Characterization of Interleukin-33 and the IL-33 receptor complex [Elektronische Ressource] / vorgelegt von Shafaqat Ali

Ali Mmadi, Shafaqat

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Characterization of Interleukin-33 and
the IL-33 Receptor Complex

Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
-Dr. rer. nat.-




Immunologie
des Fachbereiches Biologie und Chemie
der Justus-Liebig-Universität Gießen








vorlegt von
Shafaqat Ali
(M.Phil)

aus
Sialkot, Pakistan


Gießen, Juli 2009i





















Dekan
Prof. Dr. Volkmar Wolters

Gutachter
Prof. Dr. Michael U. Martin
Immunologie, Justus-Liebig-Universität Gießen

Prof. Dr. Alfred M. Pingoud
Institut für Biochemie, Justus-Liebig-Universität Gießen ii
Erklärungen
Hiermit versichere ich, die vorliegende Arbeit selbständig verfasst und keine andere
als die angegebenen Hilfsmittel benutzt zu haben. Stellen, die ich anderen Arbeiten
und Veröffentlichungen dem Wortlaut oder Sinn entsprechend entnommen habe,
sind durch Quellenangaben gekennzeichnet.


Shafaqat Ali
Gießen, den 27 Juli 2009




iii






























Dedicated to my parents
iv
Table of Contents

List of Figures....................................................................................................................................... vi
List of Tables ....................................................................................................................................... vii
List of abbreviations .......................................................................................................................... viii
Summary ............................................................................................................................................ 1
Zusammenfassung ........................................................................................................................ 3
1. Introduction.... 5
1.1. Biological activities of IL-1 family cytokines............................................................................... 5
1.2. Expression, processing and release of IL-1 family cytokines................................................... 7
1.2.1. Expression............................................................................................................................ 7
1.2.2. Processing 8
1.2.3. Release................. 8
1.3. The interleukin-1 receptor family.................................................................................................. 9
1.3.1. Regulation of the biological activity of IL-1 like cytokines............................................ 12
1.3.2. Signaling of the IL-1 family: the MyD88/IRAK/TRAF6 module....................................... 13
1.3.2.1. IRAK recruitment to the receptor complex .................................................................... 13
1.3.2.2. IRAK phosphorylation and departure from the receptor complex ................................. 14
1.3.2.3. Formation of the IRAK/TRAF6 signalosome ................................................................. 14
1.3.2.4. Activation of NF- κB and JNK/p38 MAPKs..................................................................... 14
1.4. Aims of the study ......................................................................................................................... 17
2. Materials and Methods ........................................................................................................... 18
2.1. Mammalian cell culture................................................................................................................ 18
2.2. Transient transfections................................................................................................................ 19
2.3. Cytokines and other recombinant proteins............................................................................... 20
2.4. Measurement of cytokine production ........................................................................................ 21
2.5. MTT assay / D10 assay ................................................................................................................ 21
2.6. Reporter gene assay.................................................................................................................... 22
2.7. Measurement of protein concentration from cell lysate .......................................................... 23
2.8. Western blotting ........................................................................................................................... 23
2.9. Immunoprecipitations.................................................................................................................. 25
2.10. Co-immunoprecipitation and Western blotting....................................................................... 26
2.11. Interaction of sAcP and sIL-33R α............................................................................................. 26
2.12. Measurement of signaling pathways ....................................................................................... 27
2.12.1. cJun-N-terminal kinase (JNK) assay.............................................................................. 27
2.12.2. IRAK-1 autophosphorylation activity............................................................................. 27
2.12.3. Measurement of phosphorylation and degradation of I κB α........................................ 28
2.13. Heat inactivation of IL-33........................................................................................................... 28
2.14. Pull-down assay............... 28
2.15. In vitro caspases cleavage assays........................................................................................... 28
2.16. Cleavage of IL-33 by apoptotic U937 cell lysate ..................................................................... 29
2.17. Nuclear translocation studies................................................................................................... 29
2.18. RT-PCR analysis......................................................................................................................... 29
2.18.1. RNA isolation.......... 29
2.18.2. cDNA synthesis................................................................................................................ 30
2.18.3. PCR amplification ............................................................................................................ 30
2.19. Relative quantification of gene expression............................................................................. 32
2.20. Expression plasmids........... 33
2.21. DNA isolation and purification....... 40
2.22. DNA digestion with restriction endonucleases....................................................................... 40
2.23. Agarose gel electrophoresis..................................................................................................... 41
2.24. Ligation........................................................................................................................................ 41
2.25. Bacterial transformations.......................................................................................................... 41
2.25.1. Preparation of competent cells ...................................................................................... 41
2.25.2. Transformation by heat shock........................................................................................ 42
2.26. Microarray analysis.............. 42
2.27. Radiolabelling of IL-33 and IL-1 α 43
2.27.1. Preparation of column..................................................................................................... 43
2.27.2. Iodination of IL-33 and IL-1 α ........................................................................................... 43
2.28. Crosslinking of IL-1 α and IL-33 to respective receptors........................................................ 44 v
2.29. Saturation and competition studies ......................................................................................... 44
2.30. Statistical analysis ..................................................................................................................... 45
3. Results .......................................................................................................................................... 46
3.1. Characterization of the IL-33 receptor complex........................................................................ 46
3.1.1. Cloning of the IL-33 receptor α-chain .............................................................................. 46
3.1.2. Identification of IL-33 receptor β............................................................................ 47
3.1.2.1. Murine EL-4 D6/76 cells do not respond to IL-33 due to the lack of IL-1RAcP ............ 48
3.1.2.2. IL-1RA