DOCSLIB.ORG

Proteolytic Enzymes in Grass Pollen and Their Relationship to Allergenic Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Proteolytic Enzymes in Grass Pollen and their Relationship to Allergenic Proteins By Rohit G. Saldanha A thesis submitted in fulfilment of the requirements for the degree of Masters by Research Faculty of Medicine The University of New South Wales March 2005 TABLE OF CONTENTS TABLE OF CONTENTS 1 LIST OF FIGURES 6 LIST OF TABLES 8 LIST OF TABLES 8 ABBREVIATIONS 8 ACKNOWLEDGEMENTS 11 PUBLISHED WORK FROM THIS THESIS 12 ABSTRACT 13 1. ASTHMA AND SENSITISATION IN ALLERGIC DISEASES 14 1.1 Defining Asthma and its Clinical Presentation 14 1.2 Inflammatory Responses in Asthma 15 1.2.1 The Early Phase Response 15 1.2.2 The Late Phase Reaction 16 1.3 Effects of Airway Inflammation 16 1.3.1 Respiratory Epithelium 16 1.3.2 Airway Remodelling 17 1.4 Classification of Asthma 18 1.4.1 Extrinsic Asthma 19 1.4.2 Intrinsic Asthma 19 1.5 Prevalence of Asthma 20 1.6 Immunological Sensitisation 22 1.7 Antigen Presentation and development of T cell Responses. 22 1.8 Factors Influencing T cell Activation Responses 25 1.8.1 Co-Stimulatory Interactions 25 1.8.2 Cognate Cellular Interactions 26 1.8.3 Soluble Pro-inflammatory Factors 26 1.9 Intracellular Signalling Mechanisms Regulating T cell Differentiation 30 2 POLLEN ALLERGENS AND THEIR RELATIONSHIP TO PROTEOLYTIC ENZYMES 33 1 2.1 The Role of Pollen Allergens in Asthma 33 2.2 Environmental Factors influencing Pollen Exposure 33 2.3 Classification of Pollen Sources 35 2.3.1 Taxonomy of Pollen Sources 35 2.3.2 Cross-Reactivity between different Pollen Allergens 40 2.4 Classification of Pollen Allergens 41 2.4.1 The Revised Allergen Nomenclature 42 2.4.2 Ambiguities in the Allergen Nomenclature 43 2.4.3 Allergen Isoforms 43 2.5 Localisation and Biological Functions of Pollen Allergens 45 2.6 General Features of Proteolytic enzymes 47 2.7 Proteolytic Enzyme Nomenclature 48 2.7.1 Classification of Peptidases by Catalytic Mechanism 50 2.7.1.1 Serine Peptidases 50 2.7.1.2 Cysteine Peptidases 52 2.7.1.3 Aspartic Peptidases 54 2.7.1.4 Metallopeptidases 55 2.7.2 Classification of Peptidases based on Substrate Specificity 56 2.7.2.1 Endopeptidases 56 2.7.2.2 Exopeptidases 56 2.7.2.2.1 Aminopeptidases 58 2.7.2.2.2 Carboxypeptidases 58 2.8 Classification of Peptidase Inhibitors 58 2.8.1 Natural Peptidase Enzyme Inhibitors 59 2.8.2 Synthetic Peptidase Inhibitors 62 2.9 Functional Significance of Peptidase activity in plants and pollen 64 3 IDENTIFICATION AND CHARACTERISATION OF PEPTIDASE ENZYMES 66 3.1 Proteolytic Enzyme Extraction Techniques 66 3.2 Measurement of Enzymatic Activity 68 3.2.1 Solid Phase Assays 68 3.2.2 Liquid Phase Assays 71 3.3 Protein Purification Procedures 74 2 3.3.1 General Purification Procedures 74 3.3.2 Size-Exclusion Chromatography 75 3.3.3 Ion Exchange Chromatography 76 3.3.4 Reverse-Phase Chromatography 78 3.3.5 Affinity Chromatography 79 3.4 Principles of Protein Identification by Proteomics 82 3.4.1 Why Proteomics? 83 3.4.2 Protein Separation Techniques 84 3.4.3 Proteomic Analysis by Mass Spectrometry 87 3.5 Types of Analytical Mass Spectrometers 88 3.5.1 MALDI-TOF Mass Spectrometer 88 3.5.2 Tandem Mass Spectrometers 91 3.6 Protein Identification by Mass Spectrometry 93 3.6.1 Peptide Mass Fingerprinting 93 3.6.2 Tandem Mass Spectrometry 94 3.7 Interpretation of Peptide Sequence from Tandem Mass Spectra 95 3.8 Proteomic Analysis of Allergic Diseases 97 4 MATERIALS AND METHODS 100 4.1 General Methods 100 4.1.1 Preparation of Pollen Diffusates 100 4.1.2 Development of Fluorescent Substrate Assay 100 4.1.3 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) and Western Immunoblotting 101 4.1.4 Development of Zymography 101 4.1.5 Two-Dimensional Gel Electrophoresis 103 4.2 Specific Purification Procedures 104 4.2.1 Size-Exclusion Chromatography 104 4.2.2 Ion-Exchange Chromatography 105 4.2.3 Affinity Chromatography using Benzamidine Sepharose 105 4.2.4 Affinity Chromatography using Concanavalin A Sepharose 106 4.3 Gel Destaining Protocols for the Proteomic Analysis of Proteins 107 4.3.1 Destaining of Silver Nitrate Stained gels 107 3 4.3.2 Destaining of Coomassie Stained Gels 107 4.3.3 Peptide Extraction from SDS/PAGE for Mass Spectrometry 108 4.4 Mass Spectrometric Analysis of Proteins 108 4.4.1 Peptide Mass Fingerprinting by MALDI-TOF Mass Spectrometry 108 4.4.2 Microcapillary Liquid Chromatography/Tandem Mass Spectrometric Analysis of Proteins 109 5 SERINE PEPTIDASE ACTIVITY IN POLLEN DIFFUSATES 111 5.1 Peptidase Activity in Pollen diffusates 111 5.1.1 Fluorometric Assay 111 5.1.2 Characterisation of Peptidase Activity based on the Inhibitory Profile 111 5.2 One-Dimensional Electrophoretic Analysis of Pollen Diffusates 112 5.2.1 SDS/PAGE and Western Blotting of Pollen Diffusates 112 5.2.2 Gelatin Zymography 112 5.2.3 Proteomic Analysis of Samples from One-Dimensional SDS/PAGE 118 5.3 Two-Dimensional Electrophoretic Analysis of Bermuda grass Pollen Diffusate 122 5.3.1 Two-Dimensional SDS/PAGE and Western Blotting 122 5.3.2 Two-Dimensional Gelatin Zymography 122 5.3.3 Proteomic Analysis of samples from Two-Dimensional SDS/PAGE of Bermuda grass Pollen 125 5.4 Discussion 128 6 PURIFICATION OF PROTEOLYTIC ACTIVITY IN POLLEN DIFFUSATES 137 6.1 General Chromatographic Separation Techniques 137 6.1.1 Size-Exclusion Chromatography 137 6.1.2 Ion-Exchange Chromatography 141 6.1.3 Discussion 145 6.2 Affinity Chromatography Purification Techniques 147 6.2.1 Benzamidine Sepharose Chromatography 147 6.2.2 Concanavalin A Sepharose Chromatography 149 6.2.2.1 Affinity Purification using Concanavalin A Sepharose 149 6.2.2.2 Analysis of Peptidase activity by Fluorometric Assay 149 4 6.2.2.3 Analysis by One-Dimensional SDS/PAGE and Gelatin Zymography 150 6.2.2.4 Proteomic Analysis of Con A Sepharose Purified Fractions. 154 6.2.2.5 Analysis of the proteolytically active fractions by Anion Exchange HPLC 154 6.2.3 Discussion 158 7 CONCLUDING DISCUSSION 164 BIBLIOGRAPHY 169 APPENDIX: GENERAL REAGENTS 191 5 LIST OF FIGURES Figure 2.1 - Scanning electron micrographs of fresh Poaceae pollen grains in the dry state (A&C) and in the hydrated state (B&D) 35 Figure 2.2 - Nomenclature of allergenic tree pollen sources 37 Figure 2.3 - Nomenclature of allergenic grass pollen sources 38 Figure 2.4 - Nomenclature of allergenic weed pollen sources 39 Figure 2.5 - IUIS criteria for the inclusion of allergens into allergen nomenclature 41 Figure 2.6 - Schematics of the designation of allergen names according to IUIS 42 Figure 2.7 - Revised nomenclature of allergen isoforms and variants 45 Figure 3.1 - Schematic representation of size-exclusion chromatography 76 Figure 3.2 - Schematic representation of ion-exchange chromatography 77 Figure 3.3 - Schematic of the principle of affinity chromatography. 81 Figure 3.4 - General Strategy for proteome characterisation 85 Figure 3.5 - Schematics for an orthogonal Time of Flight mass analyser 90 Figure 3.6 - Schematics for Q-Tof hybrid Tandem Mass Spectrometer 92 Figure 3.7 - The ion series produced by the fragmentation pattern of amino acid residues 96 Figure 3.8 - Typical tandem mass spectrum (MS2) of GluFibrino peptide (+2) 98 Figure 4.1 - Running parameters for isoelectric focussing of pollen diffusates 104 Figure 5.1 - Hydrolysis of fluorescent substrate by crude pollen diffusates 113 Figure 5.2 - Measurement of the hydrolysis of the fluorescent substrate by the pollen diffusates 114 Figure 5.3 - SDS/PAGE analysis of pollen diffusates 115 Figure 5.4 - SDS/PAGE and Western blot analysis of pollen diffusates 115 Figure 5.5 - Gelatin zymography of Pollen Diffusates and sensitivity to inhibitors 117 Figure 5.6 - SDS/PAGE and MALDI reflectron TOF of tryptic peptides derived from band 4 (Lolium perenne) and band 12 (Cynodon dactylon) 119 Figure 5.7 - Two-Dimensional SDS/PAGE and gelatin zymography of the pollen diffusate of Bermuda grass 123 Figure 5.8 - Two-Dimensional Western Blotting of Bermuda grass pollen diffusate 124 Figure 5.9 - Tandem mass spectrometric analysis of tryptic peptides derived from spot 4 (Cynodon dactylon) 126 Figure 5.10 - Multiple sequence alignments of selected allergens and trypsin 135 6 Figure 6.1 - Size-exclusion chromatogram of gel filtration standards 138 Figure 6.2 - Size-exclusion chromatogram of Bermuda grass pollen diffusate 139 Figure 6.3 - SDS/PAGE and Gelatin Zymography of Size-Exclusion HPLC fractions of Bermuda grass pollen diffusate 140 Figure 6.4 - Anion-exchange chromatogram of Bermuda grass pollen diffusate 142 Figure 6.5 - SDS/PAGE and Gelatin zymography of Anion-Exchange chromatography fractions of Bermuda grass pollen diffusate 143 Figure 6.6 - Analysis of the protein bands separated by SDS/PAGE of fractions of Anion exchange HPLC 144 Figure 6.7 - Fluorescent substrate assay on Benzamidine Sepharose purified Fractions of Kentucky blue and Bermuda grass 148 Figure 6.8 - Protein concentrations of the Concanavalin A Sepharose separated fractions of Bermuda grass pollen diffusates by the BCA assay 151 Figure 6.9 - Hydrolysis of the fluorescent substrate (NBAMC) by the Con A Sepharose separated fractions of Bermuda grass pollen diffusate 152 Figure 6.10 - Hydrolysis of fluorescent substrate by the Con A Sepharose separated fractions of Bermuda grass pollen diffusate 153 Figure 6.11 - SDS/PAGE and gelatin Zymography of the Con A Sepharose separated fractions of Bermuda grass pollen diffusate 155 Figure 6.12 - Proteomic analysis of Con A Sepharose separated fractions of Bermuda grass 156 Figure 6.13 - Anion Exchange chromatography of the Bound fractions of Con A Sepharose affinity chromatography of Bermuda grass pollen diffusate 157 7 LIST OF TABLES Table 1.1 - Factors influencing the polarisation of Th responses 24 Table 1.2 - Functions of cytokines influencing the Th response 28 Table 2.1 - Plant Nomenclature Sub-Divisions 36 Table 2.2: Revised nomenclature of common pollen allergens.
Recommended publications
  • Ubiquitination, Ubiquitin-Like Modifiers, and Deubiquitination in Viral Infection

    Ubiquitination, Ubiquitin-Like Modifiers, and Deubiquitination in Viral Infection

    Ubiquitination, Ubiquitin-like Modifiers, and Deubiquitination in Viral Infection The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Isaacson, Marisa K., and Hidde L. Ploegh. “Ubiquitination, Ubiquitin- like Modifiers, and Deubiquitination in Viral Infection.” Cell Host & Microbe 5, no. 6 (June 2009): 559-570. Copyright © 2009 Elsevier Inc. As Published http://dx.doi.org/10.1016/j.chom.2009.05.012 Publisher Elsevier Version Final published version Citable link http://hdl.handle.net/1721.1/84989 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Cell Host & Microbe Review Ubiquitination, Ubiquitin-like Modifiers, and Deubiquitination in Viral Infection Marisa K. Isaacson1 and Hidde L. Ploegh1,* 1Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA *Correspondence: [email protected] DOI 10.1016/j.chom.2009.05.012 Ubiquitin is important for nearly every aspect of cellular physiology. All viruses rely extensively on host machinery for replication; therefore, it is not surprising that viruses connect to the ubiquitin pathway at many levels. Viral involvement with ubiquitin occurs either adventitiously because of the unavoidable usur- pation of cellular processes, or for some specific purpose selected for by the virus to enhance viral replica- tion. Here, we review current knowledge of how the ubiquitin pathway alters viral replication and how viruses influence the ubiquitin pathway to enhance their own replication. Introduction own ubiquitin ligases or ubiquitin-specific proteases, it seems Ubiquitin is a small 76 amino acid protein widely expressed in reasonable to infer functional relevance, but even in these cases, eukaryotic cells.
  • Structure‐Based Macrocyclization of Substrate Analogue NS2B‐NS3

    Structure‐Based Macrocyclization of Substrate Analogue NS2B‐NS3

    Full Papers ChemMedChem doi.org/10.1002/cmdc.202000237 1 2 3 Structure-Based Macrocyclization of Substrate Analogue 4 5 NS2B-NS3 Protease Inhibitors of Zika, West Nile and 6 Dengue viruses 7 8 Niklas J. Braun+,[a] Jun P. Quek+,[b, c] Simon Huber+,[a] Jenny Kouretova,[a] Dorothee Rogge,[a] 9 Heike Lang-Henkel,[a] Ezekiel Z. K. Cheong,[d] Bing L. A. Chew,[b, c, e] Andreas Heine,[a] 10 [b, c, d] [a] 11 Dahai Luo,* and Torsten Steinmetzer* 12 13 14 A series of cyclic active-site-directed inhibitors of the NS2B-NS3 protease with Ki values <5 nM. Crystal structures of seven ZIKV 15 proteases from Zika (ZIKV), West Nile (WNV), and dengue-4 protease inhibitor complexes were determined to support the 16 (DENV4) viruses has been designed. The most potent com- inhibitor design. All the cyclic compounds possess high 17 pounds contain a reversely incorporated d-lysine residue in the selectivity against trypsin-like serine proteases and furin-like 18 P1 position. Its side chain is connected to the P2 backbone, its proprotein convertases. Both WNV and DENV4 proteases are 19 α-amino group is converted into a guanidine to interact with inhibited less efficiently. Nonetheless, similar structure-activity 20 the conserved Asp129 side chain in the S1 pocket, and its C relationships were observed for these enzymes, thus suggesting 21 terminus is connected to the P3 residue via different linker their potential application as pan-flaviviral protease inhibitors. 22 segments. The most potent compounds inhibit the ZIKV 23 24 Introduction subtypes (TBEV-EU and TBEV-FE) or Omsk hemorrhagic fever 25 virus.
  • Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease

    Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease

    International Journal of Molecular Sciences Review Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease Michael C. McKelvey 1 , Ryan Brown 1, Sinéad Ryan 1, Marcus A. Mall 2,3,4 , Sinéad Weldon 1 and Clifford C. Taggart 1,* 1 Airway Innate Immunity Research (AiiR) Group, Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast BT9 7BL, UK; [email protected] (M.C.M.); [email protected] (R.B.); [email protected] (S.R.); [email protected] (S.W.) 2 Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité—Universitätsmedizin Berlin, 13353 Berlin, Germany; [email protected] 3 Berlin Institute of Health (BIH), 10178 Berlin, Germany 4 German Center for Lung Research (DZL), 35392 Gießen, Germany * Correspondence: [email protected]; Tel.: +44-289097-6383 Abstract: Dysregulated protease activity has long been implicated in the pathogenesis of chronic lung diseases and especially in conditions that display mucus obstruction, such as chronic obstructive pulmonary disease, cystic fibrosis, and non-cystic fibrosis bronchiectasis. However, our appreciation of the roles of proteases in various aspects of such diseases continues to grow. Patients with muco- obstructive lung disease experience progressive spirals of inflammation, mucostasis, airway infection and lung function decline. Some therapies exist for the treatment of these symptoms, but they are unable to halt disease progression and patients may benefit from novel adjunct therapies. In this review, we highlight how proteases act as multifunctional enzymes that are vital for normal Citation: McKelvey, M.C.; Brown, R.; airway homeostasis but, when their activity becomes immoderate, also directly contribute to airway Ryan, S.; Mall, M.A.; Weldon, S.; dysfunction, and impair the processes that could resolve disease.
  • Substrate Specificities of Outer Membrane Proteases of the Omptin Family in Escherichia Coli, Salmonella Enterica, and Citrobacter Rodentium

    Substrate Specificities of Outer Membrane Proteases of the Omptin Family in Escherichia Coli, Salmonella Enterica, and Citrobacter Rodentium

    Substrate specificities of outer membrane proteases of the omptin family in Escherichia coli, Salmonella enterica, and Citrobacter rodentium Andrea Portt, McGill University, Montreal October 9th, 2010 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master’s in Microbiology © Andrea Portt 2010 Table of Contents LIST OF ABBREVIATIONS 3 ABSTRACT 6 ACKNOWLEDGEMENTS 7 CONTRIBUTIONS OF AUTHORS 8 LITERATURE REVIEW 9 2. OMPTINS 9 3. OMPTIN SUBSTRATES 13 A. ANTIMICROBIAL PEPTIDES 13 B. BLOOD CLOTTING PROTEINS AND EXTRACELLULAR MATRIX 14 C. COMPLEMENT 16 D. TROPOMYOSIN 17 4. EVOLUTION OF OMPTINS 17 5. OMPTIN REGULATION 20 6. OMPTINS OF PATHOGENIC ENTEROBACTEREACEAE 22 A. Y. PESTIS 22 B. S. ENTERICA 23 C. SHIGELLA 25 D. ATTACHING AND EFFACING BACTERIA 25 INTRODUCTION 28 MATERIALS AND METHODS 29 1. BACTERIAL GROWTH 29 2. BACTERIAL STRAINS AND CONSTRUCTION OF PLASMIDS 29 A. STRAINS 29 B. PLASMIDS 31 3. DISK INHIBITION ASSAYS 34 4. MINIMUM INHIBITORY CONCENTRATION DETERMINATIONS 34 5. PROTEOLYTIC CLEAVAGE OF AMPS BY CELLS EXPRESSING OMPTINS 34 6. OM DISRUPTION ASSAY WITH 1-N-PHENYLNAPHTHYLAMINE 35 7. DEGRADATION OF TROPOMYOSIN BY E. COLI AND C. RODENTIUM STRAINS EXPRESSING OMPTINS 35 8. REAL-TIME QUANTITATIVE PCR 35 9. PURIFICATION OF NATIVE CROP 35 10. EXPRESSION, PURIFICATION, AND REFOLDING OF HIS-PGTE AND HIS-CROP 36 A. EXPRESSION 36 B. PURIFICATION 36 C. REFOLDING 36 11. CLEAVAGE OF C2 FRET SUBSTRATE BY WHOLE CELLS OR PURIFIED OMPTINS 37 A. WHOLE CELLS 37 B. PURIFIED OMPTINS 37 1 RESULTS 38 1. GROWTH INHIBITION OF E. COLI AND C. RODENTIUM STRAINS EXPRESSING OMPTINS BY C18G.
  • (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002

    (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002

    USOO6395889B1 (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002 (54) NUCLEIC ACID MOLECULES ENCODING WO WO-98/56804 A1 * 12/1998 ........... CO7H/21/02 HUMAN PROTEASE HOMOLOGS WO WO-99/0785.0 A1 * 2/1999 ... C12N/15/12 WO WO-99/37660 A1 * 7/1999 ........... CO7H/21/04 (75) Inventor: fish E. Robison, Wilmington, MA OTHER PUBLICATIONS Vazquez, F., et al., 1999, “METH-1, a human ortholog of (73) Assignee: Millennium Pharmaceuticals, Inc., ADAMTS-1, and METH-2 are members of a new family of Cambridge, MA (US) proteins with angio-inhibitory activity', The Journal of c: - 0 Biological Chemistry, vol. 274, No. 33, pp. 23349–23357.* (*) Notice: Subject to any disclaimer, the term of this Descriptors of Protease Classes in Prosite and Pfam Data patent is extended or adjusted under 35 bases. U.S.C. 154(b) by 0 days. * cited by examiner (21) Appl. No.: 09/392, 184 Primary Examiner Ponnathapu Achutamurthy (22) Filed: Sep. 9, 1999 ASSistant Examiner William W. Moore (51) Int. Cl." C12N 15/57; C12N 15/12; (74) Attorney, Agent, or Firm-Alston & Bird LLP C12N 9/64; C12N 15/79 (57) ABSTRACT (52) U.S. Cl. .................... 536/23.2; 536/23.5; 435/69.1; 435/252.3; 435/320.1 The invention relates to polynucleotides encoding newly (58) Field of Search ............................... 536,232,235. identified protease homologs. The invention also relates to 435/6, 226, 69.1, 252.3 the proteases. The invention further relates to methods using s s s/ - - -us the protease polypeptides and polynucleotides as a target for (56) References Cited diagnosis and treatment in protease-mediated disorders.
  • What Are the Roles of Metalloproteinases in Cartilage and Bone Damage? G Murphy, M H Lee

    What Are the Roles of Metalloproteinases in Cartilage and Bone Damage? G Murphy, M H Lee

    iv44 Ann Rheum Dis: first published as 10.1136/ard.2005.042465 on 20 October 2005. Downloaded from REPORT What are the roles of metalloproteinases in cartilage and bone damage? G Murphy, M H Lee ............................................................................................................................... Ann Rheum Dis 2005;64:iv44–iv47. doi: 10.1136/ard.2005.042465 enzyme moiety into an upper and a lower subdomain. A A role for metalloproteinases in the pathological destruction common five stranded beta-sheet and two alpha-helices are in diseases such as rheumatoid arthritis and osteoarthritis, always found in the upper subdomain with a further C- and the irreversible nature of the ensuing cartilage and bone terminal helix in the lower subdomain. The catalytic sites of damage, have been the focus of much investigation for the metalloproteinases, especially the MMPs, have been several decades. This has led to the development of broad targeted for the development of low molecular weight spectrum metalloproteinase inhibitors as potential therapeu- synthetic inhibitors with a zinc chelating moiety. Inhibitors tics. More recently it has been appreciated that several able to fully differentiate between individual enzymes have families of zinc dependent proteinases play significant and not been identified thus far, although a reasonable level of varied roles in the biology of the resident cells in these tissues, discrimination is now being achieved in some cases.7 Each orchestrating development, remodelling, and subsequent family does, however, have other unique domains with pathological processes. They also play key roles in the numerous roles, including the determination of physiological activity of inflammatory cells. The task of elucidating the substrate specificity, ECM, or cell surface localisation (fig 1).
  • (12) Patent Application Publication (10) Pub. No.: US 2016/0346364 A1 BRUNS Et Al

    (12) Patent Application Publication (10) Pub. No.: US 2016/0346364 A1 BRUNS Et Al

    US 2016.0346364A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0346364 A1 BRUNS et al. (43) Pub. Date: Dec. 1, 2016 (54) MEDICAMENT AND METHOD FOR (52) U.S. Cl. TREATING INNATE IMMUNE RESPONSE CPC ........... A61K 38/488 (2013.01); A61K 38/482 DISEASES (2013.01); C12Y 304/23019 (2013.01); C12Y 304/21026 (2013.01); C12Y 304/23018 (71) Applicant: DSM IPASSETS B.V., Heerlen (NL) (2013.01); A61K 9/0053 (2013.01); C12N 9/62 (2013.01); A23L 29/06 (2016.08); A2ID 8/042 (72) Inventors: Maaike Johanna BRUINS, Kaiseraugst (2013.01); A23L 5/25 (2016.08); A23V (CH); Luppo EDENS, Kaiseraugst 2002/00 (2013.01) (CH); Lenneke NAN, Kaiseraugst (CH) (57) ABSTRACT (21) Appl. No.: 15/101,630 This invention relates to a medicament or a dietary Supple (22) PCT Filed: Dec. 11, 2014 ment comprising the Aspergillus niger aspergilloglutamic peptidase that is capable of hydrolyzing plant food allergens, (86). PCT No.: PCT/EP2014/077355 and more particularly, alpha-amylase/trypsin inhibitors, thereby treating diseases due to an innate immune response S 371 (c)(1), in humans, and/or allowing to delay the onset of said (2) Date: Jun. 3, 2016 diseases. The present invention relates to the discovery that (30) Foreign Application Priority Data the Aspergillus niger aspergilloglutamic peptidase is capable of hydrolyzing alpha-amylase/trypsin inhibitors that are Dec. 11, 2013 (EP) .................................. 13196580.8 present in wheat and related cereals said inhibitors being strong inducers of innate immune response. Furthermore, Publication Classification the present invention relates to a method for hydrolyzing alpha-amylase/trypsin inhibitors comprising incubating a (51) Int.
  • Association Between the Gut Microbiota and Blood Pressure in a Population Cohort of 6953 Individuals

    Association Between the Gut Microbiota and Blood Pressure in a Population Cohort of 6953 Individuals

    Journal of the American Heart Association ORIGINAL RESEARCH Association Between the Gut Microbiota and Blood Pressure in a Population Cohort of 6953 Individuals Joonatan Palmu , MD; Aaro Salosensaari , MSc; Aki S. Havulinna , DSc (Tech); Susan Cheng , MD, MPH; Michael Inouye, PhD; Mohit Jain, MD, PhD; Rodolfo A. Salido , BSc; Karenina Sanders , BSc; Caitriona Brennan, BSc; Gregory C. Humphrey, BSc; Jon G. Sanders , PhD; Erkki Vartiainen , MD, PhD; Tiina Laatikainen , MD, PhD; Pekka Jousilahti, MD, PhD; Veikko Salomaa , MD, PhD; Rob Knight , PhD; Leo Lahti , DSc (Tech); Teemu J. Niiranen , MD, PhD BACKGROUND: Several small-scale animal studies have suggested that gut microbiota and blood pressure (BP) are linked. However, results from human studies remain scarce and conflicting. We wanted to elucidate the multivariable-adjusted as- sociation between gut metagenome and BP in a large, representative, well-phenotyped population sample. We performed a focused analysis to examine the previously reported inverse associations between sodium intake and Lactobacillus abun- dance and between Lactobacillus abundance and BP. METHODS AND RESULTS: We studied a population sample of 6953 Finns aged 25 to 74 years (mean age, 49.2±12.9 years; 54.9% women). The participants underwent a health examination, which included BP measurement, stool collection, and 24-hour urine sampling (N=829). Gut microbiota was analyzed using shallow shotgun metagenome sequencing. In age- and sex-adjusted models, the α (within-sample) and β (between-sample) diversities of taxonomic composition were strongly re- lated to BP indexes (P<0.001 for most). In multivariable-adjusted models, β diversity was only associated with diastolic BP (P=0.032).
  • Evidence for an Active-Center Cysteine in the SH-Proteinase Cu-Clostripain Through Use of IV-Tosyl-L-Lysine Chloromethyl Ketone

    Evidence for an Active-Center Cysteine in the SH-Proteinase Cu-Clostripain Through Use of IV-Tosyl-L-Lysine Chloromethyl Ketone

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 173, number 1 FEBS 1649 July 1984 Evidence for an active-center cysteine in the SH-proteinase cu-clostripain through use of IV-tosyl-L-lysine chloromethyl ketone A.-M. Gilles and B. Keil Unitt! de Chimie des Protknes, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris CPdex 15, France Received 30 May 1984 The rapid reaction of a-clostripain with tosyl-L-lysine chloromethyl ketone results in a complete loss of activity and in the disappearance of one titratable SH group whereas the number of histidine residues is not affected. Tosyl-L-phenylalanine chloromethyl ketone and phenylmethylsulfonyl fluoride have no effect on the catalytic activity. From the molar ratio and under the assumption of 1: 1 molar interaction, the fully active enzyme has a specific activity of 650-700 units/mg [twice the value proposed by Porter et al. (J. Biol. Chem. 246 (1971) 76757682)]. Partial oxidation makes it experimentally impossible to attain this maximal value. ff-Clostripain Cysteine proteinase Active site 1. INTRODUCTION was due to the modification of a thiol group in an analogous way with other cysteine proteinases such Clostripain (EC 3.4.4.20) is a sulfhydryl protein- as papain [6] and ficin [7]. Recently [8], we eluci- ase isolated from the culture filtrate of Clostridium dated the amino acid sequence around this acces- histolyticum with a highly limited specificity sible thiol group after labelling with radioactive directed at the carboxyl bond of arginyl residues in iodoacetic acid.
  • Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens

    Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens

    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2020 Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens LU LU ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-1148-7 UPPSALA urn:nbn:se:uu:diva-435815 2021 Dissertation presented at Uppsala University to be publicly examined in Room A1:111a, BMC, Husargatan 3, Uppsala, Friday, 16 April 2021 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Christian Cambillau. Abstract Lu, L. 2021. Structural and Biochemical Characterizations of Three Potential Drug Targets from Pathogens. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2020. 91 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1148-7. As antibiotic resistance of various pathogens emerged globally, the need for new effective drugs with novel modes of action became urgent. In this thesis, we focus on infectious diseases, e.g. tuberculosis, malaria, and nosocomial infections, and the corresponding causative pathogens, Mycobacterium tuberculosis, Plasmodium falciparum, and the Gram-negative ESKAPE pathogens that underlie so many healthcare-acquired diseases. Following the same- target-other-pathogen (STOP) strategy, we attempted to comprehensively explore the properties of three promising drug targets. Signal peptidase I (SPase I), existing both in Gram-negative and Gram-positive bacteria, as well as in parasites, is vital for cell viability, due to its critical role in signal peptide cleavage, thus, protein maturation, and secreted protein transport. Three factors, comprising essentiality, a unique mode of action, and easy accessibility, make it an attractive drug target.
  • Design of a Highly Selective Quenched Activity-Based Probe

    Design of a Highly Selective Quenched Activity-Based Probe

    Article pubs.acs.org/JACS Design of a Highly Selective Quenched Activity-Based Probe and Its Application in Dual Color Imaging Studies of Cathepsin S Activity Localization † † † † ∥ Kristina Oresic Bender, Leslie Ofori, Wouter A. van der Linden, Elliot D. Mock, Gopal K. Datta, ∥ § † † ∥ # Somenath Chowdhury, Hao Li, Ehud Segal, Mateo Sanchez Lopez, Jonathan A. Ellman, , ⊥ † ‡ § † ⊥ Carl G. Figdor, Matthew Bogyo,*, , , and Martijn Verdoes*, , † ‡ § Departments of Pathology, Microbiology and Immunology, and Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305, United States ∥ Department of Chemistry, University of California-Berkeley, Berkeley, California 94720, United States ⊥ Department of Tumor Immunology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6500 HB Nijmegen, The Netherlands *S Supporting Information ABSTRACT: The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity.
  • Cloning of a Salivary Gland Metalloprotease And

    Cloning of a Salivary Gland Metalloprotease And

    University of Rhode Island DigitalCommons@URI Past Departments Faculty Publications (CELS) College of the Environment and Life Sciences 2003 Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis Ivo M.B. Francischetti Thomas N. Mather University of Rhode Island, [email protected] José M.C. Ribeiro Follow this and additional works at: https://digitalcommons.uri.edu/cels_past_depts_facpubs Citation/Publisher Attribution Francischetti, I. M.B., Mather, T. N., & Ribeiro, J. M.C. (2003). Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis. Biochemical and Biophysical Research Communications, 305(4), 869-875. doi: 10.1016/S0006-291X(03)00857-X Available at: https://doi.org/10.1016/S0006-291X(03)00857-X This Article is brought to you for free and open access by the College of the Environment and Life Sciences at DigitalCommons@URI. It has been accepted for inclusion in Past Departments Faculty Publications (CELS) by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. NIH Public Access Author Manuscript Biochem Biophys Res Commun. Author manuscript; available in PMC 2010 July 14. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Biochem Biophys Res Commun. 2003 June 13; 305(4): 869±875. Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme Disease tick vector Ixodes scapularis Ivo M.