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Understanding the Cellular Role of Prolyl Oligopeptidase

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Understanding the Cellular Role of Prolyl Oligopeptidase Understanding The Cellular Role Of Prolyl Oligopeptidase Karina McQuillan Supervisor: Prof. Adrian Harwood Cardiff University School of Biosciences And University College London MRC-LMCB PhD Thesis Submission date: May 9th 2008 UMI Number: U584306 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U584306 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Form: PG/R/06/09 CARDIFF UNIVERSITY PRIFYSGOL CaeRDY[§> CANDIDATE’S ID NUMBER 51905 CANDIDATE’S LAST NAME Mcquillan CANDIDATE’S FIRST NAME(S) KARINA DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed (candidate) Date . STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree o f . P U ?............. MCh, Md, MPhil, PhD etc, as appropriate) Signed (candidate) Date........ STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by footnotes giving explicit references. S ig n ed........................................................ (candidate) DateJ . Z l Z l z a o z . STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ... w , ................................... (candidate) Date......... STATEMENT 4 - BAR ON ACCESS APPROVED I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after exoirv of a bar on access approved bv the Graduate Development Committee. S ig n ed •..............................................(candidate) Date ..... PG/R/06/10 CARDIFF UNIVERSITY PRIFYSGOL Ca ERDY j§> STUDENT ID NUMBER: 51905 SUMMARY OF THESIS: Prolyl oligopeptidase (PO, Prolyl Endopeptidase, PE, PEP) is a member of the serine protease family and shares a good structural homology with other a/p hydrolase fold enzymes. PO specifically cleaves oligopeptides after a proline residue. The PO homologue, DpoA, was identified in the cellular slime mould Dictyostelium Discoideum following a lithium screen. Loss of DpoA confers resistance to both lithium and valproic acid (VPA), through regulation of inositol phosphate pathways. The intracellular substrate and cellular role of PO remain uncertain. However, it is known to inhibit dephosphorylation of the higher order inositol phosphates IP 5 and IP 4 to IP 3 , a reaction catalysed by Multiple Inositol Polyphosphate Phosphatase (MIPP), by an unknown mechanism. In this study it has been determined that DpoA is highly similar to the mammalian enzyme. It has also been demonstrated that while some variation was observed at the sequence level there is clear homology around the active site and the catalytic triad is conserved. Enzyme activity and inhibition studies reveal similar KM and Kf values in the presence of known specific inhibitors. Thus making it a relevant model for the mammalian enzyme. Characterisation of the direct effects of three mood stabilisers on PO activity has revealed no effect of lithium, carbamazapine or valproic acid at therapeutic concentrations. However, VPA while exerting no clear effect in vivo was able to inhibit PO in vitro at increased concentrations. This inhibition was also seen using VPA analogues lacking either the carboxylic acid domain or branching structure. Significant to the elusive intracellular role for PO is the identification of a clear inhibitory effect of PO on MIPP activity in vitro. While identification of PO presence within the nucleus as well as distributed throughout the cytoplasm may also be significant to its role in inositol signalling. Acknowledgements I would like to say a huge thank you to a number of people whose support both personal and scientific has proved invaluable throughout this PhD. A special thanks to my supervisor, Adrian Harwood, who has been constantly encouraging and optimistic, his interest and enthusiasm has been inspiring. I would also like to thank in particular all the members of the Harwood lab who have become like an extended family. They have proved an invaluable source of knowledge, encouragement, support, friendship, spare rooms and more! All of which I am extremely grateful for. I must also thank Steven Nurrish and the members of the Nurrish lab who adopted me in London during the transition from London to Cardiff and my fellow PhD students who were and remain a constant source of friendship and support. Throughout my PhD I have been extremely lucky to have the love and support of many good friends, who have pulled me through the tough times and celebrated every success. Thank You! I hope you know how much I appreciate it. To my family, especially my parents, who have supported me constantly throughout this whole experience, this is for you, I couldn’t have done it without you. Contents Acknowledgements............................................................................................................................................. i Abstract............................................................................................................................................................. vii Abbreviations..................................................................................................................................................viii 1 INTRODUCTION.......................................................................................1-1 1.1 Introduction...........................................................................................................................................1-2 1.2 Prolyl Oiigopeptidase.......................................................................................................................... 1-4 1.2.1 Enzymes Related To PO 1 -4 1.2.1.1 The Serine Protease Family 1 -4 1.2.1.2 Proline Specific Peptidases 1 -4 1.2.1.3 The Prolyl Oiigopeptidase Family 1 -9 1.2.2 Structure Of Prolyl Oiigopeptidase 1 -9 1.2.2.1 Identification Of PO.............................................................................................................. 1-9 1.2.2.2 Catalytic Domain................................................................................................................. 1-11 1.2.2.3 (3-Propeller Domain............................................................................................................. 1-12 1.2.2.4 Variations..............................................................................................................................1-17 1.2.3 Mechanism Of PO Catalysis...................................................................................................... 1-18 1.2.3.1 General Serine Protease Catalysis..................................................................................... 1-19 1.2.3.2 Catalytic Triad...................................................................................................................... 1-20 1.2.3.3 pH Dependence.................................................................................................................... 1-22 1.2.3.4 Ionic Effect............................................................................................................................1-23 1.2.3.5 Substrate Binding................................................................................................................. 1 -24 1.2.3.6 Inhibitors ...............................................................................................................................1-28 1.2.4 Localisation And Distribution....................................................................................................1 -34 1.2.4.1 Tissue Distribution...............................................................................................................1-34 1.2.4.2 Intracellular Localisation.................................................................................................... 1 -34 1.2.5 Role of Prolyl Oiigopeptidase....................................................................................................1-37 1.2.5.1 Degradation/regulation of bioactive peptides...................................................................1-37 1.2.5.2 General Proteolysis.............................................................................................................. 1 -40 1.2.5.3 Neuroprotection...................................................................................................................1 -40 1.2.5.4 Inositol Signalling................................................................................................................1-42 1.2.5.5 Mood Disorders...................................................................................................................1-43 1.2.5.6 Alzheimers Disease.............................................................................................................1
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