Cas3 Protein—A Review of a Multi-Tasking Machine
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Ribonuclease L Mediates the Cell-Lethal Phenotype of the Double-Stranded RNA Editing
1 2 Ribonuclease L mediates the cell-lethal phenotype of the double-stranded RNA editing 3 enzyme ADAR1 in a human cell line 4 5 Yize Lia,#, Shuvojit Banerjeeb,#, Stephen A. Goldsteina, Beihua Dongb, Christina Gaughanb, Sneha 6 Rathc, Jesse Donovanc, Alexei Korennykhc, Robert H. Silvermanb,* and Susan R Weissa,* 7 aDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, 8 Philadelphia, PA, USA, 19104; b Department of Cancer Biology, Lerner Research Institute, 9 Cleveland Clinic, Cleveland, OH, USA 44195; c Department of Molecular Biology, Princeton 10 University, Princeton, NJ 08544 11 12 13 14 15 16 17 18 # These authors contributed equally to this work 19 * Corresponding authors 20 21 22 23 24 25 26 27 28 29 30 Abstract 31 ADAR1 isoforms are adenosine deaminases that edit and destabilize double-stranded RNA 32 reducing its immunostimulatory activities. Mutation of ADAR1 leads to a severe neurodevelopmental 33 and inflammatory disease of children, Aicardi-Goutiéres syndrome. In mice, Adar1 mutations are 34 embryonic lethal but are rescued by mutation of the Mda5 or Mavs genes, which function in IFN 35 induction. However, the specific IFN regulated proteins responsible for the pathogenic effects of 36 ADAR1 mutation are unknown. We show that the cell-lethal phenotype of ADAR1 deletion in human 37 lung adenocarcinoma A549 cells is rescued by CRISPR/Cas9 mutagenesis of the RNASEL gene or 38 by expression of the RNase L antagonist, murine coronavirus NS2 accessory protein. Our result 39 demonstrate that ablation of RNase L activity promotes survival of ADAR1 deficient cells even in the 40 presence of MDA5 and MAVS, suggesting that the RNase L system is the primary sensor pathway 41 for endogenous dsRNA that leads to cell death. -
Chapter 1 Introduction
The Conditional Protein Splicing of Alpha-Sarcin: A model for inducible assembly of protein toxins in vivo. by Spencer C. Alford B.Sc., University of Victoria, 2004 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in the Department of Biochemistry and Microbiology Spencer C. Alford, 2007 University of Victoria All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. ii Supervisory Committee The Conditional Protein Splicing of Alpha-Sarcin: A model for inducible assembly of protein toxins in vivo. by Spencer C. Alford B.Sc, University of Victoria, 2004 Supervisory Committee Dr. Perry Howard, Supervisor (Department of Biochemistry and Microbiology) Dr. Juan Ausio, Departmental Member (Department of Biochemistry and Microbiology) Dr. Robert Chow, Outside Member (Department of Biology) iii Supervisory Committee Dr. Perry Howard, Supervisor (Department of Biochemistry and Microbiology) Dr. Juan Ausio, Departmental Member (Department of Biochemistry and Microbiology) Dr. Robert Chow, Outside Member (Department of Biology) Abstract Conditional protein splicing (CPS) is an intein-mediated post-translational modification. Inteins are intervening protein elements that autocatalytically excise themselves from precursor proteins to ligate flanking protein sequences, called exteins, with a native peptide bond. Artificially split inteins can mediate the same process by splicing proteins in trans, when intermolecular reconstitution of split intein fragments occurs. An established CPS model utilizes an artificially split Saccharomyces cerevisiae intein, called VMA. In this model, VMA intein fragments are fused to the heterodimerization domains, FKBP and FRB, which selectively form a complex with the immunosuppressive drug, rapamycin. -
Molecular Insights Into DNA Interference by CRISPR-Associated Nuclease-Helicase Cas3
Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3 Bei Gonga,1, Minsang Shinb,1, Jiali Suna, Che-Hun Junga,b, Edward L. Boltc, John van der Oostd, and Jeong-Sun Kima,b,2 aInterdisciplinary Graduate Program in Molecular Medicine, Chonnam National University, Gwangju 501-746, Korea; bDepartment of Chemistry, Chonnam National University, Gwangju 500-757, Korea; cSchool of Life Sciences, University of Nottingham Medical School, Nottingham NG72UH, United Kingdom; and dLaboratory of Microbiology, Wageningen University, 6703 HB, Wageningen, The Netherlands Edited by Wei Yang, National Institutes of Health, Bethesda, MD, and approved September 26, 2014 (received for review June 10, 2014) Mobile genetic elements in bacteria are neutralized by a system 21), Cascade complex by itself is not a nuclease for degradation based on clustered regularly interspaced short palindromic repeats of invader DNA (13). In Escherichia coli K-12 (type IE), Cascade (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas recognizes and binds crRNA to complimentary sequence in systems use a “Cascade” ribonucleoprotein complex to guide RNA target DNA, generating an RNA mediated displacement loop specifically to complementary sequence in invader double-stranded (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid DNA (dsDNA), a process called “interference.” After target recogni- within double-stranded (ds) target DNA (13). Formation of the tion by Cascade, formation of an R-loop triggers recruitment of R-loop structure induces conformational change in Cascade subunits, triggering recruitment of Cas3 protein that is a nucle- a Cas3 nuclease-helicase, completing the interference process by – destroying the invader dsDNA. -
Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy
toxins Article Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins Javier Ruiz-de-la-Herrán 1, Jaime Tomé-Amat 1,2 , Rodrigo Lázaro-Gorines 1, José G. Gavilanes 1 and Javier Lacadena 1,* 1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid 28040, Spain; [email protected] (J.R.-d.-l.-H.); [email protected] (J.T.-A.); [email protected] (R.L.-G.); [email protected] (J.G.G.) 2 Centre for Plant Biotechnology and Genomics (UPM-INIA), Universidad Politécnica de Madrid, Pozuelo de Alarcón, Madrid 28223, Spain * Correspondence: [email protected]; Tel.: +34-91-394-4266 Received: 3 September 2019; Accepted: 10 October 2019; Published: 12 October 2019 Abstract: Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. -
Molecular Insights Into DNA Interference by CRISPR-Associated Nuclease-Helicase Cas3
Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3 Bei Gonga,1, Minsang Shinb,1, Jiali Suna, Che-Hun Junga,b, Edward L. Boltc, John van der Oostd, and Jeong-Sun Kima,b,2 aInterdisciplinary Graduate Program in Molecular Medicine, Chonnam National University, Gwangju 501-746, Korea; bDepartment of Chemistry, Chonnam National University, Gwangju 500-757, Korea; cSchool of Life Sciences, University of Nottingham Medical School, Nottingham NG72UH, United Kingdom; and dLaboratory of Microbiology, Wageningen University, 6703 HB, Wageningen, The Netherlands Edited by Wei Yang, National Institutes of Health, Bethesda, MD, and approved September 26, 2014 (received for review June 10, 2014) Mobile genetic elements in bacteria are neutralized by a system 21), Cascade complex by itself is not a nuclease for degradation based on clustered regularly interspaced short palindromic repeats of invader DNA (13). In Escherichia coli K-12 (type IE), Cascade (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas recognizes and binds crRNA to complimentary sequence in systems use a “Cascade” ribonucleoprotein complex to guide RNA target DNA, generating an RNA mediated displacement loop specifically to complementary sequence in invader double-stranded (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid DNA (dsDNA), a process called “interference.” After target recogni- within double-stranded (ds) target DNA (13). Formation of the tion by Cascade, formation of an R-loop triggers recruitment of R-loop structure induces conformational change in Cascade subunits, triggering recruitment of Cas3 protein that is a nucle- a Cas3 nuclease-helicase, completing the interference process by – destroying the invader dsDNA. -
XXI Fungal Genetics Conference Abstracts
Fungal Genetics Reports Volume 48 Article 17 XXI Fungal Genetics Conference Abstracts Fungal Genetics Conference Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Fungal Genetics Conference. (2001) "XXI Fungal Genetics Conference Abstracts," Fungal Genetics Reports: Vol. 48, Article 17. https://doi.org/10.4148/1941-4765.1182 This Supplementary Material is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact [email protected]. XXI Fungal Genetics Conference Abstracts Abstract XXI Fungal Genetics Conference Abstracts This supplementary material is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol48/iss1/17 : XXI Fungal Genetics Conference Abstracts XXI Fungal Genetics Conference Abstracts Plenary sessions Cell Biology (1-87) Population and Evolutionary Biology (88-124) Genomics and Proteomics (125-179) Industrial Biology and Biotechnology (180-214) Host-Parasite Interactions (215-295) Gene Regulation (296-385) Developmental Biology (386-457) Biochemistry and Secondary Metabolism(458-492) Unclassified(493-502) Index to Abstracts Abstracts may be cited as "Fungal Genetics Newsletter 48S:abstract number" Plenary Abstracts COMPARATIVE AND FUNCTIONAL GENOMICS FUNGAL-HOST INTERACTIONS CELL BIOLOGY GENOME STRUCTURE AND MAINTENANCE COMPARATIVE AND FUNCTIONAL GENOMICS Genome reconstruction and gene expression for the rice blast fungus, Magnaporthe grisea. Ralph A. Dean. Fungal Genomics Laboratory, NC State University, Raleigh NC 27695 Rice blast disease, caused by Magnaporthe grisea, is one of the most devastating threats to food security worldwide. -
Very Fast Empirical Prediction and Rationalization of Protein Pka Values
PROTEINS: Structure, Function, and Bioinformatics 61:704–721 (2005) Very Fast Empirical Prediction and Rationalization of Protein pKa Values Hui Li,1† Andrew D. Robertson,2 and Jan H. Jensen1* 1Department of Chemistry and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 2Department of Biochemistry, The University of Iowa, Iowa City, Iowa 8,14–17 ABSTRACT A very fast empirical method is added to a model pKa value. These models usually presented for structure-based protein pKa predic- have a root-mean-square deviation (RMSD) from experi- tion and rationalization. The desolvation effects ment of Ͻ1. However, the currently available LPBE and intra-protein interactions, which cause varia- models tend to overestimate the intraprotein charge– 18,19 tions in pKa values of protein ionizable groups, are charge interactions and underestimate the hydrogen- 20 empirically related to the positions and chemical bonding and desolvation effects in calculating pKa shifts. nature of the groups proximate to the pKa sites. A The LPBE methods typically require tens of minutes to computer program is written to automatically pre- hours of computer time. dict pKa values based on these empirical relation- The pKa values of small organic acids and bases can be 21 ships within a couple of seconds. Unusual pKa val- predicted with the Hammet-Taft method based on empiri- ues at buried active sites, which are among the most cal relationship between pKa shifts and substituents. The interesting protein pKa values, are predicted very Hammet-Taft method is accurate for molecules similar to well with the empirical method. A test on 233 car- the ones included in the parameterization and is very fast. -
Computational Analysis of Protein Function Within Complete Genomes
Computational Analysis of Protein Function within Complete Genomes Anton James Enright Wolfson College A dissertation submitted to the University of Cambridge for the degree of Doctor of Philosophy European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, United Kingdom. Email: [email protected] March 7, 2002 To My Parents and Kerstin This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. This thesis does not exceed the specified length limit of 300 pages as de- fined by the Biology Degree Committee. This thesis has been typeset in 12pt font using LATEX2ε accordingtothe specifications defined by the Board of Graduate Studies and the Biology Degree Committee. ii Computational Analysis of Protein Function within Complete Genomes Summary Anton James Enright March 7, 2002 Wolfson College Since the advent of complete genome sequencing, vast amounts of nucleotide and amino acid sequence data have been produced. These data need to be effectively analysed and verified so that they may be used for biologi- cal discovery. A significant proportion of predicted protein sequences from these complete genomes have poorly characterised or unknown functional annotations. This thesis describes a number of approaches which detail the computational analysis of amino acid sequences for the prediction and analy- sis of protein function within complete genomes. The first chapter is a short introduction to computational genome analysis while the second and third chapters describe how groups of related protein sequences (termed protein families) may be characterised using sequence clustering algorithms. -
Method for Prediction of Protein Function from Sequence Using The
Article No. mb981993 J. Mol. Biol. (1998) 281, 949±968 Method for Prediction of Protein Function from Sequence using the Sequence-to-Structure-to- Function Paradigm with Application to Glutaredoxins/ Thioredoxins and T1 Ribonucleases Jacquelyn S. Fetrow1 and Jeffrey Skolnick2* 1Department of Biological The practical exploitation of the vast numbers of sequences in the gen- Sciences, Center for ome sequence databases is crucially dependent on the ability to identify Biochemistry and Biophysics the function of each sequence. Unfortunately, current methods, including University at Albany, SUNY global sequence alignment and local sequence motif identi®cation, are 1400 Washington Avenue limited by the extent of sequence similarity between sequences of Albany, NY 12222, USA unknown and known function; these methods increasingly fail as the sequence identity diverges into and beyond the twilight zone of sequence 2Department of Molecular identity. To address this problem, a novel method for identi®cation of Biology, The Scripps Institute protein function based directly on the sequence-to-structure-to-function 10550 North Torrey Pines paradigm is described. Descriptors of protein active sites, termed ``fuzzy Road, La Jolla, CA 92037, USA functional forms'' or FFFs, are created based on the geometry and confor- mation of the active site. By way of illustration, the active sites respon- sible for the disul®de oxidoreductase activity of the glutaredoxin/ thioredoxin family and the RNA hydrolytic activity of the T1 ribonuclease family are presented. First, the FFFs are shown to correctly identify their corresponding active sites in a library of exact protein models produced by crystallography or NMR spectroscopy, most of which lack the speci- ®ed activity. -
Mimicking the Action of Ribonucleases: Studies on Rnase a and Design of PNA Based Artificial Enzymes
From DEPARTMENT OF BIOSCIENCES AND NUTRITION Karolinska Institutet, Stockholm, Sweden Mimicking the action of ribonucleases: studies on RNase A and design of PNA based artificial enzymes Alice Ghidini Stockholm 2015 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Eprint AB 2015 © Alice Ghidini, 2015 ISBN 978-91-7676-039-0 Mimicking the action of ribonucleases: studies on RNase A and design of PNA based artificial enzymes THESIS FOR DOCTORAL DEGREE (Ph.D.) By Alice Ghidini Principal Supervisor: Opponent: Professor Roger Strömberg Professor Michael J. Gait Karolinska Institutet Medical Research Council Department of Bioscience and nutrition Department of Laboratory of Molecular Biology Division of Bioorganic chemistry Examination Board: Co-supervisor(s): Lars Baltzer Merita Murtola Uppsala Universitet Karolinska Institutet Department of Institutionen för kemi Department of Bioscience and nutrition Division of BMC, Fysikalisk organisk kemi Division of Bioorganic chemistry Mikael Leijon Malgorzata Honcharenko National Veterinary Institute (SVA) Karolinska Institutet Division of Virology, Immunobiology and Department of Bioscience and nutrition Parasitology (VIP) Division of Bioorganic chemistry Marcus Wilhelmsson Chalmers University of Technology Department of Chemical and Chemical Engineering/Chemistry and Biochemistry With love to my family, lontani ma sempre vicini… ABSTRACT A 3’-deoxy-3’-C-methylenephosphonate modified diribonucleotide is highly resistant to degradation by spleen phosphodiesterase and not cleaved at all by snake venom phosphodiesterase. Despite that both the vicinal 2-hydroxy nucleophile and the 5’-oxyanion leaving group are intact, the 3’-methylenephosponate RNA modification is also highly resistant towards the action of RNase A. Several different approaches were explored for conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues. -
Bacterial Retrons Function in Anti-Phage Defense
Article Bacterial Retrons Function In Anti-Phage Defense Graphical Abstract Authors Adi Millman, Aude Bernheim, Retrons appear in an operon Retrons generate an RNA-DNA hybrid via reverse transcription with additional “effector” genes Avigail Stokar-Avihail, ..., Azita Leavitt, ncRNA Reverse Transcriptase (RT) msDNA Ribosyltransferase Yaara Oppenheimer-Shaanan, (RNA-DNA hybrid) DNA-binding RT Rotem Sorek RNA Retron function 2 transmembrane 5’ G RT domains 2’-5’ 3’ was unknown 3’ RT Correspondence cDNA RT Cold-shock [email protected] 5’ G 2’ 3’ G G cDNA RT RT ATPase Nuclease In Brief reverse transcription RNase H Retrons are part of a large family of anti- Retrons protect bacteria from phage Inhibition of RecBCD by phages triggers retron Ec48 defense phage defense systems that are widespread in bacteria and confer Bacterial density during phage infection Growth resistance against a broad range of B Effector arrest RT D RT activation with retron C B phages, mediated by abortive infection. Effector no retron Ec48 “guards” the bacterial Effector activation leads RecBCD complex to abortive infection bacterial density Retron Ec48 RT 2TM time RecBCD inhibitor B B D D RT Bacteria without retron Bacteria with retron C C B B Phage proteins inhibit RecBCD Retron Ec48 senses RecBCD inhibition Highlights d Retrons are preferentially located in defense islands d Retrons, together with their effector genes, protect bacteria from phages d Protection from phage is mediated by abortive infection d Retron Ec48 guards RecBCD. Inhibition of RecBCD by phages triggers retron defense Millman et al., 2020, Cell 183, 1–11 December 10, 2020 ª 2020 Elsevier Inc. -
Contribution of SAM and HD Domains to Retroviral Restriction Mediated by Human SAMHD1
Virology 436 (2013) 81–90 Contents lists available at SciVerse ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1 Tommy E. White a,1, Alberto Brandariz-Nun˜ez a,1, Jose Carlos Valle-Casuso a, Sarah Amie b, Laura Nguyen b, Baek Kim b, Jurgen Brojatsch a, Felipe Diaz-Griffero a,n a Department of Microbiology and Immunology, Albert Einstein College of Medicine Bronx, NY 10461, USA b Department of Microbiology & Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA article info abstract Article history: The human SAMHD1 protein is a novel retroviral restriction factor expressed in myeloid cells. Previous Received 1 September 2012 work has correlated the deoxynucleotide triphosphohydrolase activity of SAMHD1 with its ability to Returned to author for revisions block HIV-1 and SIVmac infection. SAMHD1 is comprised of the sterile alpha motif (SAM) and histidine– 24 September 2012 aspartic (HD) domains; however the contribution of these domains to retroviral restriction is not Accepted 20 October 2012 understood. Mutagenesis and deletion studies revealed that expression of the sole HD domain of Available online 13 November 2012 SAMHD1 is sufficient to achieve potent restriction of HIV-1 and SIVmac. We demonstrated that the HD Keywords: domain of SAMHD1 is essential for the ability of SAMHD1 to oligomerize by using a biochemical assay. SAMHD1 In agreement with previous observations, we mapped the RNA-binding ability of SAMHD1 to the HD HIV-1 domain. We also demonstrated a direct interaction of SAMHD1 with RNA by using enzymatically-active HD domain purified SAMHD1 protein from insect cells.