Granzyme H [Elektronische Ressource] : a novel cell death inducing serine protease / vorgelegt von Edward Fellows
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Granzyme H [Elektronische Ressource] : a novel cell death inducing serine protease / vorgelegt von Edward Fellows

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________________________________________________________ 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.

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Publié par
Publié le 01 janvier 2007
Nombre de lectures 46
Langue Deutsch
Poids de l'ouvrage 8 Mo

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________________________________________________________


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 Sumary 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
Granzyme delivery: where does perforin open the door to death? 13 es: natural born killers 15
Granzyme B 16 e A
Orphan granzymes 17
II.3 Granyzme B and its multiple apoptogenic substrates 19
The caspase-dependent cell death pathway
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)
Mutant streptolysin O 25
Bacterial Streptolysin O versus human perforin
II.7 Aim 26

III Materials & Methods 27
III.1 Cloning in plasmid vectors
Plasmid DNA purification from E.coli 27
DNA amplification
DNA restriction digestion 28
Agarose gel electrophoresis
DNA purification form agarose gels 29
DNA concentration and purity determination
DNA fragment ligation 29
Construction of expression plasmids 30
Expression constructs
DNA sequencing 31
III.2 DNA transformation
Preparation of CaCl competent cells 31 2
Transformation of CaCl competent cells 2
III.3 Recombinant protein expression in E.coli 32
The pET expression system
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)
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
Recombinant granzyme H activity assays 45
Recombe H inhibition assays 46
III.7 Apoptosis assays 47
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