4.8-kb PstI/ activation of the epidermal growth factor receptor sig- HindIII subclone with the EZ::TNϽKAN-2Ͼ kit, using the naling pathway (Thackeray et al. 1998). This weak phe- conditions recommended by the manufacturer (Epicentre notype contrasts markedly with the more significant Technologies, Madison, WI). The 5Ј end of the D. pseudoob- effects resulting from loss of PLC-␥ function in verte- scura gene sequence was determined by primer walking within ␥ an overlapping 2.1-kb PstI/SacI fragment. For each species, brates: PLC- 1 knockout mice die in early embryogene- the subcloned fragments included the entire sl homolog. Se- sis (Ji et al. 1997), while mice lacking PLC-␥2 are viable, quencing reactions on purified plasmid DNA were performed but have defects in B cell development (Hashimoto et using the ABI Prism Big Dye terminator cycle sequencing kit al. 2000; Wang et al. 2000). The amino acid sequence (Applied Biosystems, Foster City, CA). Sequencing reactions of Sl is generally similar to its mammalian homologs were purified by ethanol precipitation with pellet paint (Nova- gen, Madison, WI) and separated on a Perkin-Elmer (Norwalk, (Emori et al. 1994), suggesting that its mode of activa- CT) ABI Prism 377 DNA sequencer. Sequences were edited tion and cellular function are conserved. However, sub- with Sequencher 4.1 (Gene Codes, Ann Arbor, MI) and fur- tle differences in its activation may exist; for example, ther analyzed with MacVector 6.01 (Accelrys, Madison, WI). two of the three tyrosines in mammalian PLC-␥1, which Phylogenetic analysis: Amino acid sequences were aligned become phosphorylated during activation, are missing using either ClustalW 1.4 (Thompson et al. 1994) or ClustalX 1.6 (Thompson et al. 1997) and in some cases improved by eye. in Sl. In an effort to determine the functional signifi- Unrooted trees were generated from the aligned sequences by cance of these and other differences between vertebrate PAUP (version 4.0b10; Swofford 2002) using bootstrap with and invertebrate PLC-␥, we cloned and sequenced two heuristic search and 1000 replicates; gaps were treated as miss- additional Drosophila PLC-␥ homologs. We present ing; parsimony was used as the optimality criterion; starting here a comparison of both the amino acid sequence and trees were obtained by stepwise addition; and branch swapping was by tree-bisection reconnection. the gene structure of these sl homologs in Drosophila, Tyrosine phosphorylation prediction: The probability of a as well as among a variety of other invertebrate and given tyrosine being phosphorylated was determined using vertebrate PLC-␥ homologs. Our analysis not only sheds the NetPhos algorithm (http://www.cbs.dtu.dk/services/ light on PLC-␥ function and evolution, but also provides NetPhos/), which uses a neural network method to compare tyrosines and their local context to tyrosines known to be evidence for a common origin of SH2 domains in all phosphorylated (Blom et al. 1999). A score Ͼ0.5 predicts that proteins. a tyrosine is in a context that will be phosphorylated in vivo. MATERIALS AND METHODS RESULTS ␭-library construction and screening: ␭-libraries of Drosophila Isolation of sl homologs from D. virilis and D. pseudoob- pseudoobcura and D. virilis genomic DNA were constructed in scura: We constructed ␭-libraries of D. pseudoobscura and ␭FIXII (Stratagene, La Jolla, CA) and ␭GEM11 (Promega, D. virilis genomic DNA and screened them with various Madison, WI), respectively. Briefly, genomic DNA was pre- ␥ pared from adults by standard methods, partially digested with genomic probes from the D. melanogaster PLC- homo- DpnII, and separated on a 0.5% agarose gel. Fragments in the log encoded by sl. Several clones were isolated and 10- to 20-kb range were recovered onto dialysis membrane mapped by Southern blotting and various restriction and the ends partially filled in with dCTP and dTTP. The fragments were subcloned and sequenced from each genomic fragments were then ligated overnight at 4Њ to XhoI- digested ␭DNA that had been partially filled in with dATP and species. We compared the two putative sl homologs with dGTP. The ligated DNA was packaged in vitro using Packagene the D.
Load more

Recommended publications
  • Association of Gene Ontology Categories with Decay Rate for Hepg2 Experiments These Tables Show Details for All Gene Ontology Categories
    Supplementary Table 1: Association of Gene Ontology Categories with Decay Rate for HepG2 Experiments These tables show details for all Gene Ontology categories. Inferences for manual classification scheme shown at the bottom. Those categories used in Figure 1A are highlighted in bold. Standard Deviations are shown in parentheses. P-values less than 1E-20 are indicated with a "0". Rate r (hour^-1) Half-life < 2hr. Decay % GO Number Category Name Probe Sets Group Non-Group Distribution p-value In-Group Non-Group Representation p-value GO:0006350 transcription 1523 0.221 (0.009) 0.127 (0.002) FASTER 0 13.1 (0.4) 4.5 (0.1) OVER 0 GO:0006351 transcription, DNA-dependent 1498 0.220 (0.009) 0.127 (0.002) FASTER 0 13.0 (0.4) 4.5 (0.1) OVER 0 GO:0006355 regulation of transcription, DNA-dependent 1163 0.230 (0.011) 0.128 (0.002) FASTER 5.00E-21 14.2 (0.5) 4.6 (0.1) OVER 0 GO:0006366 transcription from Pol II promoter 845 0.225 (0.012) 0.130 (0.002) FASTER 1.88E-14 13.0 (0.5) 4.8 (0.1) OVER 0 GO:0006139 nucleobase, nucleoside, nucleotide and nucleic acid metabolism3004 0.173 (0.006) 0.127 (0.002) FASTER 1.28E-12 8.4 (0.2) 4.5 (0.1) OVER 0 GO:0006357 regulation of transcription from Pol II promoter 487 0.231 (0.016) 0.132 (0.002) FASTER 6.05E-10 13.5 (0.6) 4.9 (0.1) OVER 0 GO:0008283 cell proliferation 625 0.189 (0.014) 0.132 (0.002) FASTER 1.95E-05 10.1 (0.6) 5.0 (0.1) OVER 1.50E-20 GO:0006513 monoubiquitination 36 0.305 (0.049) 0.134 (0.002) FASTER 2.69E-04 25.4 (4.4) 5.1 (0.1) OVER 2.04E-06 GO:0007050 cell cycle arrest 57 0.311 (0.054) 0.133 (0.002)
    [Show full text]
  • A Novel Resveratrol Analog: Its Cell Cycle Inhibitory, Pro-Apoptotic and Anti-Inflammatory Activities on Human Tumor Cells
    A NOVEL RESVERATROL ANALOG : ITS CELL CYCLE INHIBITORY, PRO-APOPTOTIC AND ANTI-INFLAMMATORY ACTIVITIES ON HUMAN TUMOR CELLS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Boren Lin May 2006 Dissertation written by Boren Lin B.S., Tunghai University, 1996 M.S., Kent State University, 2003 Ph. D., Kent State University, 2006 Approved by Dr. Chun-che Tsai , Chair, Doctoral Dissertation Committee Dr. Bryan R. G. Williams , Co-chair, Doctoral Dissertation Committee Dr. Johnnie W. Baker , Members, Doctoral Dissertation Committee Dr. James L. Blank , Dr. Bansidhar Datta , Dr. Gail C. Fraizer , Accepted by Dr. Robert V. Dorman , Director, School of Biomedical Sciences Dr. John R. Stalvey , Dean, College of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….………v LIST OF TABLES……………………………………………………………………….vii ACKNOWLEDGEMENTS….………………………………………………………….viii I INTRODUCTION….………………………………………………….1 Background and Significance……………………………………………………..1 Specific Aims………………………………………………………………………12 II MATERIALS AND METHODS.…………………………………………….16 Cell Culture and Compounds…….……………….…………………………….….16 MTT Cell Viability Assay………………………………………………………….16 Trypan Blue Exclusive Assay……………………………………………………...18 Flow Cytometry for Cell Cycle Analysis……………..……………....……………19 DNA Fragmentation Assay……………………………………………...…………23 Caspase-3 Activity Assay………………………………...……….….…….………24 Annexin V-FITC Staining Assay…………………………………..…...….………28 NF-kappa B p65 Activity Assay……………………………………..………….…29
    [Show full text]
  • Bayesian Analysis of Fmri Data and RNA-Seq Time Course Experiment Data
    Bayesian Analysis of fMRI Data and RNA-Seq Time Course Experiment Data A Dissertation presented to the Faculty of the Graduate School at the University of Missouri In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by YUAN CHENG Advisor: Marco A. R. Ferreira December 2015 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled: Bayesian Analysis of fMRI Data and RNA-Seq Time Course Experiment Data presented by Yuan Cheng, a candidate for the degree of Doctor of Philosophy and hereby certify that, in their opinion, it is worthy of acceptance. Dr. Marco A. R. Ferreira Dr. Paul Speckman Dr. TieMing Ji Dr. Subharup Guha Dr. Jeff Rouder ACKNOWLEDGMENTS I would never have been able to finish my dissertation without the guidance of my advisor and my committee members, help from friends, and support from my family and husband. I would like to express my sincere gratitude and thanks to my advisor, Marco A. R. Ferreira, for introducing me to the research of Bayesian statistics and all the challenging yet interesting topics. Without his continuous encouragement and inspi- ration, all the work will never be possible. I also thank him for his kind help and wise guidance through out my PhD study, which make my research life here much easier and more enjoyable. I am grateful to Dr. Rouder for his help in conquering hard background materials of functional MRI and his suggestions in the development of this work. I extend my thanks to my committee members: Dr. Paul Speckman, Dr.
    [Show full text]
  • Multiomic Analysis of the UV-Induced DNA Damage Response
    Resource Multiomic Analysis of the UV-Induced DNA Damage Response Graphical Abstract Authors Stefan Boeing, Laura Williamson, Vesela Encheva, ..., Michael Howell, Ambrosius P. Snijders, Jesper Q. Svejstrup Correspondence [email protected] In Brief Boeing et al. investigate the UV-induced DNA damage response by combining a range of proteomic and genomic screens. A function in this response for the melanoma driver STK19 as well as a number of other factors are uncovered. Highlights d A multiomic screening approach examines the UV-induced DNA damage response d Multiple factors are connected to the transcription-related DNA damage response d Melanoma gene STK19 is required for a normal DNA damage response Boeing et al., 2016, Cell Reports 15, 1597–1610 May 17, 2016 ª 2016 The Author(s) http://dx.doi.org/10.1016/j.celrep.2016.04.047 Cell Reports Resource Multiomic Analysis of the UV-Induced DNA Damage Response Stefan Boeing,1,5 Laura Williamson,1 Vesela Encheva,2 Ilaria Gori,3 Rebecca E. Saunders,3 Rachael Instrell,3 Ozan Aygun,€ 1,7 Marta Rodriguez-Martinez,1 Juston C. Weems,4 Gavin P. Kelly,5 Joan W. Conaway,4,6 Ronald C. Conaway,4,6 Aengus Stewart,5 Michael Howell,3 Ambrosius P. Snijders,2 and Jesper Q. Svejstrup1,* 1Mechanisms of Transcription Laboratory, the Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK 2Protein Analysis and Proteomics Laboratory, the Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK 3High Throughput Screening Laboratory, the Francis Crick Institute, 44 Lincoln’s
    [Show full text]
  • A Role for the RNA Pol II–Associated PAF Complex in AID-Induced Immune Diversification
    Article A role for the RNA pol II–associated PAF complex in AID-induced immune diversification Katharina L. Willmann,1,2 Sara Milosevic,4 Siim Pauklin,1 Kerstin-Maike Schmitz,2 Gopinath Rangam,1,2 Maria T. Simon,1 Sarah Maslen,3 Mark Skehel,3 Isabelle Robert,4 Vincent Heyer,4 Ebe Schiavo,4 Bernardo Reina-San-Martin,4 Svend K. Petersen-Mahrt1,2 1DNA Editing Laboratory, London Research Institute, South Mimms EN6 3LD, England, UK 2DNA Editing in Immunity and Epigenetics, IFOM-Fondazione Instituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milano, Italy 3Protein Analysis and Proteomics Laboratory, London Research Institute, South Mimms EN6 3LD, England, UK 4Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Centre National de la Recherche Scientifique (CNRS) UMR7104, Université de Strasbourg, 67404 Illkirch, France Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcrip- tion possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversi- fying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase sub- units, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin.
    [Show full text]
  • Variation in Protein Coding Genes Identifies Information Flow
    bioRxiv preprint doi: https://doi.org/10.1101/679456; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Animal complexity and information flow 1 1 2 3 4 5 Variation in protein coding genes identifies information flow as a contributor to 6 animal complexity 7 8 Jack Dean, Daniela Lopes Cardoso and Colin Sharpe* 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Institute of Biological and Biomedical Sciences 25 School of Biological Science 26 University of Portsmouth, 27 Portsmouth, UK 28 PO16 7YH 29 30 * Author for correspondence 31 [email protected] 32 33 Orcid numbers: 34 DLC: 0000-0003-2683-1745 35 CS: 0000-0002-5022-0840 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Abstract bioRxiv preprint doi: https://doi.org/10.1101/679456; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Animal complexity and information flow 2 1 Across the metazoans there is a trend towards greater organismal complexity. How 2 complexity is generated, however, is uncertain. Since C.elegans and humans have 3 approximately the same number of genes, the explanation will depend on how genes are 4 used, rather than their absolute number.
    [Show full text]
  • Using Network Inference to Discover Molecular Pathways Underlying Cytokine Synergism and Age-Related Neurodegeneration by Bryce Hwang S.B., M.I.T
    Using network inference to discover molecular pathways underlying cytokine synergism and age-related neurodegeneration by Bryce Hwang S.B., M.I.T. (2018) Submitted to the Department of Electrical Engineering and Computer Science in partial fulfillment of the requirements for the degree of Master of Engineering in Computer Science and Molecular Biology at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2018 ○c Bryce Hwang. All rights reserved. The author hereby grants to M.I.T. permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Author.............................................................. Department of Electrical Engineering and Computer Science May 25, 2018 Certified by. Ernest Fraenkel Professor Thesis Supervisor Accepted by . Katrina LaCurts Chair, Master of Engineering Thesis Committee Using network inference to discover molecular pathways underlying cytokine synergism and age-related neurodegeneration by Bryce Hwang Submitted to the Department of Electrical Engineering and Computer Science on May 25, 2018, in partial fulfillment of the requirements for the degree of Master of Engineering in Computer Science and Molecular Biology Abstract New high-throughput “omic” methods can help shed light on molecular pathways underpinning diseases ranging from cancers to neurodegenerative disorders. However, effectively integrating information across these diverse data types is challenging. Network modeling approaches can help bridge this gap. In particular, the Prize- Collecting Steiner Forest approach (PCSF) is a network modeling method that provides high-confidence subnetworks of physically interacting molecules by integrating diverse “omics” data with prior knowledge from protein-protein interaction networks (PPIs). However, PCSF is sensitive to initial parameterization and generating biological hypotheses from the resulting subnetworks can often be difficult.
    [Show full text]
  • Content Based Search in Gene Expression Databases and a Meta-Analysis of Host Responses to Infection
    Content Based Search in Gene Expression Databases and a Meta-analysis of Host Responses to Infection A Thesis Submitted to the Faculty of Drexel University by Francis X. Bell in partial fulfillment of the requirements for the degree of Doctor of Philosophy November 2015 c Copyright 2015 Francis X. Bell. All Rights Reserved. ii Acknowledgments I would like to acknowledge and thank my advisor, Dr. Ahmet Sacan. Without his advice, support, and patience I would not have been able to accomplish all that I have. I would also like to thank my committee members and the Biomed Faculty that have guided me. I would like to give a special thanks for the members of the bioinformatics lab, in particular the members of the Sacan lab: Rehman Qureshi, Daisy Heng Yang, April Chunyu Zhao, and Yiqian Zhou. Thank you for creating a pleasant and friendly environment in the lab. I give the members of my family my sincerest gratitude for all that they have done for me. I cannot begin to repay my parents for their sacrifices. I am eternally grateful for everything they have done. The support of my sisters and their encouragement gave me the strength to persevere to the end. iii Table of Contents LIST OF TABLES.......................................................................... vii LIST OF FIGURES ........................................................................ xiv ABSTRACT ................................................................................ xvii 1. A BRIEF INTRODUCTION TO GENE EXPRESSION............................. 1 1.1 Central Dogma of Molecular Biology........................................... 1 1.1.1 Basic Transfers .......................................................... 1 1.1.2 Uncommon Transfers ................................................... 3 1.2 Gene Expression ................................................................. 4 1.2.1 Estimating Gene Expression ............................................ 4 1.2.2 DNA Microarrays ......................................................
    [Show full text]
  • The Neurodegenerative Diseases ALS and SMA Are Linked at The
    Nucleic Acids Research, 2019 1 doi: 10.1093/nar/gky1093 The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex Downloaded from https://academic.oup.com/nar/advance-article-abstract/doi/10.1093/nar/gky1093/5162471 by [email protected] on 06 November 2018 Binkai Chi, Jeremy D. O’Connell, Alexander D. Iocolano, Jordan A. Coady, Yong Yu, Jaya Gangopadhyay, Steven P. Gygi and Robin Reed* Department of Cell Biology, Harvard Medical School, 240 Longwood Ave. Boston MA 02115, USA Received July 17, 2018; Revised October 16, 2018; Editorial Decision October 18, 2018; Accepted October 19, 2018 ABSTRACT Fused in Sarcoma (FUS) and TAR DNA Binding Protein (TARDBP) (9–13). FUS is one of the three members of Understanding the molecular pathways disrupted in the structurally related FET (FUS, EWSR1 and TAF15) motor neuron diseases is urgently needed. Here, we family of RNA/DNA binding proteins (14). In addition to employed CRISPR knockout (KO) to investigate the the RNA/DNA binding domains, the FET proteins also functions of four ALS-causative RNA/DNA binding contain low-complexity domains, and these domains are proteins (FUS, EWSR1, TAF15 and MATR3) within the thought to be involved in ALS pathogenesis (5,15). In light RNAP II/U1 snRNP machinery. We found that each of of the discovery that mutations in FUS are ALS-causative, these structurally related proteins has distinct roles several groups carried out studies to determine whether the with FUS KO resulting in loss of U1 snRNP and the other two members of the FET family, TATA-Box Bind- SMN complex, EWSR1 KO causing dissociation of ing Protein Associated Factor 15 (TAF15) and EWS RNA the tRNA ligase complex, and TAF15 KO resulting in Binding Protein 1 (EWSR1), have a role in ALS.
    [Show full text]
  • Product Datasheet Qprest
    Product Datasheet QPrEST PRODUCT SPECIFICATION Product Name QPrEST SPT6H Mass Spectrometry Protein Standard Product Number QPrEST29936 Protein Name Transcription elongation factor SPT6 Uniprot ID Q7KZ85 Gene SUPT6H Product Description Stable isotope-labeled standard for absolute protein quantification of Transcription elongation factor SPT6. Lys (13C and 15N) and Arg (13C and 15N) metabolically labeled recombinant human protein fragment. Application Absolute protein quantification using mass spectrometry Sequence (excluding RTTYIKRVIAHPSFHNINFKQAEKMMETMDQGDVIIRPSSKGENHLTVTW fusion tag) KVSDGIYQHVDVREEGKENAFSLGATLWINSEEFEDLDEIVARYVQPM Theoretical MW 29211 Da including N-terminal His6ABP fusion tag Fusion Tag A purification and quantification tag (QTag) consisting of a hexahistidine sequence followed by an Albumin Binding Protein (ABP) domain derived from Streptococcal Protein G. Expression Host Escherichia coli LysA ArgA BL21(DE3) Purification IMAC purification Purity >90% as determined by Bioanalyzer Protein 230 Purity Assay Isotopic Incorporation >99% Concentration >5 μM after reconstitution in 100 μl H20 Concentration Concentration determined by LC-MS/MS using a highly pure amino acid analyzed internal Determination reference (QTag), CV ≤10%. Amount >0.5 nmol per vial, two vials supplied. Formulation Lyophilized in 100 mM Tris-HCl 5% Trehalose, pH 8.0 Instructions for Spin vial before opening. Add 100 μL ultrapure H2O to the vial. Vortex thoroughly and spin Reconstitution down. For further dilution, see Application Protocol. Shipping Shipped at ambient temperature Storage Lyophilized product shall be stored at -20°C. See COA for expiry date. Reconstituted product can be stored at -20°C for up to 4 weeks. Avoid repeated freeze-thaw cycles. Notes For research use only Product of Sweden. For research use only. Not intended for pharmaceutical development, diagnostic, therapeutic or any in vivo use.
    [Show full text]
  • Quantitative Proteomic Comparison of Myofibroblasts Derived from Bone
    www.nature.com/scientificreports OPEN Quantitative proteomic comparison of myofbroblasts derived from bone marrow and cornea Paramananda Saikia1,4, Jack S. Crabb1,2,4, Luciana L. Dibbin1, Madison J. Juszczak1, Belinda Willard2, Geeng‑Fu Jang1,2, Thomas Michael Shiju1, John W. Crabb1,2,3* & Steven E. Wilson1,3* Myofbroblasts are fbroblastic cells that function in wound healing, tissue repair and fbrosis, and arise from bone marrow (BM)‑derived fbrocytes and a variety of local progenitor cells. In the cornea, myofbroblasts are derived primarily from stromal keratocytes and from BM‑derived fbrocytes after epithelial‑stromal and endothelial‑stromal injuries. Quantitative proteomic comparison of mature alpha‑smooth muscle actin (α‑SMA)+ myofbroblasts (verifed by immunocytochemistry for vimentin, α‑SMA, desmin, and vinculin) generated from rabbit corneal fbroblasts treated with transforming growth factor (TGF) beta‑1 or generated directly from cultured BM treated with TGF beta‑1 was pursued for insights into possible functional diferences. Paired cornea‑derived and BM‑derived α‑SMA+ myofbroblast primary cultures were generated from four New Zealand white rabbits and confrmed to be myofbroblasts by immunocytochemistry. Paired cornea‑ and BM‑derived myofbroblast specimens from each rabbit were analyzed by LC MS/MS iTRAQ technology using an Orbitrap Fusion Lumos Tribrid mass spectrometer, the Mascot search engine, the weighted average quantifcation method and the UniProt rabbit and human databases. From 2329 proteins quantifed with ≥ 2 unique peptides from ≥ 3 rabbits, a total of 673 diferentially expressed (DE) proteins were identifed. Bioinformatic analysis of DE proteins with Ingenuity Pathway Analysis implicate progenitor‑dependent functional diferences in myofbroblasts that could impact tissue development.
    [Show full text]
  • S41467-019-13144-Y.Pdf
    ARTICLE https://doi.org/10.1038/s41467-019-13144-y OPEN The landscape of multiscale transcriptomic networks and key regulators in Parkinson’s disease Qian Wang1,2,3,4, Yuanxi Zhang1, Minghui Wang 2,3,4, Won-Min Song 2,3,4, Qi Shen2,3,4, Andrew McKenzie2,3,4, Insup Choi1, Xianxiao Zhou 2,3,4, Ping-Yue Pan1, Zhenyu Yue1* & Bin Zhang 2,3,4* Genetic and genomic studies have advanced our knowledge of inherited Parkinson’s disease (PD), however, the etiology and pathophysiology of idiopathic PD remain unclear. Herein, we 1234567890():,; perform a meta-analysis of 8 PD postmortem brain transcriptome studies by employing a multiscale network biology approach to delineate the gene-gene regulatory structures in the substantia nigra and determine key regulators of the PD transcriptomic networks. We identify STMN2, which encodes a stathmin family protein and is down-regulated in PD brains, as a key regulator functionally connected to known PD risk genes. Our network analysis predicts a function of human STMN2 in synaptic trafficking, which is validated in Stmn2-knockdown mouse dopaminergic neurons. Stmn2 reduction in the mouse midbrain causes dopaminergic neuron degeneration, phosphorylated α-synuclein elevation, and locomotor deficits. Our integrative analysis not only begins to elucidate the global landscape of PD transcriptomic networks but also pinpoints potential key regulators of PD pathogenic pathways. 1 Department of Neurology and Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA. 2 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA.
    [Show full text]