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Granzyme H: a Novel Cell-Death-Inducing Serine Protease

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Granzyme H: a Novel Cell-Death-Inducing Serine Protease ________________________________________________________ Granzyme H: A novel cell-death-inducing serine protease ________________________________________________________ Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie der Ludwig-Maximilians-Universität München Vorgelegt von Edward Fellows aus Wesel München 2007 Eingereicht am: 8.9.2007................................................................................................................... Mitglieder der Promotionskommission: Erstgutachter: Prof. Dr. Elisabeth Weiß Zweitgutachter: Prof. Dr. Charles David Tag des Promotionskolloquiums: 4.12.2007..................................................................................... Doktorurkunde ausgehändigt am: .................................................................................................... Die Versuche zur vorgelegten Dissertation wurden in der Zeit vom April 2003 bis März 2007 in der Arbeitsgruppe von Dr. med. habil. Dieter Jenne, Abteilung für Neuroimmunologie am Max-Planck-Institut für Neurobiologie in Martinsried bei München durchgeführt. Hiermit erkläre ich, daß ich diese Arbiet selbständig und nur unter Verwendung der angegebenen Quellen und Hilfsmittel angefertigt habe. Martinsried, den 5. September 2007 Dedicated to my mother Patricia and my father Leslie A macrophage engulfes the apoptotic bodies of a diseased cell in the final stages of apoptosis…. U.S. National Library of Medicine Acknowledgements I would like to thank, primarily, PD Dr. Dieter Jenne, Dr. Florian Kurschus and Prof. Hartmut Wekerle for making this thesis, here at the Max-Planck-Institute of Neurobiology, possible. I am particularly fortunate to have been supervised and mentored by my boss, Dieter, and my colleague Florian. I am extremely grateful to Dieter for accepting me into his lab and for his continual guidance, support and advice throughout this thesis. Dieter, you are a master of your craft and a true fountain of knowledge! I also greatly acknowledge Florian. I have benefited tremendously from planning, conducting and discussing research with him; I thank him for his patience! Florian, cheers for being a most triumphant colleague but also a good friend. I am also grateful to Prof. Hartmut Wekerle for hosting me in the department of Neuroimmunology and, of course, for his continual interest in my PhD project. I express my gratitude to Prof. Elisabeth Weiß for accepting to act as my principal supervisor at the biological faculty of the Ludwig Maximilian University here in Munich. Along with Prof Weiß, I also kindly acknowledge the members of my thesis examination board from the Ludwig Maximilian University, namely Prof. Charles David, Prof. Hugo Scheer, Prof. Heinrich Leonhardt, Prof. Michael Schleicher and Prof. Michael Boshart. I would also like to thank the members of my thesis committee here at the Max-Plank-Institute, which included Dr. Klaus Dornmair and Dr. Uwe Jacobe, for their helpful discussions and ideas. I am also thankful to my co-authors. I am extremely grateful to Shirley Gil-Parrado who so willingly offered me her help and experience in the study of caspase activation. Shirley, the good times at Tiers will be remembered! Additionally, I am particularly grateful to Felipe Andrade; I thank him for a successful collaboration but also for widening my interests in the field of granzyme biology. During my time here, I have benefited greatly from my fellow students and colleagues. I thank each and every one of them for making my experience in, and outside, the department a memorable one. To those of you from the “Jenne group”, namely Elisabeth Stegmann (alias “Lisa Minnelli”), Angelika Kuhl (alias “Ange”), Kai Kessenbrock (alias “K.K”) and until recently Florian Kurschus (alias “Carlos”), it has been an pleasure working, and laughing, alongside you. In particular, I am indebted to Kai, not only for providing me with support and stimulating discussions during my thesis, but also for his invaluable friendship. Cheers mate for an unforgettable time here in Bavaria; Servus, and be true to the dunkel stuff! Very special thanks also go to some very special friends, namely all the gang from the European School and the Nuneham Courtney countryside mansion; you know who you are! Last, but not least, I thank my dear friends Sophie, Adrian, Simon and Philipp. Needless to say, this entire exercise would have been an insurmountable task if it were not for my loving family. To my parents, Patricia and Leslie, I thank you for your continual input into my education; thank you for your consistant interest and encouragement but also for your critical advice. Above all, I thank you both for always being there for me. To my sisters, Rebecca and Amelia, I thank you for your love and support. Finally, I dearly thank my fiancée Denitsa for her unceasing love and care throughout this entire endeavour. Content ____________________________________________________________________________ Table of content I Summary 1 II Introduction 3 II.1 Granzymes 3 Definition 3 II.1.1 Human and mouse granzymes: genetic organization 3 Nomenclature 3 Chromosome locations: family clusters and enzyme activities 3 GzmB, GzmH and murine homologues 5 II.1.2 Granzyme synthesis and storage 5 Proteolytic processing in three steps 5 From zymogen to active serine protease, an example chymotrypsin 6 Secretory granules store subdued active granzymes 7 II.1.3 Structural characteristics of granzymes 7 II.1.4 Enzymes specificity of granzymes 8 Serine 195 of serine proteases 8 The Schechter and Berger nomenclature 9 Enzyme specificity 9 II.2 Cytotoxic effector cells and granzyme cytotoxicity 11 The granule exocytosis model 11 Granzyme delivery: where does perforin open the door to death? 13 Granzymes: natural born killers 15 Granzyme B 16 Granzyme A 16 Orphan granzymes 17 II.3 Granyzme B and its multiple apoptogenic substrates 19 The caspase-dependent cell death pathway 19 The caspase-independent cell death pathway 20 II.4 Granzyme H 22 II.5 Granzymes and antiviral strategies 24 I Content ____________________________________________________________________________ II.6 In vitro granzyme delivery by alternative pore-forming proteins 25 Bacterial streptolysin O (SLO) 25 Mutant streptolysin O 25 Bacterial Streptolysin O versus human perforin 25 II.7 Aim 26 III Materials & Methods 27 III.1 Cloning in plasmid vectors 27 Plasmid DNA purification from E.coli 27 DNA amplification 27 DNA restriction digestion 28 Agarose gel electrophoresis 28 DNA purification form agarose gels 29 DNA concentration and purity determination 29 DNA fragment ligation 29 Construction of expression plasmids 30 Expression constructs 30 DNA sequencing 31 III.2 DNA transformation 31 Preparation of CaCl2 competent cells 31 Transformation of CaCl2 competent cells 31 III.3 Recombinant protein expression in E.coli 32 The pET expression system 32 Inclusion body (IB) isolation 34 Solubilization of inclusion bodies 35 Renaturation of IB proteins 36 FPLC protein purification 38 Granzyme activation: processing by cathepsin C 39 III.4 Protein separation 41 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 41 III.5 Protein Detection 42 Silver nitrate staining of proteins after SDS-PAGE 42 II Content ____________________________________________________________________________ SDS-polyacrylamide gel Coomassie-blue staining 42 Western blot analysis: protein detection using chemiluminesence 43 Spectrophotometric determination of protein concentration 44 Colorimetric determination of protein determination 45 III.6 Enzymatic tests 45 Recombinant granzyme H activity assays 45 Recombinant granzyme H inhibition assays 46 III.7 Apoptosis assays 47 Culture of target cells 47 III.7.1 Changes in membrane integrity 47 FITC-annexin V and propidium iodide staining 47 Trypan blue staining 48 III.7.2 Caspase activation 49 Purified recombinant procaspases as potential substrates 49 Caspase activity in living cells 49 DNA laddering 50 Inhibitor of caspase activated DNase (ICAD) cleavage 50 III.7.3 Mitochondrial damage 51 Purified recombinant Bcl-2 family protein Bid 51 Mitochondrial transmembrane potential loss 51 Reactive oxygen species (ROS) production 52 Cytochrome c release 53 III.7.4 Characterization of monoclonal GzmH antibodies 53 III.7.5 Isolation of peripheral blood leukocytes subsets 54 Peripheral blood leukocyte subset isolation 54 Natural killer (NK) cell isolation 54 III.7.6 NK cell cytotoxicity and granzyme release 55 NK cell killing of target cells 55 GzmH release from NK cells 55 III.7.7 Cellular morphology and nuclear fragmentation 56 Apoptotic nuclei 56 Nuclear fragmentation 56 3H-thymidine release assay 57 III Content ____________________________________________________________________________ IV Results 58 IV.1 Construction of the GzmH plasmids 58 pET24c(+)MKH6GzmH 58 S195A pET24c(+)MKH6GzmH 58 IV.2 Production of GzmH and variants from inclusion body material 59 GzmH expression yields high amounts of pure IB material 59 Interchain disulphide bond rich GzmH IB are efficiently solubilized 60 Renaturation and dialysis of GzmH results in correctly folded protein 61 GzmH is efficiently purified via ion exchange FPLC 63 Pure, converted GzmH variants are stored in physiological conditions 64 Difference in yields between active and inactive GzmH 65 IV.3 GzmH activity 65 GzmH has chymotrypsin-like (chymase) activity 65 GzmH cleaves AMC based substrates 66 IV.4 GzmH inhibition 68 GzmH is efficiently inhibited by the general protease inhibitor DCI 68 IV.5
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