Structural and functional characterization of pattern recognition receptors of the innate immune system [Elektronische Ressource] / Diana Angela Pippig

Diana Angela Pippig

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München



Structural and Functional Characterization
of Pattern Recognition Receptors of the
Innate Immune System


Diana Angela Pippig
aus
Plauen


München, 2010
Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Herrn Prof. Dr. Karl-Peter Hopfner betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfsmittel erarbeitet.



München, am 17.08.2010




...........................................
Diana Pippig





Dissertation eingereicht am: 17.08.2010
1. Gutachter: Prof. Dr. Karl-Peter Hopfner
2. Gutachter: Prof. Dr. Elena Conti
Mündliche Prüfung am: 19.10.2010
This thesis has been prepared from February 2007 to August 2010 in the laboratory of
Professor Dr. Karl-Peter Hopfner at the Gene Center of the Ludwig-Maximilians-University
of Munich (LMU).



Parts of this thesis have been published:

Pippig, D. A., Hellmuth, J. C., Cui, S., Kirchhofer, A., Lammens, K., Lammens, A., Schmidt,
A., Rothenfusser, S. and Hopfner, K. P. (2009). "The regulatory domain of the RIG-I family
ATPase LGP2 senses double-stranded RNA." Nucleic Acids Res 37(6): 2014-2025.


Parts of this thesis have been presented at the following international conferences:

Posters displaying the “Structural and Functional Characterization of RIG-I Like Receptors”
were presented at:

RNA 2008: Thirteenth Annual Meeting of the RNA Society; Berlin, Germany, August 2008

EMBO Conference Series: Helicases and NTP-Driven Nucleic Acid Motors – Structure,
Function, Mechanism and Roles in Human Disease; Les Diablerets, Switzerland, June 2009
































„Dass ich erkenne, was die Welt im Innersten zusammenhält“
Goethe, Faust I Table of Contents
1. Introduction ........................................................................................................................... 1
1.1. The Immune System .......................................................................................................... 1
1.1.1. Surface Barriers as First Primitive Stage of Immune Defense ... 1
1.1.2. Innate Immunity – The Second Stage .......... 2
1.1.3. Adaptive Immunity – A Third Stage in Vertebrate Immunity ..................................... 3
1.2. Pattern Recognition Receptors of the Innate Immune System .......... 4
1.2.1. Nucleic Acid Responsive PRRs ................................................................................... 6
1.2.2. PRR’s Knowing Friend from Foe ............... 8
1.3. RIG-I-like Receptors ......................................................................................................... 9
1.3.1. LGP2 – The Odd Member of the RLR Family .......................... 11
1.3.2. RD – Regulatory or Repressor Domain? .. 11
1.4. Inflammasomes – Stress and Infection Inducible Multi Protein Platforms .................... 14
1.4.1. Types of Inflammasomes ........................................................................................... 14
1.4.2. RLR Signaling and Inflammasomes – a Possible Intersection ................................. 16
1.4.3. The AIM2 Inflammasome – a Cytosolic DNA sensor ............... 16
1.4.4. AIM2 and the Interferon-Inducible p200 Protein Family ........ 17
1.4.5. ASC – A Versatile Adaptor in Inflammation and Innate Immunity .......................... 19
1.5. Objectives ....................................................................................................................... 21
2. Material and Methods ......... 22
2.1. Materials ......................... 22
2.1.1. Chemicals.................................................................................................................. 22
2.1.2. Media and Supplements ............................ 22
2.1.3. Bacterial Strains ....................................................................................................... 23
2.1.4. Plasmids .................... 23
2.1.5. Cloning and Mutagenesis Primer ............. 24
2.1.6. RNA and DNA Oligonucleotides .............................................................................. 25
2.2. Methods .......................................................... 27
2.2.1. Molecular Biological Methods ................. 27
2.2.1.1. Molecular Cloning .............................................................................................. 27
2.2.1.2. Site Directed Mutagenesis .................. 28
2.2.1.3. Transformation ................................... 28
2.2.1.4. Plasmid Preparation ........................................................................................... 28
2.2.1.5. Bacmid Preparation ............................ 28
2.2.2. Protein Biochemical Methods ................... 29
2.2.2.1. Protein Expression in Insect Cells ...................................................................... 29
2.2.2.2. Protein Expression in E.coli ............... 29 Table of Contents
2.2.2.3. Protein Purification ............................................................................................ 30
2.2.2.3.1. Glutathione-S-Transferase Affinity Chromatography .................................. 30
2.2.2.3.2. Nickel Affinity Chromatography ... 32
2.2.2.3.3. Heparin Affinity Chromatography ................................ 32
2.2.2.3.4. Dialysis .......................................................................... 32
2.2.2.3.5. An- and Cation Exchange Chromatography ................. 32
2.2.2.3.6. Size Exclusion Chromatography (SEC or Gelfiltration) ............................... 33
2.2.3. Crystallographic Methods ........................................................................................ 33
2.2.3.1. Crystallization ..................................... 33
2.2.3.2. Crystallographic Data Collection of LGP2 RD .................. 34
2.2.3.3. Structure Determination of LGP2 RD ................................ 34
2.2.3.3.1. Theoretical Background ................................................................................ 34
2.2.3.3.2. Solution of the LGP2 RD Structure ............................... 35
2.2.4. RNA and DNA Biochemistry ..................... 36
2.2.4.1. RNA Preparation ................................................................................................ 36
2.2.4.2. Ribozymes and DNAzymes .................. 37
2.2.5. Biochemical Assays... 37
2.2.5.1. Fluorescence Anisotropy Measurements ............................................................ 37
2.2.5.2. Electrophoretic Mobility Shift Assays ................................. 38
2.2.5.3. Pulldown Assays ................................. 38
2.2.5.4. Western Blots and Immunostaining .................................... 39
2.2.6. Bioinformatic Methods ............................. 39
2.2.6.1. Sequence Alignments .......................................................................................... 39
2.2.6.2. Calculation of Protein Parameters ..................................... 39
2.2.6.3. Structure Visualization and Analysis .. 39
2.2.6.4. Protein Profile Search ........................................................................................ 40
2.2.6.5. Structural Homology Modeling .......... 40
2.2.6.6. Secondary Structure Predictions ........................................................................ 40
2.2.7. Analytical Methods ................................... 40
2.2.7.1. Mass Spectrometry .............................................................. 40
2.2.7.2. Edman-Sequencing ............................. 40
3. LGP2 – Results .................................................................................................................... 41
3.1. Full Length LGP2 ........... 41
3.2. The Regulatory Domain of LGP2 ................... 42
3.2.1. Constructs and Purification ...................................................................................... 42
3.2.2. Crystallization and Structure Determination of LGP2 RD ...................................... 43 Table of Contents
3.2.3. Overall Structure ...................................................................................................... 44
3.2.4. Comparison of LGP2 RD to RIG-I and MDA5 RDs ................. 47
3.2.5. LGP2 RD Binds to dsRNA in a 5’-Triphosphate Independent Manner .................... 50
3.2.6. The dsRNA Binding Site of LGP2 ............................................................................. 52
3.2.6.1. Study of LGP2 RD’s RNA Interaction by Fluorescence Anisotropy ................... 53
3.2.6.2. Electrophoretic Mobility Shift Assays of LGP2 RD – RNA complexes .............. 54
3.3. RD – RNA Complex Crystallization Attempts ....................................