Characterization of a Heat-Stable Enzyme Possessing GTP-Dependent RNA Ligase Activity from a Hyperthermophilic Archaeon, Pyrococcus Furiosus
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Characterization and evolution of artificial RNA ligases A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Aleardo Morelli IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Adviser: Burckhard Seelig June 2015 © Aleardo Morelli, 2015 Abstract Enzymes enable biocatalysis with minimal by-products, high regio- and enantioselectivity, and can operate under mild conditions. These properties facilitate numerous applications of enzymes in both industry and research. Great progress has been made in protein engineering to modify properties such as stability and catalytic activity of an enzyme to suit specific processes. On the contrary, the generation of artificial enzymes de novo is still challenging, and only few examples have been reported. The study and characterization of artificial enzymes will not only expand our knowledge of protein chemistry and catalysis, but ultimately improve our ability to generate novel biocatalysts and engineer those found in nature. My thesis focused on the characterization of an artificial RNA ligase previously selected from a library of polypeptide variants based on a non-catalytic protein scaffold. The selection employed mRNA display, a technique to isolate de novo enzymes in vitro from large libraries of 1013 protein variants. The artificial RNA ligase catalyzes the formation of a phosphodiester bond between two RNA substrates by joining a 5'- triphospate to a 3'-hydroxyl, with the release of pyrophosphate. This activity has not been observed in nature. An initial selection carried out at 23°C yielded variants that were poorly suitable for biochemical and biophysical characterization due do their low solubility and poor folding. -
Phosphodiesterase (PDE)
Phosphodiesterase (PDE) Phosphodiesterase (PDE) is any enzyme that breaks a phosphodiester bond. Usually, people speaking of phosphodiesterase are referring to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below. However, there are many other families of phosphodiesterases, including phospholipases C and D, autotaxin, sphingomyelin phosphodiesterase, DNases, RNases, and restriction endonucleases, as well as numerous less-well-characterized small-molecule phosphodiesterases. The cyclic nucleotide phosphodiesterases comprise a group of enzymes that degrade the phosphodiester bond in the second messenger molecules cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling within subcellular domains. PDEs are therefore important regulators ofsignal transduction mediated by these second messenger molecules. www.MedChemExpress.com 1 Phosphodiesterase (PDE) Inhibitors, Activators & Modulators (+)-Medioresinol Di-O-β-D-glucopyranoside (R)-(-)-Rolipram Cat. No.: HY-N8209 ((R)-Rolipram; (-)-Rolipram) Cat. No.: HY-16900A (+)-Medioresinol Di-O-β-D-glucopyranoside is a (R)-(-)-Rolipram is the R-enantiomer of Rolipram. lignan glucoside with strong inhibitory activity Rolipram is a selective inhibitor of of 3', 5'-cyclic monophosphate (cyclic AMP) phosphodiesterases PDE4 with IC50 of 3 nM, 130 nM phosphodiesterase. and 240 nM for PDE4A, PDE4B, and PDE4D, respectively. Purity: >98% Purity: 99.91% Clinical Data: No Development Reported Clinical Data: No Development Reported Size: 1 mg, 5 mg Size: 10 mM × 1 mL, 10 mg, 50 mg (R)-DNMDP (S)-(+)-Rolipram Cat. No.: HY-122751 ((+)-Rolipram; (S)-Rolipram) Cat. No.: HY-B0392 (R)-DNMDP is a potent and selective cancer cell (S)-(+)-Rolipram ((+)-Rolipram) is a cyclic cytotoxic agent. (R)-DNMDP, the R-form of DNMDP, AMP(cAMP)-specific phosphodiesterase (PDE) binds PDE3A directly. -
Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase -
Lipid Related Genes Altered in NASH Connect Inflammation in Liver Pathogenesis Progression to HCC: a Canonical Pathway
Lipid related genes altered in NASH connect inflammation in liver pathogenesis progression to HCC: a canonical pathway Christophe Desterke1, Franck Chiappini2* 1 Inserm, U935, Villejuif, F-94800, France 2 Cell Growth and Tissue Repair (CRRET) Laboratory, Université Paris-Est Créteil (UPEC), EA 4397 / ERL CNRS 9215, F-94010, Créteil, France. Corresponding author: *Franck Chiappini. Laboratoire du CRRET (Croissance, Réparation et Régénération Tissulaires), Université Paris-Est Créteil, 61 avenue du Général de Gaulle F- 94010, Créteil Cedex, Val de Marne, France. Email address: [email protected]; Tel: +33(0)145177080; Fax: +33(0)145171816 Supplementary Information Supplementary Datasets Table S1: Text-mining list of genes associated in PubMed literature with lipid related keywords. Supplementary Datasets Table S2: Expression fold change of lipid related genes found differentially expressed between NASH and healthy obese liver samples. Supplementary Datasets Table S3: Liver as principal filter for prioritization of lipid related genes found differentially expressed in NASH. Supplementary Datasets Table S4: Gene prioritization secondary filters (immunological, inflammation, liver pathogenesis progression) table found with lipid related genes differentially expressed in NASH. Supplementary Datasets Table S5: Identification of protein partners of YWHAZ gene using InnateDB database. Supplementary Datasets Table S1: Text-mining list of genes associated in PubMed literature with lipid related keywords. Ranking of "lipidic" textmining Gene symbol -
Supplementary Information Genomice and Transcriptomic Analysis
Supplementary Information Genomice and Transcriptomic Analysis for Identification of Genes and Interlinked Pathways Mediating Artemisinin Resistance in Leishmania donovani Sushmita Ghosh1,2, Aditya Verma1, Vinay Kumar1, Dibyabhaba Pradhan3, Angamuthu Selvapandiyan 2, Poonam Salotra1, Ruchi Singh1* 1. ICMR- National Institute of Pathology, Safdarjung Hospital Campus, New Delhi-110029, India 2. Jamia Hamdard University-Institute of Molecular Medicine, New Delhi-110062, India 3. ICMR-AIIMS Computational Genomics Centre, Indian Council of Medical Research, New Delhi- 110029 *Correspondence: [email protected] Supplementary Figures and Tables: Figure S1. Figure S1: Comparative transcriptional responses following ART adaptation in L. donovani. Overlap of log2 transformed K133 AS-R and K133 WT expression ratio plotted as a function of chromosomal location of probes representing the full genome microarray. The plot represents the average values of three independent hybridizations for each isolate. Table S1: List of genes validated for their modulated expression by Quantitative real time- PCR S.N Primer Gene Name/ Function/relevance Primer Sequence o. Name Gene ID 1 AQP1 Aquaglyceropor Metal ion F- in (LinJ.31.0030) transmembrane 5’CAGGGACAGCTCGAGGGTAA transporter activity, AA3’ integral to membrane; transmembrane R- transport; transporter 5’GTTACCGGCGTGAAAGACAG activity; water TG3’ transport. 2 A2 A2 protein Cellular response to F- (LinJ.22.0670) stress 5’GTTGGCCCGCTTTCTGTTGG3’ R- 5’ACCAACGTCAACAGAGAGA GGG3’ 3 ABCG1 ATP-binding ATP binding, ATPase -
T4 RNA Ligase Catalyzes the Synthesis of Dinucleoside Polyphosphates
Eur. J. Biochem. 261, 802±811 (1999) q FEBS 1999 T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates Eva Ana Atencia, Olga Madrid, MarõÂaA.GuÈnther Sillero and Antonio Sillero Instituto de Investigaciones BiomeÂdicas Alberto Sols, UAM/CSIC, Departamento de BioquõÂmica, Facultad de Medicina, Madrid, Spain T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 50-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme 32 was incubated in the presence of 0.02 mm [a- P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, mm) and acceptor (Km,mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm)of[a-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. -
Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in -
RNA Ligase Structures Reveal the Basis for RNA Specificity And
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector RNA Ligase Structures Reveal the Basis for RNA Specificity and Conformational Changes that Drive Ligation Forward Jayakrishnan Nandakumar,1,2,3 Stewart Shuman,2 and Christopher D. Lima1,* 1 Structural Biology Program 2 Molecular Biology Program 3 Training Program in Chemical Biology Sloan-Kettering Institute, New York, NY 10021, USA *Contact: [email protected] DOI 10.1016/j.cell.2006.08.038 0 SUMMARY 5 PO4 strand is DNA or RNA (Nandakumar and Shuman, 2004). Thus, ligase specificity might be dictated by dis- T4 RNA ligase 2 (Rnl2) and kinetoplastid RNA crimination of A form and B form secondary structures in 0 0 editing ligases exemplify a family of RNA repair duplex nucleic acid on either the 3 OH or 5 PO4 sides of 0 0 enzymes that seal 3 OH/5 PO4 nicks in duplex a nick. This model is difficult to extend to RNA ligases RNAs via ligase adenylylation (step 1), AMP that join single-stranded polynucleotide ends. 0 Two families of RNA ligases, named Rnl1 and Rnl2, transfer to the nick 5 PO4 (step 2), and attack by the nick 30OH on the 50-adenylylated strand function in the repair of ‘‘purposeful’’ RNA breaks. The Rnl1 family includes bacteriophage T4 RNA ligase 1 (Silber to form a phosphodiester (step 3). Crystal struc- et al., 1972; Uhlenbeck and Gumport, 1982; Wang et al., tures are reported for Rnl2 at discrete steps 2003) and tRNA ligases of fungi and plants (Wang and along this pathway: the covalent Rnl2-AMP Shuman, 2005; Englert and Beier, 2005). -
Systematic Evaluation and Optimization of the Experimental
www.nature.com/scientificreports Corrected: Author Correction OPEN Systematic evaluation and optimization of the experimental steps in RNA G-quadruplex Received: 11 December 2018 Accepted: 20 May 2019 structure sequencing Published online: 30 May 2019 Pui Yan Yeung 1, Jieyu Zhao 1, Eugene Yui-Ching Chow 2, Xi Mou 1, HuiQi Hong3,4, Leilei Chen 4,5, Ting-Fung Chan 2 & Chun Kit Kwok 1 cDNA library preparation is important for many high-throughput sequencing applications, such as RNA G-quadruplex structure sequencing (rG4-seq). A systematic evaluation of the procedures of the experimental pipeline, however, is lacking. Herein, we perform a comprehensive assessment of the 5 key experimental steps involved in the cDNA library preparation of rG4-seq, and identify better reaction conditions and/or enzymes to carry out each of these key steps. Notably, we apply the improved methods to fragmented cellular RNA, and show reduced RNA input requirement, lower transcript abundance variations between biological replicates, as well as lower transcript coverage bias when compared to prior arts. In addition, the time to perform these steps is substantially reduced to hours. Our method and results can be directly applied in protocols that require cDNA library preparation, and provide insights to the further development of simple and efcient cDNA library preparation for diferent biological applications. Multitudes of cDNA library preparation methods have been developed as a result of the recent exploration and investigation of the novel features of RNA, such as RNA structures1–4. However, most of these methods require high RNA input (microgram of RNA), numerous processing and purifcation steps, and thus long cDNA library preparation time (few days)5–9. -
Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA
G C A T T A C G G C A T genes Review Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA Suguru Morimoto 1, Masataka Tsuda 1, Heeyoun Bunch 2 , Hiroyuki Sasanuma 1, Caroline Austin 3 and Shunichi Takeda 1,* 1 Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan; [email protected] (S.M.); [email protected] (M.T.); [email protected] (H.S.) 2 Department of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Korea; [email protected] 3 The Institute for Cell and Molecular Biosciences, the Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; [email protected] * Correspondence: [email protected]; Tel.: +81-(075)-753-4411 Received: 30 September 2019; Accepted: 26 October 2019; Published: 30 October 2019 Abstract: Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this ‘abortive catalysis’ can be a major source of spontaneously arising DNA double-strand breaks (DSBs). -
Potent Lipolytic Activity of Lactoferrin in Mature Adipocytes
Biosci. Biotechnol. Biochem., 77 (3), 566–571, 2013 Potent Lipolytic Activity of Lactoferrin in Mature Adipocytes y Tomoji ONO,1;2; Chikako FUJISAKI,1 Yasuharu ISHIHARA,1 Keiko IKOMA,1;2 Satoru MORISHITA,1;3 Michiaki MURAKOSHI,1;4 Keikichi SUGIYAMA,1;5 Hisanori KATO,3 Kazuo MIYASHITA,6 Toshihide YOSHIDA,4;7 and Hoyoku NISHINO4;5 1Research and Development Headquarters, Lion Corporation, 100 Tajima, Odawara, Kanagawa 256-0811, Japan 2Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan 3Food for Life, Organization for Interdisciplinary Research Projects, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 4Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyou-ku, Kyoto 602-8566, Japan 5Research Organization of Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan 6Department of Marine Bioresources Chemistry, Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minatocho, Hakodate, Hokkaido 041-8611, Japan 7Kyoto City Hospital, 1-2 Higashi-takada-cho, Mibu, Nakagyou-ku, Kyoto 604-8845, Japan Received October 22, 2012; Accepted November 26, 2012; Online Publication, March 7, 2013 [doi:10.1271/bbb.120817] Lactoferrin (LF) is a multifunctional glycoprotein resistance, high blood pressure, and dyslipidemia. To found in mammalian milk. We have shown in a previous prevent progression of metabolic syndrome, lifestyle clinical study that enteric-coated bovine LF tablets habits must be improved to achieve a balance between decreased visceral fat accumulation. To address the energy intake and consumption. In addition, the use of underlying mechanism, we conducted in vitro studies specific food factors as helpful supplements is attracting and revealed the anti-adipogenic action of LF in pre- increasing attention. -
Isolation of a Ribozyme with 5'-5' Ligase Activity
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Isolation of a ribozyme with 5’-5’ ligase activity Karen B Chapman+ and Jack W Szostak* Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA Background: Many new ribozymes, including sequ- linkage. Deletion analysis of one of the selected ence-specific nucleases, ligases and kinases, have been sequences revealed that a 54-nucleotide RNA retained isolated by in vitro selection from large pools of random- activity; this small ribozyme folds into a pseudoknot sec- sequence RNAs.We are attempting to use in vitro selec- ondary structure with an internal binding site for the tion to isolate new ribozymes that have, or can be substrate oligonucleotide.The ribozyme can also synthe- evolved to have, RNA polymerase-like activities. As size 5’-5’ triphosphate and 5’-5’ pyrophosphate linkages. phosphorimidazolide-activated nucleosides are exten- Conclusions: The emergence of ribozymes that acceler- sively used to study non-enzymatic RNA replication, we ate an unexpected 5’-5’ ligation reaction from a selection wished to select for a ribozyme that would accelerate the designed to yield template-dependent 3’-5’ ligases template-directed ligation of 5’-phosphorimidazolide- suggests that it may be much easier for RNA to catalyze activated oligonucleotides. the synthesis of 5’-5’ linkages than 3’-5’ linkages. 5’-5’ Results: Ribozymes selected to perform the desired linkages are found in a variety of contexts in present-day template-directed ligation reaction instead ligated them- biology. The ribozyme-catalyzed synthesis of such selves to the activated substrate oligonucleotide via their linkages raises the possibility that these 5’-5’ linkages 5’-triphosphate, generating a 5’-5’ P’,P4-tetraphosphate originated in the biochemistry of the RNA world.