DOCSLIB.ORG

Cranfield University Glaxosmithkline JESSICA BROTHWOOD

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cranfield University Glaxosmithkline JESSICA BROTHWOOD Cranfield University GlaxoSmithKline JESSICA BROTHWOOD DRUGGABLE AND BIOPHARMABLE GENOME ANNOTATION PIPELINE DEVELOPMENT Cranfield Health Applied Bioinformatics MSc Thesis Academic year: 2011-12 Supervisors: Dr Michael Cauchi (Cranfield) Dr Hannah Tipney (GSK) September 2012 Cranfield University GlaxoSmithKline Research & Development Ltd Cranfield Health Applied Bioinformatics MSc Thesis Academic Year 2011-12 Jessica Brothwood Druggable and biopharmable genome annotation pipeline development Supervisors: Dr Michael Cauchi (Cranfield University) Dr Hannah Tipney (GlaxoSmithKline) September 2012 This thesis is submitted in partial fulfilment of the requirements for the degree of Master of Science. © Cranfield University 2012. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder. Abstract The identification of proteins which could be potential targets for new pharmaceutical products is invaluable for the continued improvement people’s quality of life and expansion of available treatment options. In order to aid the discovery of new drug targets, predictions of every human gene likely to be exploitable by compounds and biotechnology were generated using open source tools and publicly available data. An automated pipeline was produced in order to minimise the effort required to reproduce, update and expand this work. In total, using various different prediction techniques, over 15,000 genes were predicted to code potential targets. An optimistic estimate of the druggable genome at 5,097 genes was produced. These genes contain one or more of the same Pfam protein domains as a drug target (a protein displaying significant activity with a phase four drug from ChEMBL database). The preliminary techniques explored here estimate the biopharmable genome to encompass between 3,169 and 8,117 genes. However, as they failed to identify many of the known approved targets, it is likely this is not an accurate representation. An easy to run, updated and expandable prediction pipeline, which annotates genes with a predicted druggable target class as well as a ChEMBL target class, if available, has been produced in Perl and implemented within GlaxoSmithKline. i Acknowledgements This dissertation would not have been possible without advice, guidance and support of Dr Hannah Tipney, to whom I am extremely grateful. I would also like to thank Peter Woollard for his recommendations and help with Perl and Dr Michael Cauchi for his advice and support and for his assistance in producing the ROC curves. I am obliged to Kieran Todd for his invaluable work on the biopharmable genome. I would also like to thank Dr David Michalovich, for information on drug target classes, and Stefan Senger, for his explanations of compound activities, as well as everyone else in the Computational Biology department at GlaxoSmithKline for their warm welcome and help. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115191, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution (www.imi.europa.eu). ii Table of Contents Abstract .............................................................................................................................. i Acknowledgements .......................................................................................................... ii List of Figures .................................................................................................................. vi List of Tables ................................................................................................................... ix Abbreviations ................................................................................................................... x 1 Introduction ............................................................................................................... 1 1.1 Drug Discovery and Development .................................................................... 1 1.1.1 Target Identification and Validation........................................................... 1 1.1.2 Lead Compound Selection and Optimisation ............................................. 3 1.1.3 Preclinical Testing ...................................................................................... 3 1.1.4 Clinical Trials ............................................................................................. 3 1.2 Challenges Facing the Pharmaceutical Industry ................................................ 5 1.3 Drug Types ........................................................................................................ 6 1.3.1 Estimates of Drug-likeness ......................................................................... 7 1.3.2 Small Molecular Drug Targets ................................................................... 9 1.3.3 Biological Therapeutics ............................................................................ 10 1.4 The Druggable and Biopharmable Genome .................................................... 11 1.4.1 Genomic Sequencing ................................................................................ 11 1.4.2 Determining Target Druggability ............................................................. 13 1.4.3 Known Drug Targets ................................................................................ 15 1.4.4 The Druggable Genome............................................................................ 16 1.4.5 The Secretome and Biopharmable Genome ............................................. 18 1.5 Strategy to New Drug Discovery ..................................................................... 20 2 Aims and Objectives ............................................................................................... 22 3 Materials and Methods ............................................................................................ 23 3.1 Programming Languages ................................................................................. 23 3.2 Mutual Resources ............................................................................................ 23 3.3 Chemically Tractable Resources and Filters ................................................... 23 3.4 Chemically Tractable Genome Outputs ........................................................... 26 3.5 Biopharmable Resources and Filters ............................................................... 28 3.6 Biopharmable Genome Outputs ....................................................................... 29 iii 3.7 Prediction Method Evaluation ......................................................................... 33 4 Results ..................................................................................................................... 34 4.1 Druggable Genome .......................................................................................... 35 4.1.1 Prediction of ChEMBL database small molecule targets ......................... 36 4.1.2 Prediction of DrugBank database small molecule targets ........................ 38 4.1.3 Druggable Predictions .............................................................................. 39 4.1.4 Sensitivity and Specificity of methods predicting ChEMBL small molecule targets....................................................................................................... 42 4.1.5 Sensitivity and Specificity of methods predicting DrugBank small molecule targets....................................................................................................... 43 4.1.6 Receiver operating characteristic curves for methods predicting small molecule targets....................................................................................................... 44 4.1.7 Druggable Predictive Power ..................................................................... 48 4.1.8 Hopkins and Groom comparison .............................................................. 48 4.1.8 Predicted Druggable Target Classes ......................................................... 50 4.2 Biopharmable Genome .................................................................................... 52 4.2.1 Prediction of ChEMBL database biotechnology targets .......................... 53 4.2.2 Prediction of DrugBank database biotechnology targets ......................... 54 4.2.3 Biopharmable Predictions ........................................................................ 55 4.2.4 Sensitivity and Specificity of methods predicting ChEMBL biotechnology targets .................................................................................................................. 56 4.2.5 Sensitivity and Specificity of methods predicting DrugBank biotechnology targets .............................................................................................. 57 4.2.6 Receiver operating characteristic curves for predicting targets of biotechnology .......................................................................................................... 58 4.2.7 Biopharmable Predictive Power ............................................................... 60 4.2.8 Predicted Biopharmable Target Classes ................................................... 61 4.3 Results Summary ............................................................................................
Load more

Recommended publications
  • Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane- Proximal Regions of VAMP2 and SNAP25
    bioRxiv preprint doi: https://doi.org/10.1101/849885; this version posted November 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Complexin suppresses spontaneous exocytosis by capturing the membrane- proximal regions of VAMP2 and SNAP25 Authors: J. Malsam1,6, S. Bärfuss1,6, T. Trimbuch2, F. Zarebidaki2, A.F.-P. Sonnen3,5, K. Wild1, A. Scheutzow1, I. Sinning1, J.A.G. Briggs3,4, C. Rosenmund2, and T.H. Söllner1,7,* Author Affiliations: 1Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. 2Neuroscience Research Center, Charité Universitätsmedizin Berlin, Chariteplatz 1, 10117 Berlin, Germany. 3European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany. 4MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK. 5Present address: Department of Pathology, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands 6These authors contributed equally 7Lead Contact *Correspondence: [email protected]. Summary The neuronal protein complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor
    [Show full text]
  • C-Terminal Domain of Mammalian Complexin-1 Localizes to Highly Curved Membranes
    C-terminal domain of mammalian complexin-1 localizes to highly curved membranes Jihong Gonga,b,c,d,1, Ying Laie,1, Xiaohong Lia,b,c,d, Mengxian Wanga,b,c,d, Jeremy Leitze,f, Yachong Hug, Yunxiang Zhange,UcheorB.Choie, Daniel Ciprianoe,f, Richard A. Pfuetznere,f, Thomas C. Südhofe,f, Xiaofei Yanga,b,c,d,2, Axel T. Brungere,f,h,i,j,2, and Jiajie Diaoe,f,2,3 aKey Laboratory of Cognitive Science, College of Biomedical Engineering, South-Central University for Nationalities, Wuhan 430074, China; bHubei Key Laboratory of Medical Information Analysis and Tumor Diagnosis & Treatment, College of Biomedical Engineering, South-Central University for Nationalities, Wuhan 430074, China; cLaboratory of Membrane Ion Channels and Medicine, College of Biomedical Engineering, South-Central University for Nationalities, Wuhan 430074, China; dCollege of Life Science, South-Central University for Nationalities, Wuhan 430074, China; eDepartment of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; fHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305; gCenter for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China; hDepartment of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305; iDepartment of Structural Biology, Stanford University, Stanford, CA 94305; and jDepartment of Photon Science, Stanford University, Stanford, CA 94305 Contributed by Axel T. Brunger, August 25, 2016 (sent for review June 21, 2016; reviewed by Jeremy S. Dittman and Erdem Karatekin) In presynaptic nerve terminals, complexin regulates spontaneous membranes via a membrane-binding motif (20, 21).
    [Show full text]
  • The Role of Complexin in Neurotransmitter Release
    THE ROLE OF COMPLEXIN IN NEUROTRANSMITTER RELEASE Neurons communicate using specialized con- Student Researcher: Joel Fernandez nections called synapses by releasing synaptic Mentor: Jeremy Dittman, Weill Cornell Medical College, New York, NY vesicles (SVs) containing neurotransmitters. How this process is regulated is not completely understood, although some of the key mole- cules have been identified. Complexins are a INTRODUCTION proteins in vivo. Furthermore, many family of protein found primarily in the synaptic mutants have already been nervous system of all animals. We investigated Chemical synapses play a critical identified (a complexin null mutant the role of complexin I (cpx-1) in the nematode role in the function of an animal's among them). C. elegans. We found that cpx-1 null mutants Using this organism, we tested paralyzed far quicker in aldicarb than wild- nervous system. They are the means type animals. Furthermore, the null mutants through which neurons transfer infor- whether complexin plays an inhibitory displayed severe locomotory defects; the null mation. The presynaptic terminal is or facilitating role in neurotransmitter mutants showed about a 70% decrease in pivotal in this exchange. It is responsible release. Furthermore, we wanted to body bends per 20 seconds. Introducing a for the regulated release of vesicles know if a null mutant can be rescued complexin GFP fusion protein into GABA by reintroducing complexin into the motor neurons alone did not rescue the containing neurotransmitters, the chem- mutants; these animals displayed the exact icals which relay messages across synap- animal. defects as the null mutants, suggesting that null ses. How this vital and rapid process is defects are independent of the GABA motor regulated is still a mystery, although neurons.
    [Show full text]
  • The Molecular Machinery of Neurotransmitter Release Nobel Lecture, 7 December 2013
    The Molecular Machinery of Neurotransmitter Release Nobel Lecture, 7 December 2013 by Thomas C. Südhof Dept. of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University, USA. 1. THE NEUROTRANSMITTER RELEASE ENIGMA Synapses have a long history in science. Synapses were frst functionally demon- strated by Emil duBois-Reymond (1818–1896), were morphologically identifed by classical neuroanatomists such as Rudolf von Kölliker (1817–1905) and San- tiago Ramon y Cajal (1852–1934), and named in 1897 by Michael Foster (1836– 1907). Although the chemical nature of synaptic transmission was already sug- gested by duBois-Reymond, it was long disputed because of its incredible speed. Over time, however, overwhelming evidence established that most synapses use chemical messengers called neurotransmitters, most notably with the pioneer- ing contributions by Otto Loewi (1873–1961), Henry Dale (1875–1968), Ulf von Euler (1905–1983), and Julius Axelrod (1912–2004). In parallel, arguably the most important advance to understanding how synapses work was provided by Bernard Katz (1911–2003), who elucidated the principal mechanism of syn- aptic transmission (Katz, 1969). Most initial studies on synapses were carried out on the neuromuscular junction, and central synapses have only come to the fore in recent decades. Here, major contributions by many scientists, including George Palade, Rodolfo Llinas, Chuck Stevens, Bert Sakmann, Eric Kandel, and Victor Whittaker, to name just a few, not only confrmed the principal results obtained in the neuromuscular junction by Katz, but also revealed that synapses 259 6490_Book.indb 259 11/4/14 2:29 PM 260 The Nobel Prizes exhibit an enormous diversity of properties as well as an unexpected capacity for plasticity.
    [Show full text]
  • Complexin II Plays a Positive Role in Ca -Triggered Exocytosis By
    Complexin II plays a positive role in Ca2؉-triggered exocytosis by facilitating vesicle priming Haijiang Caia,1, Kerstin Reimb, Frederique Varoqueauxb, Sompol Tapechuma,2, Kerstin Hillc,3, Jakob B. Sørensenc, Nils Broseb, and Robert H. Chowa,4 aDepartment of Physiology and Biophysics, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA 90089; bDepartment of Molecular Neurobiology and Center for the Molecular Physiology of the Brain, Max Planck Institute of Experimental Medicine, D-37075 Go¨ttingen, Germany; and cDepartment of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, D-37077 Go¨ttingen, Germany Communicated by Clay M. Armstrong, University of Pennsylvania, Philadelphia, PA, October 10, 2008 (received for review August 3, 2008) SNARE-mediated exocytosis is a multistage process central to II antibody into Aplysia buccal ganglia neurons stimulated neuro- synaptic transmission and hormone release. Complexins (CPXs) are transmitter release (14), and overexpression of CPX I or CPX II in small proteins that bind very rapidly and with a high affinity to the PC12 cells was reported to suppress acetylcholine (ACh) release SNARE core complex, where they have been proposed recently to (15). More recent studies showed that cell–cell fusion of cells that inhibit exocytosis by clamping the complex and inhibiting mem- express SNAREs facing extracellularly is inhibited by CPX (16–18) brane fusion. However, several other studies also suggest that and that overexpression of a synaptobrevin CPX fusion protein, CPXs are positive regulators of neurotransmitter release. Thus, which is thought to cause high presynaptic levels of CPX, inhibits whether CPXs are positive or negative regulators of exocytosis is synaptic transmission (19).
    [Show full text]
  • SNARE Complex Oligomerization by Synaphin/Complexin Is Essential for Synaptic Vesicle Exocytosis
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 104, 421±432, February 9, 2001, Copyright 2001 by Cell Press SNARE Complex Oligomerization by Synaphin/Complexin Is Essential for Synaptic Vesicle Exocytosis Hiroshi Tokumaru,*² Keiko Umayahara,*² al., 1994; Broadie et al., 1995; Sweeney et al., 1995). Lorenzo L. Pellegrini,²§ Toru Ishizuka,³ Reconstituting SNARE proteins into liposomes demon- Hideo Saisu,³ Heinrich Betz,²§ strates that these proteins can serve as a minimal molec- George J. Augustine,*²k and Teruo Abe²³ ular machinery to fuse membranes (Weber et al., 1998), *Department of Neurobiology though it is presently unclear whether SNAREs directly Duke University Medical Center mediate membrane fusion or act at a step that immedi- Box 3209 ately precedes fusion (Coorssen et al., 1998; Ungermann Durham, North Carolina 27710 et al., 1998). ² Marine Biological Laboratory In vitro, SNARE proteins bind to each other to form Woods Hole, Massachusetts 02543 binary and heterotrimeric complexes (Hayashi et al., ³ Department of Cellular Neurobiology 1994; Pevsner et al., 1994). The ternary SNARE complex Brain Research Institute is highly stable and resists denaturation by detergents Niigata University such as SDS (Hayashi et al., 1994). It consists of a parallel Niigata 951-8585 four-helix bundle containing one coiled-coil domain Japan from syntaxin, another from synaptobrevin 2, and two § Department of Neurochemistry from SNAP-25 (Sutton et al., 1998). Dissociation of the Max-Planck-Institute for Brain Research complex appears to require NSF, an ATPase that atta- 60528 Frankfurt ches to the SNARE complex via ␣-SNAP (SoÈ llner et al., Germany 1993a, 1993b).
    [Show full text]
  • Structural and Functional Study of SNARE Machinery in Neurotransmitter Release Shuang Song Iowa State University
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2013 Structural and functional study of SNARE machinery in neurotransmitter release Shuang Song Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons, Biology Commons, and the Biophysics Commons Recommended Citation Song, Shuang, "Structural and functional study of SNARE machinery in neurotransmitter release" (2013). Graduate Theses and Dissertations. 13082. https://lib.dr.iastate.edu/etd/13082 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Structural and functional study of SNARE machinery in neurotransmitter release by Shuang Song A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Molecular, Cellular, and Developmental Biology Program of Study Committee: Yeon-Kyun Shin, Major Professor Linda Ambrosio Edward Yu Alan DiSpirito Diane Bassham Iowa State University Ames, Iowa 2013 Copyright © Shuang Song, 2013. All rights reserved. ii TABLE OF CONTENTS ABSTRACT .............................................................................................................................
    [Show full text]
  • Subcloning and Expression of Complexin Isoforms Involved in Mast Cell Degranulation
    The University of Southern Mississippi The Aquila Digital Community Honors Theses Honors College Spring 5-2014 Subcloning and Expression of Complexin Isoforms Involved in Mast Cell Degranulation Cameron Blake King University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/honors_theses Part of the Cell and Developmental Biology Commons Recommended Citation King, Cameron Blake, "Subcloning and Expression of Complexin Isoforms Involved in Mast Cell Degranulation" (2014). Honors Theses. 226. https://aquila.usm.edu/honors_theses/226 This Honors College Thesis is brought to you for free and open access by the Honors College at The Aquila Digital Community. It has been accepted for inclusion in Honors Theses by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. The University of Southern Mississippi Subcloning and Expression of Complexin Isoforms Involved in Mast Cell Degranulation by Cameron King A Thesis Submitted to the Honors College of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in the Department of Biological Sciences May 2014 ii Approved by ______________________________ Hao Xu, Ph.D., Thesis Adviser Assistant Professor of Biology ______________________________ Shiao Wang, Ph.D., Interim Chair Department of Biological Sciences ________________________________________ David R. Davies, Ph.D., Dean Honors College iii Abstract Mast cells play an important role in the immune system by releasing chemicals such as chemokines and cytokines once they are stimulated. These products are released after stimulation by a process called mast cell degranulation. Mast cell degranulation is accomplished when vesicles containing the chemicals inside the mast cell fuse with the mast cell membrane via SNARE-mediated (Soluble NSF Attachment Protein Receptors) membrane fusion.
    [Show full text]
  • Complexin Stabilizes Newly Primed Synaptic Vesicles and Prevents Their Premature Fusion at the Mouse Calyx of Held Synapse
    8272 • The Journal of Neuroscience, May 27, 2015 • 35(21):8272–8290 Cellular/Molecular Complexin Stabilizes Newly Primed Synaptic Vesicles and Prevents Their Premature Fusion at the Mouse Calyx of Held Synapse X Shuwen Chang,1 Kerstin Reim,2 Meike Pedersen,1 XErwin Neher,1,3 Nils Brose,2,3 and Holger Taschenberger1,2,3 1Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, 37077 Go¨ttingen, Germany, 2Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Go¨ttingen, Germany, and 3DFG-Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain, 37073 Go¨ttingen, Germany Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in “clamping” SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca 2ϩ influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca 2ϩ channels and docked SV.
    [Show full text]
  • Competition Between Synaptotagmin 1 and Complexin for Snare
    COMPETITION BETWEEN SYNAPTOTAGMIN 1 AND COMPLEXIN FOR SNARE COMPLEX BINDING, CONTROLS FAST SYNAPTIC VESICLE EXOCYTOSIS APPROVED BY SUPERVISORY COMMITTEE Thomas C. Südhof, M.D., Advisor Jane Johnson, Ph.D., Committee Chair Jose Rizo-Rey, Ph.D. Leon Avery, Ph.D. Gang Yu, Ph.D. DEDICATION I would like to thank the members of my Graduate Committee and my parents. COMPETITION BETWEEN SYNAPTOTAGMIN 1 AND COMPLEXIN FOR SNARE COMPLEX BINDING, CONTROLS FAST SYNAPTIC VESICLE EXOCYTOSIS by JIONG TANG DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas May, 2007 Copyright by Jiong Tang, 2007 All Rights Reserved Copyright ACKNOWLEDGEMENTS First, I would like to thank my mentor, Dr. Thomas C. Südhof, who gave me enormous instructions and support during the past five years. What impressed me most is his passion and enthusiasm for science that have always encouraged me to pursue my goals. From him, I learned not only how to design and perform experiments, but also to address science in a systematic way and in different angles. It would have been impossible to finish my training and research work without him. I would like to give my thanks to all the people in Tom’s lab. They have always been friendly and generous to help me. Dr. Ok-Ho Shin, a bench mate for 5 years, gave me a lot of advice on details of experiments.
    [Show full text]
  • A Mechanism for Exocytotic Arrest by the Complexin C-Terminus
    RESEARCH ARTICLE A mechanism for exocytotic arrest by the Complexin C-terminus Mazen Makke1†, Maria Mantero Martinez1, Surya Gaya1, Yvonne Schwarz1, Walentina Frisch1, Lina Silva-Bermudez1, Martin Jung2, Ralf Mohrmann3, Madhurima Dhara1†*, Dieter Bruns1†* 1Institute for Physiology, Center of Integrative Physiology and Molecular Medicine, University of Saarland, Homburg, Germany; 2Institute for Medical Biochemistry and Molecular Biology, University of Saarland, Homburg, Germany; 3Institute for Physiology, Otto-von-Guericke University, Magdeburg, Germany Abstract ComplexinII (CpxII) inhibits non-synchronized vesicle fusion, but the underlying mechanisms have remained unclear. Here, we provide evidence that the far C-terminal domain (CTD) of CpxII interferes with SNARE assembly, thereby arresting tonic exocytosis. Acute infusion of a CTD-derived peptide into mouse chromaffin cells enhances synchronous release by diminishing premature vesicle fusion like full-length CpxII, indicating a direct, inhibitory function of the CTD that sets the magnitude of the primed vesicle pool. We describe a high degree of structural similarity between the CpxII CTD and the SNAP25-SN1 domain (C-terminal half) and show that the CTD peptide lowers the rate of SDS-resistant SNARE complex formation in vitro. Moreover, corresponding CpxII:SNAP25 chimeras do restore complexin’s function and even ‘superclamp’ tonic secretion. Collectively, these results support a so far unrecognized clamping mechanism wherein the CpxII C-terminus hinders spontaneous SNARE complex assembly, enabling the build- up of a release-ready pool of vesicles for synchronized Ca2+-triggered exocytosis. *For correspondence: DOI: https://doi.org/10.7554/eLife.38981.001 [email protected] (MD); [email protected] (DB) †These authors contributed equally to this work Introduction The accumulation of vesicles in a release-ready state is essential for fast transmitter release from Competing interests: The secretory cells.
    [Show full text]
  • The Carboxy-Terminal Domain of Complexin I Stimulates Liposome Fusion
    The carboxy-terminal domain of complexin I stimulates liposome fusion Jo¨ rg Malsam1, Florian Seiler1, Yvette Schollmeier, Patricia Rusu, Jean Michel Krause, and Thomas H. So¨ llner2 Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany Communicated by James E. Rothman, Columbia University Medical Center, New York, NY, December 17, 2008 (received for review August 8, 2008) Regulated exocytosis requires tight coupling of the membrane CpxI/II double- and CpxI/II/III triple-knockout mice (15). Other fusion machinery to a triggering signal and a fast response time. studies, including complexin overexpression, in vitro liposome Complexins are part of this regulation and, together with synap- fusion assays, and cell-cell fusion experiments, indicate that totagmins, control calcium-dependent exocytosis. Stimulatory and complexins can also function as fusion clamps (16–19). The inhibitory functions have been reported for complexins. To test if clamp is released by synaptotagmin 1 in a calcium-dependent complexins directly affect membrane fusion, we analyzed the 4 manner. Recent analysis revealed that distinct domains of com- known mammalian complexin isoforms in a reconstituted fusion plexins differently control neurotransmitter release and mem- assay. In contrast to complexin III (CpxIII) and CpxIV, CpxI and CpxII brane fusion. The amino-terminal 26 amino acids of CpxI stimulated soluble N-ethylmaleimide-sensitive factor attachment contain a stimulatory function followed by an inhibitory ␣-helix protein receptor (SNARE)-pin assembly and membrane fusion. This (amino acids 29–48) and the SNARE binding helix (amino acids stimulatory effect required a preincubation at low temperature 48–70) (20, 21). Although low-affinity binding of CpxI to and was specific for neuronal t-SNAREs.
    [Show full text]