Maturation of Escherichia Coli Maltose-Binding Protein by Signal Peptidase I in Vivo. Sequence Requirements for Efficient Proces
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Foreign Gene Expression in Yeast: a Review
YEAST VOL. 8: 423-488 (1992) Foreign Gene Expression in Yeast: a Review MICHAEL A. ROMANOS, CAROL A. SCORER AND JEFFREY J. CLARE Department of Cell Biology, Wellcome Research Laboratories. Beckenham, Kent BR3 3BS, U.K. Received 14 February 1992; accepted 22 February 1992 CONTENTS Expression in non-Saccharomyces yeasts Introduction Introduction Pichia pastoris Transformation and selectable markers Hansenula polymorpha Auxotrophic selection markers Kluyveromyces lactis Dominant selectable markers Yarrowia lipolytica Autoselection Schizosaccharomyces pombe Episomal vectors Physiology of foreign gene expression ARS vectors Mechanisms of toxicity 2pbased vectors Generation of low-expressingvariants Regulated copy number vectors Large-scale fermentation and Integrating vectors optimization YIP vectors Concluding remarks Transplacement Acknowledgements Integration into reiterated DNA References Transposition vectors Transcriptional promoters and terminators Foreign versus yeast promoters INTRODUCTION Glycolytic promoters Galactose-regulated promoters The yeast Saccharomyces cerevisiae has several Phosphate-regulated promoters properties which have established it as an important Glucose-repressible promoters tool in theexpressionofforeign protein sfor research, Other regulated promoter systems industrial or medical use. As a food organism, it Selection of novel yeast promoters is highly acceptable for the production of pharma- Foreign promoter systems ceutical proteins. In contrast, Escherichia coli has Yeast terminators toxic cell wall pyrogens and mammalian cells may contain oncogenic or viral DNA, so that products Factors affecting intracellular expression Initiation of transcription from these organisms must be tested more exten- RNA elongation sively. Yeast can be grown rapidly on simple media RNA stability and to high cell density, and its genetics are more Initiation of translation advanced than any other eukaryote, so that it can be Translational elongation manipulated almost as readily as E.coli. -
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. -
Serine Proteases with Altered Sensitivity to Activity-Modulating
(19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants. -
Proteolytic Enzymes in Grass Pollen and Their Relationship to Allergenic Proteins
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 -
Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage
bioRxiv preprint doi: https://doi.org/10.1101/2020.11.11.378711; this version posted November 11, 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-NC-ND 4.0 International license. Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage A. Manuel Liaci1, Barbara Steigenberger2,3, Sem Tamara2,3, Paulo Cesar Telles de Souza4, Mariska Gröllers-Mulderij1, Patrick Ogrissek1,5, Siewert J. Marrink4, Richard A. Scheltema2,3, Friedrich Förster1* Abstract The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo- microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments. Keywords Signal Peptidase Complex, Signal Peptide, Protein Maturation, Membrane Thinning, cryo-EM, Crosslinking Mass Spectrometry, Molecular Dynamics Simulations, Protein Secretion, ER Translocon 1Cryo-Electron Microscopy, Bijvoet Centre for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. -
Sequence and Functional Analysis of Schistosoma
MINING FOR CONSERVED MOTIFS AND SIGNIFICANT FUNCTIONS IN S. MANSONI CERCARIAL SECRETIONS Amy L. Schmidbauer Submitted to the faculty of the School of Informatics in partial fulfillment of the requirements for the degree Master of Science in Bioinformatics, Indiana University December 30, 2006 ii Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Master of Science in Bioinformatics. (Committee Chair’s signature)_______________________ Sean D. Mooney, Ph.D., Chair Master’s Thesis Committee (Second member’s signature)________________________ Xiaoman Shawn Li, Ph.D. (Third member’s signature)__________ _______________ William J. Sullivan, Ph.D. ii © 2006 Amy L. Schmidbauer All Rights Reserved iii ACKNOWLEDGMENTS This project would not have been possible without the guidance and support of many people including faculty, advisors, colleagues, friends and family. I am extremely grateful to my advisor, Dr. Sean Mooney, for welcoming me into his laboratory as a graduate student, and for providing, not only computing resources, but continued support, suggestions, and guidance as, what started out as an independent study project, grew into what became this thesis. I extend my sincere appreciation to Dr. Giselle Knudsen, an honorary member of my thesis committee, for the original project inspiration, for her enthusiastic encouragement, insight, and direction throughout the project, and for her thoughtful review of this thesis. I would like also like to extend a heartfelt thank you to Dr. William Sullivan and Dr. Xiaoman Li for their willingness to lend their time to reviewing my thesis and for the insightful feedback they provided. For statistical expertise and support I would like to extend my deepest appreciation to Dr. -
Bunyamwera Orthobunyavirus Glycoprotein Precursor Is Processed by Cellular Signal Peptidase and Signal Peptide Peptidase
Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase Xiaohong Shia,1, Catherine H. Bottingb, Ping Lia, Mark Niglasb, Benjamin Brennana, Sally L. Shirranb, Agnieszka M. Szemiela, and Richard M. Elliotta,2 aMedical Research Council–University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow G61 1QH, United Kingdom; and bBiomedical Sciences Research Complex, University of St. Andrews, St. Andrews KY16 9ST, United Kingdom Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved June 17, 2016 (received for review February 29, 2016) The M genome segment of Bunyamwera virus (BUNV)—the pro- (II and IV) (Fig. S1A), and its N-terminal domain (I) is required totype of both the Bunyaviridae family and the Orthobunyavirus for BUNV replication (8). genus—encodes the glycoprotein precursor (GPC) that is proteo- Cleavage of BUNV GPC is mediated by host proteases, but the lytically cleaved to yield two viral structural glycoproteins, Gn and Gc, details of which proteases are involved and the precise cleavage sites and a nonstructural protein, NSm. The cleavage mechanism of ortho- have not been clarified. Experimental data on GPC processing have bunyavirus GPCs and the host proteases involved have not been only been reported for snowshoe hare orthobunyavirus (SSHV); the clarified. In this study, we investigated the processing of BUNV GPC C terminus of SSHV Gn was determined by C-terminal amino acid and found that both NSm and Gc proteins were cleaved at their own sequencing to be an arginine (R) residue at position 299 (9) (Fig. -
Structures of Lipoprotein Signal Peptidase II From
University of Birmingham Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin Olatunji, Samir; Yu, Xiaoxiao; Bailey, Jonathan; Huang, Chia-Ying; Zapotoczna, Marta; Bowen, Katherine; Remškar, Maja; Müller, Rolf; Scanlan, Eoin M; Geoghegan, Joan A; Olieric, Vincent; Caffrey, Martin DOI: 10.1038/s41467-019-13724-y License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Olatunji, S, Yu, X, Bailey, J, Huang, C-Y, Zapotoczna, M, Bowen, K, Remškar, M, Müller, R, Scanlan, EM, Geoghegan, JA, Olieric, V & Caffrey, M 2020, 'Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin', Nature Communications, vol. 11, no. 1, 140. https://doi.org/10.1038/s41467-019-13724-y Link to publication on Research at Birmingham portal General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. -
Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases
Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases Alan J. Barrett Neil D. Rawlings J. Fred Woessner Editor biographies xxi Contributors xxiii Preface xxxi Introduction ' Abbreviations xxxvii ASPARTIC PEPTIDASES Introduction 1 Aspartic peptidases and their clans 3 2 Catalytic pathway of aspartic peptidases 12 Clan AA Family Al 3 Pepsin A 19 4 Pepsin B 28 5 Chymosin 29 6 Cathepsin E 33 7 Gastricsin 38 8 Cathepsin D 43 9 Napsin A 52 10 Renin 54 11 Mouse submandibular renin 62 12 Memapsin 1 64 13 Memapsin 2 66 14 Plasmepsins 70 15 Plasmepsin II 73 16 Tick heme-binding aspartic proteinase 76 17 Phytepsin 77 18 Nepenthesin 85 19 Saccharopepsin 87 20 Neurosporapepsin 90 21 Acrocylindropepsin 9 1 22 Aspergillopepsin I 92 23 Penicillopepsin 99 24 Endothiapepsin 104 25 Rhizopuspepsin 108 26 Mucorpepsin 11 1 27 Polyporopepsin 113 28 Candidapepsin 115 29 Candiparapsin 120 30 Canditropsin 123 31 Syncephapepsin 125 32 Barrierpepsin 126 33 Yapsin 1 128 34 Yapsin 2 132 35 Yapsin A 133 36 Pregnancy-associated glycoproteins 135 37 Pepsin F 137 38 Rhodotorulapepsin 139 39 Cladosporopepsin 140 40 Pycnoporopepsin 141 Family A2 and others 41 Human immunodeficiency virus 1 retropepsin 144 42 Human immunodeficiency virus 2 retropepsin 154 43 Simian immunodeficiency virus retropepsin 158 44 Equine infectious anemia virus retropepsin 160 45 Rous sarcoma virus retropepsin and avian myeloblastosis virus retropepsin 163 46 Human T-cell leukemia virus type I (HTLV-I) retropepsin 166 47 Bovine leukemia virus retropepsin 169 48 -
Intrinsic Evolutionary Constraints on Protease Structure, Enzyme
Intrinsic evolutionary constraints on protease PNAS PLUS structure, enzyme acylation, and the identity of the catalytic triad Andrew R. Buller and Craig A. Townsend1 Departments of Biophysics and Chemistry, The Johns Hopkins University, Baltimore MD 21218 Edited by David Baker, University of Washington, Seattle, WA, and approved January 11, 2013 (received for review December 6, 2012) The study of proteolysis lies at the heart of our understanding of enzyme evolution remain unanswered. Because evolution oper- biocatalysis, enzyme evolution, and drug development. To un- ates through random forces, rationalizing why a particular out- derstand the degree of natural variation in protease active sites, come occurs is a difficult challenge. For example, the hydroxyl we systematically evaluated simple active site features from all nucleophile of a Ser protease was swapped for the thiol of Cys at serine, cysteine and threonine proteases of independent lineage. least twice in evolutionary history (9). However, there is not This convergent evolutionary analysis revealed several interre- a single example of Thr naturally substituting for Ser in the lated and previously unrecognized relationships. The reactive protease catalytic triad, despite its greater chemical similarity rotamer of the nucleophile determines which neighboring amide (9). Instead, the Thr proteases generate their N-terminal nu- can be used in the local oxyanion hole. Each rotamer–oxyanion cleophile through a posttranslational modification: cis-autopro- hole combination limits the location of the moiety facilitating pro- teolysis (10, 11). These facts constitute clear evidence that there ton transfer and, combined together, fixes the stereochemistry of is a strong selective pressure against Thr in the catalytic triad that catalysis. -
Secretory Signal Peptide Modification for Optimized Antibody-Fragment Expression-Secretion in Leishmania Tarentolae Stephan Klatt1,2 and Zoltán Konthur1*
Klatt and Konthur Microbial Cell Factories 2012, 11:97 http://www.microbialcellfactories.com/content/11/1/97 RESEARCH Open Access Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae Stephan Klatt1,2 and Zoltán Konthur1* Abstract Background: Secretory signal peptides (SPs) are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1) of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s). The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results: We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. -
Supporting Information
Comparative genomic and transcriptomic analysis of Wangiella dermatitidis, a major cause of phaeohyphomycosis and a model black yeast human pathogen Zehua Chen*, Diego A. Martinez*, Sharvari Gujja*, Sean M. Sykes*, Qiandong Zeng*, Paul J. Szaniszlo§, Zheng Wang†,1, Christina A. Cuomo*,1 *Broad Institute of MIT and Harvard, Cambridge, MA 02142. §The Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712. †Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C. 20375. 1Corresponding authors: [email protected] and [email protected] Data availability: The assembly and annotation of Wangiella dermatitidis are available at the NCBI nucleotide database under the accession number AFPA01000000; RNA-Seq differential expression analysis of pH stress is available at the NCBI GEO database under record GSE51646. DOI: 10.1534/g3.113.009241 Figure S1 Independent expansion of MFS and APC transporter families in W. dermatitidis and selected aspergilli. (A) Average number of genes per genome for different category of MFS and APC families (Core families are the ortholog clusters shared by all four fungal groups; Shared, present in at least two out of the four fungal groups; Unique, unique to each group, including species-specific paralogous clusters and unclustered genes). (B) Enrichment of different category of MFS and APC transporters under different stress conditions (low pH or radiation). A positive normalized enrichment score (NES) indicates enrichment under stress conditions (pH 2.5 or with radiation), and a negative score indicates enrichment underChen normal et al, Figure conditions S1 (pH 6 or no radiation). Significant enrichments noted with **: q-value < 0.05; ***: q-value < 0.01.