Biosynthetic Mechanism of the Antibiotic Capuramycin

Total Page:16

File Type:pdf, Size:1020Kb

Biosynthetic Mechanism of the Antibiotic Capuramycin University of Kentucky UKnowledge Theses and Dissertations--Pharmacy College of Pharmacy 2018 BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN Erfu Yan University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0002-5545-0995 Digital Object Identifier: https://doi.org/10.13023/etd.2018.360 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Yan, Erfu, "BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN" (2018). Theses and Dissertations--Pharmacy. 92. https://uknowledge.uky.edu/pharmacy_etds/92 This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Erfu Yan, Student Dr. Steven Van Lanen, Major Professor Dr. David Feola, Director of Graduate Studies BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky By Erfu Yan Lexington, Kentucky Director: Dr. Steven Van Lanen, Professor of Pharmaceutical Science Lexington, Kentucky 2018 Copyright © Erfu Yan 2018 ABSTRACT OF DISSERTATION BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN A-102395 is a member of the capuramycin family of antibiotics which was isolated from the culture broth of Amycolatopsis sp. SANK 60206. A-102339 is structurally classified as a nucleoside antibiotic, which like all members of the capuramycin family, inhibits bacterial MraY (translocase I) with IC50 of 11 nM which is the lowest among the capuramycin family. A semisynthetic derivative of capuramycin is currently in clinical trials as an antituberculosis antibiotic, suggesting high potential for using A- 102395 as a starting point for new antibiotic discovery. In contrast to other capuramycins, A-102395 has a unique arylamine-containing polyamide side chain. The biosynthetic gene cluster of A-102395 was previously identified and includes 35 putative open reading frames responsible for biosynthesis and resistance. Presently, there are no reports focused on the biosynthesis of this polyamide chain. Here we present the functional assignment and biochemical characterization of seven proteins, Cpr33-38 and Cpr12, that initiate the biosynthesis of the polyamide. Functional characterization of Cpr38, which has sequence similarity to the gene products encoded by pabA and pabB from E. coli, revealed that it functions as a 4- amino-4-deoxychorismate (ADC) synthase catalyzing a two-step reaction involving amidohydrolysis of L-Gln with ammonia channeled and incorporated into chorismic acid to generate ADC. Cpr12, encoded by a gene that was originally proposed to be outside the gene cluster and sharing similarity to proteins annotated as ADC lyase, was revealed to catalyze the elimination of pyruvate to form PABA. Cpr36 is demonstrated to function as a free-standingpeptidyl carrier protein (PCP), which is activated to form holo-protein from the apo-form. Cpr37, which belongs to the adenylation domain protein in the nonribosomal peptide synthase (NRPS), subsequently activates PABA and loads it to holo-Cpr36 Two proteins Cpr34 and Cpr35 work in concert to catalyze decarboxylative condensation between a thioester linked PABA and malonyl-S-acyl carrier protein (ACP) during aromatic polyketide biosynthesis catalyzed by type II polyketide synthases. Following condensation, Cpr33 acts as 3-oxoacyl-ACP reductase that catalyzes reduction to the β-hydroxythioester intermediate. In this scenario, hydride is predicted to be added to the re face to generate the S configuration resulting in the same stereochemical outcome as other 3-oxoacyl-ACP reductase (FabG) from bacterial type II fatty acid synthases.These findings are critical advancement for interrogating the biosynthesis of the unusual chemical components of the family of antibiotics of capuramycin. KEWWORDS: capuramycins, peptidoglycan cell wall, polyamide assembly Erfu Yan Student’s Signature 08/28/2018 Date BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN By Erfu Yan Steven Van Lanen Director of Dissertation David Feola Director of Graduate Studies 08/28/2018 Date 献给我的妻子和女儿,婷婷和雯潇,谢谢你们一路相濡以沫 This dissertation is dedicated to my dear wife and daughter, Tingting and Liliana, whose love and support made this journey through graduate school possible. ACKNOWLEDGMENTS I would like to take this special moment to express my gratitude to my mentor, Dr. Steven Van Lanen, for his guidance, support, and inspiration throughout my study in his group. I am grateful for the chance to seek refuge in this lab following departure of Dr. Guo’s lab in my second PhD year. His expertise and insightful discussions have benefited me greatly and extensively. I would also like to thank my committee members, Dr. Chang-guo Zhan, Dr. Oleg Tsodikov, and Dr. Chris Richards, for their invaluable advice and comments over the years, and I would like to sincerely thank Dr. Zhenyu Li for being the external department examiner for my dissertation defense. Special thanks to all current and previous members from the Van Lanen group: Dr., Dr. Zhaoyong Yang, Dr. Sandra Barnard, Dr. Xiuling Chi, Dr. Anwesha Goswami, Dr. Xiaodong Liu, Dr. Zheng Cui, Dr. Ying Huang, Matthew McErlean, Ashley Arlinghaus, and Jonathon Overbay. Also, a special thanks to Dr. Bert C. Lynn and Dr. Fang Huang in Department of Chemistry, University of Kentucky. I would also like to thank Dr. Minakshi Bhardwaj for analyzing all the NMR data showed in this thesis. It was a great honor to work with and learn from you all! Last but not least, I would like to acknowledge my family and friends for their constant support and encouragement. I need to thank all my friends and coworkers who have been my teachers in different aspects of my life in graduate school. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................................................................... iii LIST OF TABLES ....................................................................................................... vii LIST OF FIGURES ................................................................................................... viii LIST OF ABBREVIATIONS ....................................................................................... xi Chapter 1: Introduction and Background ....................................................................... 1 1.1 Natural Products – Significance ........................................................................... 1 1.2 Need for New Anti-tuberculosis Antibiotics ........................................................ 2 1.3 Biosynthesis of Peptidoglycan Cell Wall ............................................................. 4 1.4 MraY – Structure and Function ........................................................................... 7 1.5 Inhibitors of MraY-Translocase I ....................................................................... 12 1.6 Non-ribosomal Peptides and Their Synthesis .................................................... 15 1.7 Mass Spectrometry and Protein Analysis .......................................................... 18 1.8 Discovery of Capuramycins ............................................................................... 23 1.9 Current Understanding of the Biosynthetic Pathways of A-102395 .................. 26 1.10 Aims of this Study ............................................................................................ 29 Chapter 2: Elucidating the mechanism of Cpr38 and Cpr12 ....................................... 30 2.1 Background ........................................................................................................ 30 2.2 Materials and Methods ......................................................................................
Recommended publications
  • Part One Amino Acids As Building Blocks
    Part One Amino Acids as Building Blocks Amino Acids, Peptides and Proteins in Organic Chemistry. Vol.3 – Building Blocks, Catalysis and Coupling Chemistry. Edited by Andrew B. Hughes Copyright Ó 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32102-5 j3 1 Amino Acid Biosynthesis Emily J. Parker and Andrew J. Pratt 1.1 Introduction The ribosomal synthesis of proteins utilizes a family of 20 a-amino acids that are universally coded by the translation machinery; in addition, two further a-amino acids, selenocysteine and pyrrolysine, are now believed to be incorporated into proteins via ribosomal synthesis in some organisms. More than 300 other amino acid residues have been identified in proteins, but most are of restricted distribution and produced via post-translational modification of the ubiquitous protein amino acids [1]. The ribosomally encoded a-amino acids described here ultimately derive from a-keto acids by a process corresponding to reductive amination. The most important biosynthetic distinction relates to whether appropriate carbon skeletons are pre-existing in basic metabolism or whether they have to be synthesized de novo and this division underpins the structure of this chapter. There are a small number of a-keto acids ubiquitously found in core metabolism, notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis), together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon skeletons for unbranched a-amino acids of three, four, and five carbons, respectively. a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight chains are made by alternative pathways depending on the available raw materials.
    [Show full text]
  • Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics
    toxins Article Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics Bryan John J. Subong 1,2,* , Arturo O. Lluisma 1, Rhodora V. Azanza 1 and Lilibeth A. Salvador-Reyes 1,* 1 Marine Science Institute, University of the Philippines- Diliman, Velasquez Street, Quezon City 1101, Philippines; [email protected] (A.O.L.); [email protected] (R.V.A.) 2 Department of Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo City, Tokyo 113-8654, Japan * Correspondence: [email protected] (B.J.J.S.); [email protected] (L.A.S.-R.) Abstract: Alexandrium minutum and Alexandrium tamutum are two closely related harmful algal bloom (HAB)-causing species with different toxicity. Using isobaric tags for relative and absolute quantita- tion (iTRAQ)-based quantitative proteomics and two-dimensional differential gel electrophoresis (2D-DIGE), a comprehensive characterization of the proteomes of A. minutum and A. tamutum was performed to identify the cellular and molecular underpinnings for the dissimilarity between these two species. A total of 1436 proteins and 420 protein spots were identified using iTRAQ-based proteomics and 2D-DIGE, respectively. Both methods revealed little difference (10–12%) between the proteomes of A. minutum and A. tamutum, highlighting that these organisms follow similar cellular and biological processes at the exponential stage. Toxin biosynthetic enzymes were present in both organisms. However, the gonyautoxin-producing A. minutum showed higher levels of osmotic growth proteins, Zn-dependent alcohol dehydrogenase and type-I polyketide synthase compared to the non-toxic A. tamutum. Further, A. tamutum had increased S-adenosylmethionine transferase that may potentially have a negative feedback mechanism to toxin biosynthesis.
    [Show full text]
  • Infant Antibiotic Exposure Search EMBASE 1. Exp Antibiotic Agent/ 2
    Infant Antibiotic Exposure Search EMBASE 1. exp antibiotic agent/ 2. (Acedapsone or Alamethicin or Amdinocillin or Amdinocillin Pivoxil or Amikacin or Aminosalicylic Acid or Amoxicillin or Amoxicillin-Potassium Clavulanate Combination or Amphotericin B or Ampicillin or Anisomycin or Antimycin A or Arsphenamine or Aurodox or Azithromycin or Azlocillin or Aztreonam or Bacitracin or Bacteriocins or Bambermycins or beta-Lactams or Bongkrekic Acid or Brefeldin A or Butirosin Sulfate or Calcimycin or Candicidin or Capreomycin or Carbenicillin or Carfecillin or Cefaclor or Cefadroxil or Cefamandole or Cefatrizine or Cefazolin or Cefixime or Cefmenoxime or Cefmetazole or Cefonicid or Cefoperazone or Cefotaxime or Cefotetan or Cefotiam or Cefoxitin or Cefsulodin or Ceftazidime or Ceftizoxime or Ceftriaxone or Cefuroxime or Cephacetrile or Cephalexin or Cephaloglycin or Cephaloridine or Cephalosporins or Cephalothin or Cephamycins or Cephapirin or Cephradine or Chloramphenicol or Chlortetracycline or Ciprofloxacin or Citrinin or Clarithromycin or Clavulanic Acid or Clavulanic Acids or clindamycin or Clofazimine or Cloxacillin or Colistin or Cyclacillin or Cycloserine or Dactinomycin or Dapsone or Daptomycin or Demeclocycline or Diarylquinolines or Dibekacin or Dicloxacillin or Dihydrostreptomycin Sulfate or Diketopiperazines or Distamycins or Doxycycline or Echinomycin or Edeine or Enoxacin or Enviomycin or Erythromycin or Erythromycin Estolate or Erythromycin Ethylsuccinate or Ethambutol or Ethionamide or Filipin or Floxacillin or Fluoroquinolones
    [Show full text]
  • N-Carbamoylation of 2,4-Diaminobutyrate Reroutes the Outcome in Padanamide Biosynthesis
    Chemistry & Biology Article N-Carbamoylation of 2,4-Diaminobutyrate Reroutes the Outcome in Padanamide Biosynthesis Yi-Ling Du,1 Doralyn S. Dalisay,1 Raymond J. Andersen,1,2 and Katherine S. Ryan1,* 1Department of Chemistry 2Department of Earth, Ocean and Atmospheric Sciences University of British Columbia, Vancouver, BC V6T 1Z1, Canada *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2013.06.013 SUMMARY literature. It is interesting that a compound identical to padana- mide A, named actinoramide A (Nam et al., 2011), was indepen- Padanamides are linear tetrapeptides notable for the dently reported from Streptomyces sp. CNQ-027. This actinomy- absence of proteinogenic amino acids in their struc- cete strain was isolated from sediment on the opposite side of the tures. In particular, two unusual heterocycles, (S)- Pacific Ocean, near San Diego, CA, suggesting a potentially wide 3-amino-2-oxopyrrolidine-1-carboxamide (S-Aopc) distribution of the padanamides/actinoramides. It is intriguing and (S)-3-aminopiperidine-2,6-dione (S-Apd), are that, whereas padanamide A and actinoramide A are identical, found at the C-termini of padanamides A and B, the minor compounds (actinoramides B and C) co-isolated from Streptomyces sp. CNQ-027 are unique (Figure 1A). Total respectively. Here we identify the padanamide synthesis of padanamides A and B was recently reported (Long biosynthetic gene cluster and carry out systematic et al., 2013), confirming the previous structural elucidations. gene inactivation studies. Our results show that The padanamides attracted our attention for their many padanamides are synthesized by highly dissociated unusual chemical features.
    [Show full text]
  • Yeast Genome Gazetteer P35-65
    gazetteer Metabolism 35 tRNA modification mitochondrial transport amino-acid metabolism other tRNA-transcription activities vesicular transport (Golgi network, etc.) nitrogen and sulphur metabolism mRNA synthesis peroxisomal transport nucleotide metabolism mRNA processing (splicing) vacuolar transport phosphate metabolism mRNA processing (5’-end, 3’-end processing extracellular transport carbohydrate metabolism and mRNA degradation) cellular import lipid, fatty-acid and sterol metabolism other mRNA-transcription activities other intracellular-transport activities biosynthesis of vitamins, cofactors and RNA transport prosthetic groups other transcription activities Cellular organization and biogenesis 54 ionic homeostasis organization and biogenesis of cell wall and Protein synthesis 48 plasma membrane Energy 40 ribosomal proteins organization and biogenesis of glycolysis translation (initiation,elongation and cytoskeleton gluconeogenesis termination) organization and biogenesis of endoplasmic pentose-phosphate pathway translational control reticulum and Golgi tricarboxylic-acid pathway tRNA synthetases organization and biogenesis of chromosome respiration other protein-synthesis activities structure fermentation mitochondrial organization and biogenesis metabolism of energy reserves (glycogen Protein destination 49 peroxisomal organization and biogenesis and trehalose) protein folding and stabilization endosomal organization and biogenesis other energy-generation activities protein targeting, sorting and translocation vacuolar and lysosomal
    [Show full text]
  • Polyketide Biosynthesis Beyond the Type I, II and III Polyketide Synthase Paradigms Ben Shen
    285 Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms Ben Shen Recent literature on polyketide biosynthesis suggests that Three types of bacterial PKSs are known to date. First, polyketide synthases have much greater diversity in both type I PKSs are multifunctional enzymes that are orga- mechanism and structure than the current type I, II and III nized into modules, each of which harbors a set of distinct, paradigms. These examples serve as an inspiration for searching non-iteratively acting activities responsible for the cata- novel polyketide synthases to give new insights into polyketide lysis of one cycle of polyketide chain elongation, as biosynthesis and to provide new opportunities for combinatorial exemplified by the 6-deoxyerythromycin B synthase biosynthesis. (DEBS) for the biosynthesis of reduced polyketides (i.e. macrolides, polyethers and polyene) such as erythro- Addresses mycin A (1)(Figure 1a) [1]. Second, type II PKSs are Division of Pharmaceutical Sciences and Department of Chemistry, multienzyme complexes that carry a single set of iteratively University of Wisconsin, Madison, WI 53705, USA acting activities, as exemplified by the tetracenomycin e-mail: [email protected] PKS for the biosynthesis of aromatic polyketides (often polycyclic) such as tetracenomycin C (2)(Figure 1b) [2]. Current Opinion in Chemical Biology 2003, 7:285–295 Third, type III PKSs, also known as chalcone synthase- like PKSs, are homodimeric enzymes that essentially are This review comes from a themed section on iteratively acting condensing enzymes, as exemplified by Biocatalysis and biotransformation Edited by Tadhg Begley and Ming-Daw Tsai the RppA synthase for the biosynthesis of aromatic poly- ketides (often monocyclic or bicyclic), such as flavolin (3) 1367-5931/03/$ – see front matter (Figure 1c) [3].
    [Show full text]
  • On Glyphosate
    Ecocycles 2(2): 1-8 (2016) ISSN 2416-2140 DOI: 10.19040/ecocycles.v2i2.60 EDITORIAL On glyphosate Tamas Komives1 * and Peter Schröder2 1Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Herman Otto 15, 1022 Budapest, Hungary and Department of Environmental Science, Esterhazy Karoly University, 3200 Gyongyos, Hungary 2Helmholtz Zentrum München, German Research Centre for Environmental Health, GmbH, Research Unit Environmental Genomics, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany *E-mail: [email protected] Abstract – This Editorial briefly discusses the current issues surrounding glyphosate - the most controversial pesticide active ingredient of our time. The paper pays special attention to the effects of glyphosate on plant-pathogen interactions. Keywords – glyphosate, plant-pathogen interactions, environment, human health, ecocycles, sustainability Received: October 14, 2016 Accepted: November 10, 2016 ———————————————————————————————————————————————— - In nature nothing exists alone. researchers of the company missed to identify the Rachel Carson in “Silent Spring” (Carson, 1962) molecule as a potential herbicide because of the short duration of the company's standardized biological - Alle Dinge sind Gift, und nichts ist ohne Gift; allein assays (only five days, while the first, glyphosate- die Dosis machts, daß ein Ding kein Gift sei. (All induced phytotoxic symptoms usually appear after things are poison, and nothing is without poison: the about one week) (F. M. Pallos,
    [Show full text]
  • Multiplex De Novo Sequencing of Peptide Antibiotics
    JOURNAL OF COMPUTATIONAL BIOLOGY Volume 18, Number 11, 2011 Research Articles # Mary Ann Liebert, Inc. Pp. 1371–1381 DOI: 10.1089/cmb.2011.0158 Multiplex De Novo Sequencing of Peptide Antibiotics HOSEIN MOHIMANI,1 WEI-TING LIU,2 YU-LIANG YANG,3 SUSANA P. GAUDEˆ NCIO,4 WILLIAM FENICAL,4 PIETER C. DORRESTEIN,2,3 and PAVEL A. PEVZNER5 ABSTRACT Proliferation of drug-resistant diseases raises the challenge of searching for new, more efficient antibiotics. Currently, some of the most effective antibiotics (i.e., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. The isolation and sequencing of cyclic peptide antibiotics, unlike the same activity with linear peptides, is time-consuming and error-prone. The dominant technique for sequencing cyclic peptides is nuclear magnetic resonance (NMR)–based and requires large amounts (milli- grams) of purified materials that, for most compounds, are not possible to obtain. Given these facts, there is a need for new tools to sequence cyclic non-ribosomal peptides (NRPs) using picograms of material. Since nearly all cyclic NRPs are produced along with related analogs, we develop a mass spectrometry approach for sequencing all related peptides at once (in contrast to the existing approach that analyzes individual peptides). Our results suggest that instead of attempting to isolate and NMR-sequence the most abundant com- pound, one should acquire spectra of many related compounds and sequence all of them simultaneously using tandem mass spectrometry. We illustrate applications of this approach by sequencing new variants of cyclic peptide antibiotics from Bacillus brevis, as well as sequencing a previously unknown family of cyclic NRPs produced by marine bacteria.
    [Show full text]
  • Genome-Scale Fitness Profile of Caulobacter Crescentus Grown in Natural Freshwater
    Supplemental Material Genome-scale fitness profile of Caulobacter crescentus grown in natural freshwater Kristy L. Hentchel, Leila M. Reyes Ruiz, Aretha Fiebig, Patrick D. Curtis, Maureen L. Coleman, Sean Crosson Tn5 and Tn-Himar: comparing gene essentiality and the effects of gene disruption on fitness across studies A previous analysis of a highly saturated Caulobacter Tn5 transposon library revealed a set of genes that are required for growth in complex PYE medium [1]; approximately 14% of genes in the genome were deemed essential. The total genome insertion coverage was lower in the Himar library described here than in the Tn5 dataset of Christen et al (2011), as Tn-Himar inserts specifically into TA dinucleotide sites (with 67% GC content, TA sites are relatively limited in the Caulobacter genome). Genes for which we failed to detect Tn-Himar insertions (Table S13) were largely consistent with essential genes reported by Christen et al [1], with exceptions likely due to differential coverage of Tn5 versus Tn-Himar mutagenesis and differences in metrics used to define essentiality. A comparison of the essential genes defined by Christen et al and by our Tn5-seq and Tn-Himar fitness studies is presented in Table S4. We have uncovered evidence for gene disruptions that both enhanced or reduced strain fitness in lake water and M2X relative to PYE. Such results are consistent for a number of genes across both the Tn5 and Tn-Himar datasets. Disruption of genes encoding three metabolic enzymes, a class C β-lactamase family protein (CCNA_00255), transaldolase (CCNA_03729), and methylcrotonyl-CoA carboxylase (CCNA_02250), enhanced Caulobacter fitness in Lake Michigan water relative to PYE using both Tn5 and Tn-Himar approaches (Table S7).
    [Show full text]
  • Supplemental Methods
    Supplemental Methods: Sample Collection Duplicate surface samples were collected from the Amazon River plume aboard the R/V Knorr in June 2010 (4 52.71’N, 51 21.59’W) during a period of high river discharge. The collection site (Station 10, 4° 52.71’N, 51° 21.59’W; S = 21.0; T = 29.6°C), located ~ 500 Km to the north of the Amazon River mouth, was characterized by the presence of coastal diatoms in the top 8 m of the water column. Sampling was conducted between 0700 and 0900 local time by gently impeller pumping (modified Rule 1800 submersible sump pump) surface water through 10 m of tygon tubing (3 cm) to the ship's deck where it then flowed through a 156 µm mesh into 20 L carboys. In the lab, cells were partitioned into two size fractions by sequential filtration (using a Masterflex peristaltic pump) of the pre-filtered seawater through a 2.0 µm pore-size, 142 mm diameter polycarbonate (PCTE) membrane filter (Sterlitech Corporation, Kent, CWA) and a 0.22 µm pore-size, 142 mm diameter Supor membrane filter (Pall, Port Washington, NY). Metagenomic and non-selective metatranscriptomic analyses were conducted on both pore-size filters; poly(A)-selected (eukaryote-dominated) metatranscriptomic analyses were conducted only on the larger pore-size filter (2.0 µm pore-size). All filters were immediately submerged in RNAlater (Applied Biosystems, Austin, TX) in sterile 50 mL conical tubes, incubated at room temperature overnight and then stored at -80oC until extraction. Filtration and stabilization of each sample was completed within 30 min of water collection.
    [Show full text]
  • The UBE2L3 Ubiquitin Conjugating Enzyme: Interplay with Inflammasome Signalling and Bacterial Ubiquitin Ligases
    The UBE2L3 ubiquitin conjugating enzyme: interplay with inflammasome signalling and bacterial ubiquitin ligases Matthew James George Eldridge 2018 Imperial College London Department of Medicine Submitted to Imperial College London for the degree of Doctor of Philosophy 1 Abstract Inflammasome-controlled immune responses such as IL-1β release and pyroptosis play key roles in antimicrobial immunity and are heavily implicated in multiple hereditary autoimmune diseases. Despite extensive knowledge of the mechanisms regulating inflammasome activation, many downstream responses remain poorly understood or uncharacterised. The cysteine protease caspase-1 is the executor of inflammasome responses, therefore identifying and characterising its substrates is vital for better understanding of inflammasome-mediated effector mechanisms. Using unbiased proteomics, the Shenoy grouped identified the ubiquitin conjugating enzyme UBE2L3 as a target of caspase-1. In this work, I have confirmed UBE2L3 as an indirect target of caspase-1 and characterised its role in inflammasomes-mediated immune responses. I show that UBE2L3 functions in the negative regulation of cellular pro-IL-1 via the ubiquitin- proteasome system. Following inflammatory stimuli, UBE2L3 assists in the ubiquitylation and degradation of newly produced pro-IL-1. However, in response to caspase-1 activation, UBE2L3 is itself targeted for degradation by the proteasome in a caspase-1-dependent manner, thereby liberating an additional pool of IL-1 which may be processed and released. UBE2L3 therefore acts a molecular rheostat, conferring caspase-1 an additional level of control over this potent cytokine, ensuring that it is efficiently secreted only in appropriate circumstances. These findings on UBE2L3 have implications for IL-1- driven pathology in hereditary fever syndromes, and autoinflammatory conditions associated with UBE2L3 polymorphisms.
    [Show full text]
  • Journal of Chromatography
    aphy & S r ep og a t r a a t m i o o r n Lilla et al., J Chromatograph Separat Techniq 2012, 3:2 h T e C c f Journal of Chromatography h DOI: 10.4172/2157-7064.1000122 o n l i a q ISSN:n 2157-7064 u r e u s o J Separation Techniques Research Article OpenOpen Access Access Structural Characterization of Transglutaminase-Catalyzed Casein Cross- Linking Sergio Lilla1,2, Gianfranco Mamone2, Maria Adalgisa Nicolai1, Lina Chianese1, Gianluca Picariello2, Simonetta Caira2 and Francesco Addeo1,2* 1Dipartimento di Scienza degli Alimenti, University of Naples “Federico II”, Parco Gussone, Portici 80055, Italy 2Istituto di Scienze dell’Alimentazione (ISA) – CNR, Via Roma 64, 83100 Avellino, Italy Abstract Microbial transglutaminase is used in the food industry to improve texture by catalyzing protein cross-linking. Casein is a well-known transglutaminase substrate, but the complete role of glutamine (Q) and lysine (K) residues in its cross-linking is not fully understood. In this study, we describe the characterization of microbial Transglutaminase -modified casein using a combination of immunological and proteomic techniques. Using 5-(biotinamido)pentylamine as an acyl acceptor probe, three Q residues of β-casein and one of αs1-casein were found to participate as acyl donors. However, no Q-residues were involved in network formation with κ-casein or αs2-casein. Q and K residues in the ε-(γ-glutamyl)lysine-isopeptide bonds β-casein were identified by nanoelectrospray tandem mass spectrometry of the proteolytic digests. This work reports our progress toward a better understanding of the function and mechanism of action of microbial transglutaminase-mediated proteins.
    [Show full text]