DOCSLIB.ORG

Asca (Yeca) Is a Molecular Chaperone Involved in Sec-Dependent Protein Translocation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Asca (Yeca) Is a Molecular Chaperone Involved in Sec-Dependent Protein Translocation bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 1 1 AscA (YecA) is a molecular chaperone involved in Sec-dependent protein translocation 2 in Escherichia coli 3 4 Running title: AscA is a Sec chaperone 5 6 Tamar Cranford Smith1, Max Wynne1, Cailean Carter, Chen Jiang, Mohammed Jamshad, 7 Mathew T. Milner, Yousra Djouider, Emily Hutchinson, Peter A. Lund, Ian Henderson2 and 8 Damon Huber* 9 10 Institute for Microbiology and Infection; University of Birmingham; Edgbaston, 11 Birmingham, UK 12 13 1These authors contributed equally to this work 14 15 2Current address: Institute for Molecular Bioscience; University of Queensland; Brisbane, 16 Australia 17 18 *To whom correspondence should be addressed: [email protected] 19 20 Keywords: protein translocation, Sec pathway, molecular chaperone, SecB, metal binding 21 domain 22 23 ABSTRACT. 24 Proteins that are translocated across the cytoplasmic membrane by Sec 25 machinery must be in an unfolded conformation in order to pass through the protein- bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 2 26 conducting channel during translocation. Molecular chaperones assist Sec-dependent 27 protein translocation by holding substrate proteins in an unfolded conformation in the 28 cytoplasm until they can be delivered to the membrane-embedded Sec machinery. For 29 example, in Escherichia coli, SecB binds to a subset of unfolded Sec substrates and 30 delivers them to the Sec machinery by interacting with the metal-binding domain 31 (MBD) of SecA, an ATPase required for translocation in bacteria. Here, we describe a 32 novel molecular chaperone involved Sec-dependent protein translocation, which we 33 have named AscA (for accessory Sec component). AscA contains a metal-binding 34 domain (MBD) that is nearly identical to the MBD of SecA. In vitro binding studies 35 indicated that AscA binds to SecB and ribosomes in an MBD-dependent fashion. 36 Saturated transposon mutagenesis and genetics studies suggested that AscA is involved 37 in cell-envelope biogenesis and that its function overlaps with that of SecB. In support of 38 this idea, AscA copurified with a range of proteins and prevented the aggregation of 39 citrate synthase in vitro. Our results suggest that AscA is molecular chaperone and that 40 it enhances Sec-dependent protein translocation by delivering its substrate proteins to 41 SecB. 42 43 IMPORTANCE. 44 This research describes the discovery of a novel molecular chaperone, AscA (YecA). 45 The function of AscA was previously unknown. However, it contains a small domain, 46 known as the MBD, suggesting it could interact with the bacterial Sec machinery, which 47 is responsible for transporting proteins across the cytoplasmic membrane. The work 48 described this study indicates that the MBD allows AscA to bind to both the protein 49 synthesis machinery and the Sec machinery. The previously function of the previously 50 uncharacterised N-terminal domain is that of a molecular chaperone, which binds to bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. 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-ND 4.0 International license. 3 51 unfolded substrate proteins. We propose that AscA binds to protein substrates as they 52 are still be synthesised by ribosomes in order to channel them into the Sec pathway. 53 54 INTRODUCTION. 55 In Escherichia coli, most newly synthesized periplasmic and outer membrane proteins 56 are transported across the cytoplasmic membrane by the Sec machinery. During 57 translocation, protein substrates of the Sec machinery pass through an evolutionarily 58 conserved channel in the cytoplasmic membrane (composed of the integral membrane 59 proteins SecY, -E and -G) in an unfolded conformation (1, 2). In addition, translocation 60 usually requires the activity of SecA (3), an ATPase that facilitates translocation through 61 SecYEG (4). The translocation of periplasmic and outer membrane proteins typically begins 62 only after the substrate protein is fully (or nearly fully) synthesised (i.e. “posttranslationally”) 63 (5, 6). 64 Because proteins must be unfolded to pass through SecYEG, folding of substrate 65 proteins in the cytoplasm blocks Sec-dependent protein translocation, causing a protein to 66 become irreversibly trapped in the cytoplasm (7). Furthermore, partially folded proteins that 67 engage SecYEG can clog (or “jam”) the Sec machinery, which is toxic (8). As a result, cells 68 have evolved multiple mechanisms to prevent premature folding of substrate proteins. For 69 example, molecular chaperones can bind to unfolded Sec substrate proteins and hold them in 70 an unfolded conformation until they can be delivered to the membrane-embedded Sec 71 machinery. One such chaperone is SecB, which binds to a subset of unfolded Sec substrate 72 proteins and delivers them to SecA for translocation across the membrane (9-13). 73 Recognition of nascent substrates by SecB is dependent on SecA (14), suggesting that SecB 74 requires an intermediary to recognise its substrate proteins. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. 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-ND 4.0 International license. 4 75 The interaction of SecA with SecB is mediated by a small (~20 amino acid) metal- 76 binding domain (MBD) near the extreme C-terminus of SecA (13, 15, 16). Recent work 77 indicates that the MBD also binds to ribosomes and that ribosome binding is involved in 78 coordinating binding of SecA to nascent polypeptides (17). As its name indicates, the MBD 79 binds to a transition metal (zinc and/or iron) (15, 18), and binding to the metal ion is required 80 for stable folding of the MBD (15). The amino acids responsible for metal binding are highly 81 conserved (CXCXSX6CH or CXCXSX6CC) (15, 17, 19). 82 We recently described a protein of unknown function in E. coli that contains a MBD 83 that is nearly identical to the MBD of SecA (18), YecA, which we have re-named AscA (for 84 accessory Sec component). AscA also contains a UPF0149-family domain at its N-terminus, 85 the function of which has not been described. In this work, we investigated the function of 86 AscA. The similarity of the AscA and SecA MBDs led us to investigate the interaction of 87 AscA with SecB and ribosomes and the dependence of these interactions on the MBD. 88 Genetic analysis suggested that AscA is involved in cell-envelope biogenesis and that AscA 89 could be a molecular chaperone. Further studies indicated that AscA binds to cytoplasmic Sec 90 substrate proteins and that it carries out its function in coordination with SecB in vivo. Our 91 results suggest a potential model for how AscA could facilitate Sec-dependent protein 92 translocation in E. coli. 93 94 RESULTS. 95 Binding of AscA to SecB. Many of the amino acids that mediate the interaction 96 between the SecA MBD and SecB from Haemophilus influenzae are conserved in the MBD 97 of AscA (supplemental figure S1) (18, 20). To investigate whether AscA can also bind to 98 SecB, we examined the effect of AscA on the thermophoretic mobility of SecB using 99 microscale thermophoresis. To this end, we fluorescently labelled SecB and incubated it with bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. 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-ND 4.0 International license. 5 100 unlabelled AscA. There was a large change in the thermophoretic properties of fluorescently 101 labelled SecB at saturating concentrations of AscA (figure 1A), suggesting that SecB binds 102 to AscA. However, the presence of a truncated variant of AscA, which lacks the MBD 103 (AscAΔMBD), did not affect thermophoresis of SecB (figure 1A). Purified AscAΔMBD was 104 fully folded even in the absence of its MBD (18), indicating that the interaction between 105 SecB and AscA is dependent on the MBD. Analysis of the effect of increasing concentrations 106 of AscA on the thermophoresis of suggested an equilibrium dissociation constant (KD) of 107 approximately 150 nM (figure 1B). 108 Binding of AscA to ribosomes. We next investigated the interaction of AscA with 109 ribosomes. To this end, we incubated AscA or AscAΔMBD with purified non-translating 70S 110 ribosomes and then separated ribosome-bound AscA from unbound AscA by sedimenting 111 ribosomes through a 30% sucrose cushion by ultracentrifugation. Full-length AscA 112 cosedimented with the 70S ribosomes, indicating that it can bind to ribosomes (figure 1C). 113 Truncation of the MBD in AscAΔMBD greatly reduced its ability to cosediment with 114 ribosomes, indicating that binding is dependent on the MBD.
Load more

Recommended publications
  • Intrinsically Disordered Proteins As Crucial Constituents of Cellular Aqueous Two Phase Systems and Coacervates ⇑ Vladimir N
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 589 (2015) 15–22 journal homepage: www.FEBSLetters.org Hypothesis Intrinsically disordered proteins as crucial constituents of cellular aqueous two phase systems and coacervates ⇑ Vladimir N. Uversky a,b,c,d, , Irina M. Kuznetsova d,e, Konstantin K. Turoverov d,e, Boris Zaslavsky f a Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA b Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation c Biology Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia d Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russian Federation e St. Petersburg State Polytechnical University, St. Petersburg, Russian Federation f AnalizaDx Inc., 3615 Superior Ave., Suite 4407B, Cleveland, OH 44114, USA article info abstract Article history: Here, we hypothesize that intrinsically disordered proteins (IDPs) serve as important drivers of the Received 17 August 2014 intracellular liquid–liquid phase separations that generate various membrane-less organelles. This Revised 10 October 2014 hypothesis is supported by the overwhelming abundance of IDPs in these organelles. Assembly and Accepted 19 November 2014 disassembly of these organelles are controlled by changes in the concentrations of IDPs, their post- Available online 29 November 2014 translational modifications, binding of specific partners, and changes in the pH and/or temperature Edited by A. Valencia of the solution.
    [Show full text]
  • Dissertation Inês Silva ITQB.Pdf
    The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium Inês de Jesus de Almeida e Silva Insert here an image with rounded corners Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Oeiras, December, 2012 The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium Inês de Jesus de Almeida e Silva Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa Oeiras, December, 2012 Financial Support from Fundação para a Ciência e Tecnologia (FCT) – Ph.D: grant - SFRH / BD / 43211 / 2008. Work performed at: Control of Gene Expression Laboratory Instituto de Tecnologia Química e Biológica Av. da República (EAN) 2781-901 Oeiras – Portugal Tel: +351-21-4469548 Fax: +351-21-4469549 Supervisor : Professora Doutora Cecília Maria Pais de Faria de Andrade Arraiano – Investigadora Coordenadora, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa. (Head of the Laboratory of Control of Gene Expression, where the work of this Dissertation was performed) Co-supervisor : Doutora Sandra Cristina de Oliveira Viegas – Investigadora Pós-Doutorada, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa. (Post-doc Fellow in the Laboratory of Control of Gene Expression, where the work of this Dissertation was performed) President of the Jury : Doutora Claudina Amélia Marques Rodrigues Pousada – Professora Catedrática Convidada do Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, por delegação; iii Examiners: Professor Doutor Iñigo Lasa Uzcudun – Head of Microbial Biofilm Research Group, Instituto de Agrobiotecnología, Pamplona (Principal Examiner).
    [Show full text]
  • Generated by SRI International Pathway Tools Version 25.0, Authors S
    An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Gcf_005222025Cyc: Escherichia coli 121 Cellular Overview Connections between pathways are omitted for legibility. pro a dicarboxylate a dicarboxylate an amino putrescine putrescine thiosulfate a hexose 6- glycine betaine glycine succinate γ-butyrobetaine L-ascorbate acid a dipeptide a dipeptide a dipeptide poly-β-1,6- 1-(β-D O-acetyl-L-serine O-acetyl-L-serine spermidine spermidine D-mannitol galactitol sulfate phosphate phosphate phosphate betaine Fe 3+ an amino an amino D-cellobiose an aromatic an aromatic a dipeptide 6 phosphate phosphate N-acetyl-D- thiamine cys cys spermidine spermidine protoheme 2+ citrate N -(D-fructosyl)-L-lysine L-proline betaine glutamate L-proline betaine L-carnitine acid an amino acid an amino an amino an amino an amino a tripeptide a tripeptide a tripeptide amino acid amino acid ribofuranosyl) (R)- putrescine Fe N-acetyl-D- molybdate phosphate a hydroxamate 3+ 3+ arbutin shikimate enterobactin 2-oxoglutarate N 6 -(D-psicosyl)-L-lysine D-sorbitol D-glucosamine D-mannitol galactitol N-acetylneuraminate N-acetylneuraminate sulfate sulfate sulfate glycine betaine
    [Show full text]
  • Effect of Curcumin, Mixture of Curcumin and Piperine and Curcum (Turmeric) on Lipid Profile of Normal and Hyperlipidemic Rats
    The Egyptian Journal of Hospital Medicine Vol., 21: 145 – 161 December 2005 I.S.S.N: 12084 2002–1687 Effect of Curcumin, Mixture of Curcumin and Piperine and Curcum (Turmeric) on Lipid Profile of Normal and Hyperlipidemic Rats GHADA, Z. A. Soliman Lecturer of Biochemistry, Biochemistry Department, National Nutrition Institute, Cairo Abstract Curcumin is a polyphenolic, yellow pigment obtained from rhizomes of Curcuma longa (curcum), used as a spice and food colouring. The extracts have several pharmacological effects. We evaluated the effect of curcum, curcumin, and mixture of curcumin and piperine on plasma lipids in normal and hypercholesterolemic rats. A total of 270 rats, divided into 27 groups, were used. G1, G11: control, G2-G11: normal rats fed control diet supplemented with different levels of curcumin and curcum (G2-G6: 0.1%, 0.25%, 0.5%, 1.0%, 2.0% respectively, G7-G11: 1.67%, 4.167%, 8.34%, 16.67%, and 33.34). G12-G26: at first fed control diet supplemented with 2% cholesterol then G13-17, 21-25 fed a control diet supplemented with different levels of curcumin, and curcum [the same levels as G2-G11; G18-20 fed control diet supplemented with mixture of curcumin (0.1, 0.25, 0.5%) and piperine (20 mg/kg BW)], G12 was sacrificed before addition of studied materials, G26 were fed control diet. Lipid profile, triacylglycerol and phospholipids of plasma and organs as liver and heart were measured. Serum cholesterol (total, LDL-C, VLDL-C), triacylglycerol and phospholipids contents were elevated in cholesterol-fed rats, while HDL-C were decreased.
    [Show full text]
  • SUPLEMENTARY MATERIAL 1) Comparative Analysis of The
    1 SUPLEMENTARY MATERIAL 2 1) Comparative analysis of the different differential expression methods applied to the 3 transcriptome of Vanilla planifolia Jacks. 4 5 6 7 8 9 10 11 12 13 14 2dpi 10dpi 15 16 Supplemental Figure S1. Venn diagram showing the comparison of the differentially 17 expressed unigenes obtained with the methods DESeq2, EdgeR, NOISeq, and DESeq. At the 18 center of the diagram we observed that the EdgeR method comprises the great majority of 19 genes determined by the other methods. The right panel corresponds to 2 dpi, while the left 20 panel corresponds to 10 dpi. 21 22 23 24 25 26 2) Global expression profiles in response to infection caused by Fusarium 27 oxysporum f. sp. vanillae in vanilla. 28 . 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 2dpi 10dpi 51 Supplemental Figure S2. Heat map that contrasts the global vanilla response to 52 Fusarium oxysporum f. sp. vanillae. On the right we observe the early response 53 (2dpi); while in the left panel it presents the response to 10dpi. All differentially 54 expressed unigenes are included. 55 56 3) Expression profiles related to biotic stress, in the late response (10dpi) of 57 vanilla to Fusarium oxysporum f. sp. vanillae 58 59 60 61 62 63 64 65 66 67 68 69 70 71 Supplemental Figure S3. Heat map indicating the expression profiles of the annotated DEG 72 unigenes, corresponding to 10dpi. The numbers in the figure correspond to different 73 categories of gene ontology, as described below: 25 C1-metabolism, 11 lipid metabolism, 3 74 minor CHO metabolism, 13 amino acid metabolism, 16 secondary metabolism, 26 misc, 17 75 hormone metabolism, 30 signalling, 31 cell, 23 nucleotide metabolism, 27 RNA, 28 DNA, 76 33 development, 24 Biodegradation of Xenobiotics, 18 Co-factor and vitamine metabolism, 77 35 not assigned, 34 transport, 29 protein, 20 stress, 2 major CHO metabolism, 10 cell wall.
    [Show full text]
  • Mechanistic Study of Physicochemical and Biochemical Processes
    Mechanistic study of physicochemical and biochemical processes affecting intestinal absorption of the sesquiterpene lactone nobilin from multi-component systems in the Caco-2 model. Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von URSULA STEPHANIE THORMANN aus Bern (BE) Basel, 2015 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Dieses Werk ist unter dem Vertrag „Creative Commons Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 3.0 Schweiz“ (CC BY-NC-ND 3.0 CH) lizenziert. Die vollständige Lizenz kann unter creativecommons.org/licenses/by-nc-nd/3.0/ch/eingesehen werden. Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. G. Imanidis und Prof. Dr. H. E. U. Meyer zu Schwabedissen Basel, den 18. Februar 2014 Prof. Dr. J. Schibler Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 3.0 Schweiz (CC BY-NC-ND 3.0 CH) Sie dürfen: Teilen — den Inhalt kopieren, verbreiten und zugänglich machen Unter den folgenden Bedingungen: Namensnennung — Sie müssen den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen. Keine kommerzielle Nutzung — Sie dürfen diesen Inhalt nicht für kommerzielle Zwecke nutzen. Keine Bearbeitung erlaubt — Sie dürfen diesen Inhalt nicht bearbeiten, abwandeln oder in anderer Weise verändern. Wobei gilt: Verzichtserklärung — Jede der vorgenannten Bedingungen kann aufgehoben werden, sofern Sie die
    [Show full text]
  • Understanding and Exploiting Post-Translational Modifications for Plant Disease Resistance
    biomolecules Review Understanding and Exploiting Post-Translational Modifications for Plant Disease Resistance Catherine Gough and Ari Sadanandom * Department of Biosciences, Durham University, Stockton Road, Durham DH1 3LE, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-1913341263 Abstract: Plants are constantly threatened by pathogens, so have evolved complex defence signalling networks to overcome pathogen attacks. Post-translational modifications (PTMs) are fundamental to plant immunity, allowing rapid and dynamic responses at the appropriate time. PTM regulation is essential; pathogen effectors often disrupt PTMs in an attempt to evade immune responses. Here, we cover the mechanisms of disease resistance to pathogens, and how growth is balanced with defence, with a focus on the essential roles of PTMs. Alteration of defence-related PTMs has the potential to fine-tune molecular interactions to produce disease-resistant crops, without trade-offs in growth and fitness. Keywords: post-translational modifications; plant immunity; phosphorylation; ubiquitination; SUMOylation; defence Citation: Gough, C.; Sadanandom, A. 1. Introduction Understanding and Exploiting Plant growth and survival are constantly threatened by biotic stress, including plant Post-Translational Modifications for pathogens consisting of viruses, bacteria, fungi, and chromista. In the context of agriculture, Plant Disease Resistance. Biomolecules crop yield losses due to pathogens are estimated to be around 20% worldwide in staple 2021, 11, 1122. https://doi.org/ crops [1]. The spread of pests and diseases into new environments is increasing: more 10.3390/biom11081122 extreme weather events associated with climate change create favourable environments for food- and water-borne pathogens [2,3]. Academic Editors: Giovanna Serino The significant estimates of crop losses from pathogens highlight the need to de- and Daisuke Todaka velop crops with disease-resistance traits against current and emerging pathogens.
    [Show full text]
  • Ginkgolic Acid, a Sumoylation Inhibitor, Promotes Adipocyte
    www.nature.com/scientificreports OPEN Ginkgolic acid, a sumoylation inhibitor, promotes adipocyte commitment but suppresses Received: 25 October 2017 Accepted: 15 January 2018 adipocyte terminal diferentiation Published: xx xx xxxx of mouse bone marrow stromal cells Huadie Liu1,2, Jianshuang Li2, Di Lu2, Jie Li1,2, Minmin Liu 3, Yuanzheng He4, Bart O. Williams2, Jiada Li1 & Tao Yang 2 Sumoylation is a post-translational modifcation process having an important infuence in mesenchymal stem cell (MSC) diferentiation. Thus, sumoylation-modulating chemicals might be used to control MSC diferentiation for skeletal tissue engineering. In this work, we studied how the diferentiation of mouse bone marrow stromal cells (mBMSCs) is afected by ginkgolic acid (GA), a potent sumoylation inhibitor also reported to inhibit histone acetylation transferase (HAT). Our results show that GA promoted the diferentiation of mBMSCs into adipocytes when cultured in osteogenic medium. Moreover, mBMSCs pre-treated with GA showed enhanced pre-adipogenic gene expression and were more efciently diferentiated into adipocytes when subsequently cultured in the adipogenic medium. However, when GA was added at a later stage of adipogenesis, adipocyte maturation was markedly inhibited, with a dramatic down-regulation of multiple lipogenesis genes. Moreover, we found that the efects of garcinol, a HAT inhibitor, difered from those of GA in regulating adipocyte commitment and adipocyte maturation of mBMSCs, implying that the GA function in adipogenesis is likely through its activity as a sumoylation inhibitor, not as a HAT inhibitor. Overall, our studies revealed an unprecedented role of GA in MSC diferentiation and provide new mechanistic insights into the use of GA in clinical applications.
    [Show full text]
  • Ribonuclease E Organizes the Protein Interactions in the Escherichia Coli RNA Degradosome
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome Nathalie F. Vanzo,1 Yeun Shan Li,2 Be´atrice Py,2,3 Erwin Blum,2 Christopher F. Higgins,2,4 Lelia C. Raynal,1 Henry M. Krisch,1 and Agamemnon J. Carpousis1,5 1Laboratoire de Microbiologie et Ge´ne´tique Mole´culaire, UPR 9007, Centre National de la Recherche Scientifique (CNRS), 31062 Toulouse Cedex, France; 2Nuffield Department of Clinical Biochemistry and Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented.
    [Show full text]
  • Impact of Lipid Sources on Quality Traits of Medical Cannabis-Based Oil Preparations
    Article Impact of Lipid Sources on Quality Traits of Medical Cannabis-Based Oil Preparations Alberto Ramella 1, Gabriella Roda 2, Radmila Pavlovic 3,*, Michele Dei Cas 4, Eleonora Casagni 2, Giacomo Mosconi 3, Francisco Cecati 5, Paola Minghetti 2 and Carlo Grizzetti 6 1 Farmacia Dott.ri Giuliana e Alberto Ramella–SAS, Via A. Diaz 1, 21021 Angera (VA), Italy; [email protected] 2 Department of Pharmaceutical Sciences, Università degli Studi di Milano, Via L. Mangiagalli 25, 20133 Milan, Italy; [email protected] (G.R.); [email protected] (E.C.); [email protected] (P.M.) 3 Department of Health, Animal Science and Food Safety, University of Milan, 20133 Milan, Italy; [email protected] 4 Department of Health Sciences, Università degli Studi di Milano, Via A.di Rudinì 8, 20142 Milan, Italy; [email protected] 5 INTEQUI-CONICET, Faculty of Chemistry, Biochemistry and Pharmacy, National University of San Luis, Almirante Brown 1455, CP 5700 San Luis, Argentina; [email protected] 6 S.S.D. Cure Palliative e Terapia del Dolore, Ospedale di Circolo–Fondazione Macchi, ASST Sette Laghi, Viale L. Borri 57, 21100 Varese, Italy; [email protected] * Correspondence: [email protected] Academic Editor: Maria Carla Marcotullio Received: 2 June 2020; Accepted: 29 June 2020; Published: 30 June 2020 Abstract: The feasibility of the use of two lipid sources and their impact on the cannabinoid profile, terpene fingerprint, and degradation products in medical cannabis oil preparations during 3 months of refrigerated storage time were investigated. LCHRMS-Orbitrap® and HS-SPME coupled to GC- MS for the investigation of targeted and untargeted cannabinoids, terpenes, and lipid degradation products in Bedrocan® and Bediol® macerated oils were used as analytical approaches.
    [Show full text]
  • Phospholipid Metabolism in Stimulated Human Platelets: CHANGES in PHOSPHATIDYLINOSITOL, PHOSPHATIDIC ACID, and LYSOPHOSPHOLIPIDS
    Phospholipid Metabolism in Stimulated Human Platelets: CHANGES IN PHOSPHATIDYLINOSITOL, PHOSPHATIDIC ACID, AND LYSOPHOSPHOLIPIDS M. Johan Broekman, … , Jean W. Ward, Aaron J. Marcus J Clin Invest. 1980;66(2):275-283. https://doi.org/10.1172/JCI109854. Endogenous phospholipid metabolism in stimulated human platelets was studied by phosphorus assay of major and minor components following separation by two-dimensional thin-layer chromatography. This procedure obviated the use of radioactive labels. Extensive changes were found in quantities of phosphatidylinositol (PI) and phosphatidic acid (PA) as a consequence of thrombin or collagen stimulation. Thrombin addition was followed by rapid alterations in the amount of endogenous PI and PA. The decrease in PI was not precisely reciprocated by an increase in PA when thrombin was the stimulus. This apparent discrepancy could be explained by removal of a transient intermediate in PI metabolism, such as diglyceride, formed by PI-specific phospholipase C (Rittenhouse-Simmons, S., J. Clin. Invest.63: 580-587, 1979). Diglyceride would be unavailable for PA formation by diglyceride kinase, if hydrolyzed by diglyceride lipase (Bell, R. L., D. A. Kennerly, N. Stanford, and P. W. Majerus. Proc. Natl. Acad. Sci. U. S. A.76: 3238-3241, 1979) to yield arachidonate for prostaglandin endoperoxide formation. Thrombin-treated platelets also accumulated lysophospho-glycerides. Specifically, lysophosphatidyl ethanolamines accumulated within 15s following thrombin addition. Fatty acid and aldehyde analysis indicated phospholipase A2 activity, with an apparent preference for diacyl ethanolamine phosphoglycerides. In the case of collagen, these changes occurred concomitantly with aggregation and consumption of oxygen for prostaglandin endoperoxide formation. These studies of endogenous phospholipid metabolism provide information supporting the existence of […] Find the latest version: https://jci.me/109854/pdf Phospholipid Metabolism in Stimulated Human Platelets CHANGES IN PHOSPHATIDYLINOSITOL, PHOSPHATIDIC ACID, AND LYSOPHOSPHOLIPIDS NI.
    [Show full text]
  • Positive Allosteric Modulators (Pams) in Mouse Models of Overt Cannabimimetic Activity, Subjective Drug Effects, and Neuropathic Pain
    Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2021 Investigating Cannabinoid Type-1 Receptor (CB1R) Positive Allosteric Modulators (PAMs) in Mouse Models of Overt Cannabimimetic Activity, Subjective Drug Effects, and Neuropathic Pain Jayden Elmer Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Behavioral Neurobiology Commons, Behavior and Behavior Mechanisms Commons, Medicinal and Pharmaceutical Chemistry Commons, Nervous System Diseases Commons, and the Pharmacology Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/6777 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. 2021 Investigating Cannabinoid Type-1 Receptor (CB1R) Positive Allosteric Modulators (PAMs) in Mouse Models of Overt Cannabimimetic Activity, Subjective Drug Effects and Neuropathic Pain Jayden A. Elmer Investigating Cannabinoid Type-1 Receptor (CB1R) Positive Allosteric Modulators (PAMs) in Mouse Models of Overt Cannabimimetic Activity, Subjective Drug Effects and Neuropathic Pain A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University By Jayden Aric Elmer Bachelor of Science, University of Virginia, 2018 Director: Dr. Aron Lichtman, Professor, Department of Pharmacology & Toxicology; Associate Dean of Research and Graduate Studies, School of Pharmacy Virginia Commonwealth University Richmond, Virginia July 2021 Acknowledgements I would first like to extend my gratitude towards the CERT program at VCU. The CERT program opened the doors for me to get involved in graduate research.
    [Show full text]