2019-20 Enzyme & Biochemical Catalog
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Endogenous Reverse Transcriptase and Rnase H-Mediated Antiviral Mechanism in Embryonic Stem Cells
www.nature.com/cr www.cell-research.com ARTICLE Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells Junyu Wu1, Chunyan Wu1, Fan Xing1, Liu Cao1, Weijie Zeng1, Liping Guo1, Ping Li1, Yongheng Zhong1, Hualian Jiang1, Manhui Luo1, Guang Shi2, Lang Bu1, Yanxi Ji1, Panpan Hou1, Hong Peng1, Junjiu Huang2, Chunmei Li1 and Deyin Guo 1 Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates. -
Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences
molecules Review Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences Yaroslav Staroseletz 1,†, Svetlana Gaponova 1,†, Olga Patutina 1, Elena Bichenkova 2 , Bahareh Amirloo 2, Thomas Heyman 2, Daria Chiglintseva 1 and Marina Zenkova 1,* 1 Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev’s Ave. 8, 630090 Novosibirsk, Russia; [email protected] (Y.S.); [email protected] (S.G.); [email protected] (O.P.); [email protected] (D.C.) 2 School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Rd., Manchester M13 9PT, UK; [email protected] (E.B.); [email protected] (B.A.); [email protected] (T.H.) * Correspondence: [email protected]; Tel.: +7-383-363-51-60 † These authors contributed equally to this work. Abstract: RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleav- age, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes Citation: Staroseletz, Y.; Gaponova, and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonu- S.; Patutina, O.; Bichenkova, E.; cleotide recognition motifs (or their structural -
Ribonuclease A: Disulfide Bonds, Conformational Stability, and Cytotoxicity
Ribonuclease A: Disulfide Bonds, Conformational Stability, and Cytotoxicity by Tony A. Klink A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 2()()() r OJ A dissertation entitled Ribonuclease A: Disulfide Bonds, Conformational Stability and Cytotoxicity submitted to the Graduate School of the University of Wisconsin-Madison in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Tony Anthony Klink Date of Final Oral Examination: August 4, 2000 Month & Year Degree to be awarded: December May August 2000 • * * * * * * • * * • * • • • • • * • • • * • • • • • • • • • • • • • * * • * • * * * • * • * • • • • • • * App oval Signature. f Dissertation Readers: Signature, Dean of Graduate School ---..., v.C~vJ,1A. 5. lJ,~k;at 1 Abstract Disulfide bonds between the side chains of cysteine residues are the only common cross links in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-CysllO, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the confonnational stability and catalytic activity of RNase A was detennined using variants in which each cystine was replaced independently with a pair of alanine residues. Of the four disulfide bonds, the Cys40-Cys95 and Cys65-Cys72 cross-links are the least important to confonnational stability. Removing these disulfide bonds leads to RNase A variants that have Tm values below that of the wild-type enzyme but above physiological temperature. Unlike wild-type RNase A. G88R RNase A is toxic to cancer cells. To investigate the relationship between conformational stability and cytotoxicity, the C40AlC95A and C65A1C72A variants were made in the G88R background. -
Propranolol-Mediated Attenuation of MMP-9 Excretion in Infants with Hemangiomas
Supplementary Online Content Thaivalappil S, Bauman N, Saieg A, Movius E, Brown KJ, Preciado D. Propranolol-mediated attenuation of MMP-9 excretion in infants with hemangiomas. JAMA Otolaryngol Head Neck Surg. doi:10.1001/jamaoto.2013.4773 eTable. List of All of the Proteins Identified by Proteomics This supplementary material has been provided by the authors to give readers additional information about their work. © 2013 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 eTable. List of All of the Proteins Identified by Proteomics Protein Name Prop 12 mo/4 Pred 12 mo/4 Δ Prop to Pred mo mo Myeloperoxidase OS=Homo sapiens GN=MPO 26.00 143.00 ‐117.00 Lactotransferrin OS=Homo sapiens GN=LTF 114.00 205.50 ‐91.50 Matrix metalloproteinase‐9 OS=Homo sapiens GN=MMP9 5.00 36.00 ‐31.00 Neutrophil elastase OS=Homo sapiens GN=ELANE 24.00 48.00 ‐24.00 Bleomycin hydrolase OS=Homo sapiens GN=BLMH 3.00 25.00 ‐22.00 CAP7_HUMAN Azurocidin OS=Homo sapiens GN=AZU1 PE=1 SV=3 4.00 26.00 ‐22.00 S10A8_HUMAN Protein S100‐A8 OS=Homo sapiens GN=S100A8 PE=1 14.67 30.50 ‐15.83 SV=1 IL1F9_HUMAN Interleukin‐1 family member 9 OS=Homo sapiens 1.00 15.00 ‐14.00 GN=IL1F9 PE=1 SV=1 MUC5B_HUMAN Mucin‐5B OS=Homo sapiens GN=MUC5B PE=1 SV=3 2.00 14.00 ‐12.00 MUC4_HUMAN Mucin‐4 OS=Homo sapiens GN=MUC4 PE=1 SV=3 1.00 12.00 ‐11.00 HRG_HUMAN Histidine‐rich glycoprotein OS=Homo sapiens GN=HRG 1.00 12.00 ‐11.00 PE=1 SV=1 TKT_HUMAN Transketolase OS=Homo sapiens GN=TKT PE=1 SV=3 17.00 28.00 ‐11.00 CATG_HUMAN Cathepsin G OS=Homo -
Specific Principles of Genome-Wide RNA-Chromatin Interactions
ARTICLE There are amendments to this paper https://doi.org/10.1038/s41467-020-14337-6 OPEN RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions Alessandro Bonetti 1,2,18*, Federico Agostini 3,18, Ana Maria Suzuki1,4, Kosuke Hashimoto1, Giovanni Pascarella1, Juliette Gimenez 5, Leonie Roos6,7, Alex J. Nash6,7, Marco Ghilotti1, Christopher J. F. Cameron8,9, Matthew Valentine 1, Yulia A. Medvedeva10,11,12, Shuhei Noguchi1, Eneritz Agirre 2, Kaori Kashi1, Samudyata2, Joachim Luginbühl1, Riccardo Cazzoli13, Saumya Agrawal1, Nicholas M. Luscombe 3,14,15, Mathieu Blanchette8, Takeya Kasukawa 1, Michiel de Hoon1, Erik Arner1, 1234567890():,; Boris Lenhard 6,7,16, Charles Plessy 1, Gonçalo Castelo-Branco 2, Valerio Orlando5,17* & Piero Carninci 1* Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding tran- scripts exhibit nuclear localization and several have been shown to play a role in the reg- ulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA–chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of tran- scripts as well as cell type–specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure. -
Determination of Pk, Values of the Histidine Side
Protein Science (1997), 6:1937-1944. Cambridge University Press. Printed in the USA Copyright 0 1997 The Protein Society Determination of pK, values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis TUN LIU, MARGRET RYAN, FREDERICK W. DAHLQUIST, AND 0. HAYES GRIFFITH Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403 (RECEIVEDDecember 4, 1996: ACCEPTEDMay 19, 1997) Abstract Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pK,, values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selec- tively labeled with '3C"-histidine. A series of 'H-I3C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 'H-"C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pK,, values of 7.6 and 6.9, respectively. His92 and His227 exhibit pK, values of 5.4 and 6.9. -
Novel Missense Mutation in PTPN22 in a Chinese Pedigree With
Gong et al. BMC Endocrine Disorders (2018) 18:76 https://doi.org/10.1186/s12902-018-0305-8 RESEARCHARTICLE Open Access Novel missense mutation in PTPN22 in a Chinese pedigree with Hashimoto’s thyroiditis Licheng Gong1†, Beihong Liu2,3†, Jing Wang4, Hong Pan3, Anhui Qi2,3, Siyang Zhang2,3, Jinyi Wu1, Ping Yang1* and Binbin Wang3,4,5* Abstract Background: Hashimoto’s thyroiditis is a complex autoimmune thyroid disease, the onset of which is associated with environmental exposures and specific susceptibility genes. Its incidence in females is higher than its incidence in males. Thus far, although some susceptibility loci have been elaborated, including PTPN22, FOXP3, and CD25, the aetiology and pathogenesis of Hashimoto’s thyroiditis remains unclear. Methods: Four affected members from a Chinese family with Hashimoto’s thyroiditis were selected for whole-exome sequencing. Missense, nonsense, frameshift, or splicing-site variants shared by all affected members were identified after frequency filtering against public and internal exome databases. Segregation analysis was performed by Sanger sequencing among all members with available DNA. Results: We identified a missense mutation in PTPN22 (NM_015967.5; c. 77A > G; p.Asn26Ser) using whole-exome sequencing. PTPN22 is a known susceptibility gene associated with increased risks of multiple autoimmune diseases. Cosegregation analysis confirmed that all patients in this family, all of whom were female, carried the mutation. All public and private databases showed that the missense mutation was extremely rare. Conclusions: We found a missense mutation in PTPN22 in a Chinese HT pedigree using whole-exome sequencing. Our study, for the first time, linked a rare variant of PTPN22 to Hashimoto’s thyroiditis, providing further evidence of the disease-causing or susceptibility role of PTPN22 in autoimmune thyroid disease. -
Specific Binding of a Hela Cell Nuclear Protein to RNA Sequences in The
Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4858-4862, July 1989 Biochemistry Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region (chemical nuclease imprinting/untranslated RNA binding protein 1) RICHARD GAYNOR*tt, EMMANUEL SOULTANAKIS*t, MICHIo KUWABARA*§, JOSEPH GARCIA*t, AND DAVID S. SIGMAN*§ *Molecular Biology Institute, and tDivision of Hematology-Oncology, Department of Medicine, and §Department of Biological Chemistry, School of Medicine, and tWadsworth Veterans Hospital, University of California, Los Angeles, CA 90024 Communicated by Paul D. Boyer, March 27, 1989 ABSTRACT The transactivator protein, tat, encoded by been shown to be involved in increasing the steady-state level the human immunodeficiency virus is a key regulator of viral of RNA, it has also been reported to increase the translation transcription. Activation by the tat protein requires sequences of RNA (15, 17, 22). The possibility that increased levels of downstream of the transcription initiation site called the trans- RNA are attributable to an antitermination mechanism has activating region (TAR). RNA derived from the TAR is capable also been raised (5, 18-20, 27). In contrast to other transac- offorming a stable stem-oop structure and the maintenance of tivating proteins, such as ElA (28), which activate a number both the stem structure and the loop sequences located between of viral and cellular promoters, tat activation is specific for + 19 and +44 is required for complete in vivo activation by tat. the HIV LTR. It is of interest to identify features ofthe TAR Gel retardation assays with RNA from both wild-type and responsible for this selectivity. -
Enhanced Phosphorylation of High-Mobility-Group Proteins In
Proc. Nati. Acad. Sci. USA Vol. 78, No. 4, pp. 2189-2193, April 1981 Biochemistry Enhanced phosphorylation of high-mobility-group proteins in nuclease-sensitive mononucleosomes from butyrate-treated HeLa cells (hyperphosphorylation/transcriptionally active chromatin/sodium butyrate) BEATRIZ LEVY-WILSON Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717 Communicated by James F. Bonner, December 29, 1980 ABSTRACT The protein composition of nucleosome fractions MATERIALS AND METHODS differing in their sensitivity to micrococcal nuclease and derived from butyrate-treated or untreated HeLa cells has been com- Cell Cultures. HeLa cells, line S3, were grown in monolay- pared. Most of the high-mobility-group-14 (HMG-14) and HMG- ers, either in T-150 culture flasks or in roller bottles, at 370C 17 content of HeLa cell chromatin is associated with those nu- in minimal essential medium (Flow Laboratories, Rockville, cleosomes that are preferentially sensitive to micrococcal nu- MD) supplemented with 7% bovine calfserum (KC Biologicals) clease. Furthermore, electrophoresis of these two HMG proteins and penicillin (100 units/ml) and streptomycin (100 Ag/ml) from the transcriptionally active chromatin fraction MN, of bu- (Sigma). In experiments involving sodium butyrate, this com- tyrate-treated cells resolves them into a series ofbands. The mul- pound was added to the cultures at a final concentration of5 mM tiple band pattern of HMG-14 and -17 from butyrate-treated cells about 20 hr prior to harvesting of the cells. results from hyperphosphorylation rather than hyperacetylation. Preparation of Nuclei. HeLa cell nuclei were prepared as Phosphorylation of these two small nonhistone proteins may play described by Milcarek et al. -
Rnase 2 Sirna (H): Sc-92235
SANTA CRUZ BIOTECHNOLOGY, INC. RNase 2 siRNA (h): sc-92235 BACKGROUND PRODUCT RNase 2 [ribonuclease, RNase A family, 2 (liver, eosinophil-derived neuro- RNase 2 siRNA (h) is a pool of 2 target-specific 19-25 nt siRNAs designed toxin)], also known as non-secretory ribonuclease, EDN (eosinophil-derived to knock down gene expression. Each vial contains 3.3 nmol of lyophilized neurotoxin), RNase UpI-2 or RNS2, is a 161 amino acid protein that belongs siRNA, sufficient for a 10 µM solution once resuspended using protocol to the pancreatic ribonuclease family. Localizing to lysosome and cytoplasmic below. Suitable for 50-100 transfections. Also see RNase 2 shRNA granules, RNase 2 is expressed in leukocytes, liver, spleen, lung and body Plasmid (h): sc-92235-SH and RNase 2 shRNA (h) Lentiviral Particles: fluids. RNase 2 functions as a pyrimidine specific nuclease, and has a slight sc-92235-V as alternate gene silencing products. preference for uracil. RNase 2 is capable of various biological activities, For independent verification of RNase 2 (h) gene silencing results, we including mediation of chemotactic activity and endonucleolytic cleavage of also provide the individual siRNA duplex components. Each is available as nucleoside 3'-phosphates and 3'-phosphooligonucleotides. The gene encoding 3.3 nmol of lyophilized siRNA. These include: sc-92235A and sc-92235B. RNase 2 maps to human chromosome 14q11.2. STORAGE AND RESUSPENSION REFERENCES Store lyophilized siRNA duplex at -20° C with desiccant. Stable for at least 1. Yasuda, T., Sato, W., Mizuta, K. and Kishi, K. 1988. Genetic polymorphism one year from the date of shipment. -
Pharmacokinetics, Pharmacodynamics and Metabolism Of
PHARMACOKINETICS, PHARMACODYNAMICS AND METABOLISM OF GTI-2040, A PHOSPHOROTHIOATE OLIGONUCLEOTIDE TARGETING R2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaohui Wei, M.S. * * * * * * The Ohio State University 2006 Approved by Dissertation Committee: Dr. Kenneth K. Chan, Adviser Adviser Dr. Guido Marcucci, Co-adviser Graduate Program in Pharmacy Dr. Thomas D. Schmittgen Dr. Robert J. Lee Co-Adviser Graduate Program in Pharmacy ABSTRACT Over the last several decades, antisense therapy has been developed into a promising gene-targeted strategy to specifically inhibit the gene expression. Ribonucleotide reductase (RNR), composing of subunits R1 and R2, is an important enzyme involved in the synthesis of all of the precursors used in DNA replication. Over- expression of R2 has been found in almost every type of cancer studied. GTI-2040 is a 20-mer phosphorothioate oligonucleotide targeting the coding region in mRNA of the R2 component of human RNR. In this project, clinical pharamcokinetics (PK), pharmacodynamics (PD) and metabolism of this novel therapeutics were investigated in patients with acute myeloid leukemia (AML). A picomolar specific hybridization-ligation ELISA method has been developed and validated for quantification of GTI-2040. GTI-2040 and neophectin complex was found to enhance drug cellular uptake and exhibited sequence- and dose-dependent down-regulation of R2 mRNA and protein in K562 cells. Robust intracellular concentrations (ICs) of GTI-2040 were achieved in peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from treated AML patients. GTI-2040 concentrations in the nucleus of BM cells were found to correlate with the R2 mRNA down-regulation and disease response. -
Carcinogen-Induced Early Molecular Events and Its Implication In
Biochimica et Biophysica Acta 1772 (2007) 48–59 www.elsevier.com/locate/bbadis Carcinogen-induced early molecular events and its implication in the initiation of chemical hepatocarcinogenesis in rats: Chemopreventive role of vanadium on this process Tridib Chakraborty a, Amrita Chatterjee a, Ajay Rana b, Duraisami Dhachinamoorthi a, ⁎ Ashok Kumar P a, Malay Chatterjee a, a Division of Biochemistry, Department of Pharmaceutical Technology, Jadavpur University, PO Box 17028, Calcutta-700032, India b Division of Molecular Cardiology, Cardiovascular Research Institute, College of Medicine, The Texas A&M University System HSC, Temple, TX 76504, USA Received 21 May 2006; received in revised form 19 September 2006; accepted 16 October 2006 Available online 10 November 2006 Abstract Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molecular events in the initiation of chemical carcinogenesis and modulation of such events by chemopreventive agents could be an important step in limiting neoplastic transformation in vivo. Vanadium, a dietary micronutrient has been found to be effective in several types of cancers both in vivo and in vitro and also possesses profound anticarcinogenicity against rat models of mammary, colon and hepatocarcinogenesis. Presently, we report the chemopreventive potential of vanadium on diethylnitrosamine (DEN)-induced early DNA damages in rat liver. Hepatocarcinogenesis was induced in male Sprague–Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of DEN (200 mg/kg body weight) at week 4. There was a significant induction of tissue- specific ethylguanines, steady elevation of modified DNA bases 8-hydroxy-2′-deoxyguanosines (8-OHdGs) (P<0.0001; 89.93%) along with substantial increment of the extent of single-strand breaks (SSBs) (P<0.0001) following DEN exposure.