N-terminal regulatory domains of phosphodiesterases 1, 4, 5 and 10 examined with an adenylyl cyclase as a reporter [Elektronische Ressource] / vorgelegt von Ana Banjac

Schutz11

Sujets

• Naturwissenschaften

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2011
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

N-terminal Regulatory Domains of Phosphodiesterases 1,
4, 5 and 10 examined with
an Adenylyl Cyclase as a Reporter







Dissertation
der Mathematisch-Naturwissenschaftlichen Fakultät
der Eberhard Karls Universität Tübingen
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)






vorgelegt von
Ana Banjac




Tübingen
2011































Tag der mündlichen Qualifikation: 10. 3. 2011
Dekan: Prof. Dr. Wolfgang Rosenstiel
1. Berichterstatter: Prof. Dr. Joachim E. Schultz
2. Berichterstatter: Prof. Dr. Peter Ruth Acknowledgement



The experimental part of this thesis work was done within the period from November 2005 to
July 2010 under the supervision of Prof. Dr. J. E. Schultz at the Pharmaceutical Institute of
Tübingen University.
I would like to thank Prof. Dr. J. E. Schultz for his support and encouragement as well as for
the beneficial scientific discussions. His kind support and guidance have been of great value
in this study.
I am grateful to Prof. Dr. Peter Ruth for taking part in the evaluation of my thesis.
I would like to show my gratitude to Prof. Dr. Klaus Hantke and Priv. Doz. Dr. Martina Düfer
for participation in the thesis examination.
Prof. Dr. Frank Böckler and Markus Zimmermann, I want to thank you for your valuable
suggestions.
I appreciate the help of Ursula Kurz and Anita Schultz for cloning and training me on
handling different methods. Special thanks to Karina Hofbauer, Sandra Bruder and Iman
Mansi for the interest they showed at the beginning of my work in the lab. I would also like to
thank Jürgen Linder for his detailed and constructive comments.
And not to forget my lab colleagues Laura García Mondéjar, Kajal Kanchan, Karin Winkler
and Janani Natarajan for creating a nice working atmosphere and very interesting discussions.

My special thanks to my family for their patience and support during these five years.
Without their encouragement and understanding it would have been impossible for me to
finish this work.

















Content
Content
Content ........................................................................................................................................I 
Abbreviations ................................................................................................... III 
1  Introduction ......................................................................... 1 
1.1  Signal transduction ..................................................... 1 
1.1.1  Cyclic nucleotides as second messengers ............................................ 2 
1.2  Phosphodiesterases .............................................................................. 2 
1.2.1  Catalytic Domain Structures ........................................................ 2 
1.2.2  Regulatory Domain Structures............. 3 
1.3  Small molecule binding domains (SMBD) - GAF domains........................................ 5 
1.4  PDE families........................................................................................ 7 
1.4.1  PDE1 .................................................................... 7 
1.4.2  PDE2 ............................ 7 
1.4.3  PDE4 ............................................ 8 
1.4.4  PDE5 .................................................... 9 
1.4.5  PDE10 ........................................................................................ 10 
1.4.6  PDEs in cNMP signaling compartments...................... 10 
1.5  Class III Adenylyl cyclases.............................................................. 10 
1.6  Aim of work............................................................................................................... 11 
2  Materials and equipments........................................................... 13 
2.1  Chemicals and materials.................................................................... 13 
2.2  Equipments ......................... 14 
2.3  Buffers and Solution................................................... 14 
2.3.1  Buffers and solutions for molecular biology....................................... 15 
2.3.2 nd solutions for protein chemistry....................................... 15 
2.4  Oligonucleotides........................................................................................................ 22 
2.4.1  Cloning primers........................................................... 22 
2.4.2  Sequencing primers..................... 26 
2.5  Plasmids..................................................................................................................... 27 
2.6  Bacterial strains ................... 28 
3  Methods............................................................................................................................ 29 
3.1  Molecular biology methods............................................................... 29 
3.2  Protein chemistry................................................................. 35 
3.2.1  Protein expression in E.coli................................................................................ 35 
3.2.2  Cell harvesting and lysis .................................................................................... 36 
3.2.3  Protein purification............................... 36 
3.2.4  SDS-PAGE gel electrophoresis and Western blotting ....................................... 37 
3.3  Densitometry of the SDS-PAGE gels or Western blots ...... 38 
3.4  Chromatography methods.......................................................................................... 38 
3.5  Adenylyl cyclase assay........................................................ 39 
3.6  Crystallization........................................................... 40 
3.7  Cloning ...................................................................................................................... 40 
3.7.1  Starting clones................................................... 41 
3.7.2  PDE5-CyaB1 AC Chimeras......................................... 43 
3.7.3  PDE10, PDE5 linker-CyaB1 AC chimeras ................................ 45 
3.7.4  PDE1-CyaB1 AC chimeras................................................................................ 49 
3.7.5  PDE4-Cyhimeras 50 
3.7.6  Crystal constructs ................................................ 57 
I
Content
4  Results .............................................................................................................................. 59 
4.1  hPDE5 N-terminus in tandem GAF signaling................................... 59 
4.1.1  Expression and characterization of shortened PDE5-CyaB1 AC chimeras....... 59 
4.2  Insertion of NAAIRS between hPDE5 GAF-B and CyaB1 AC ............................... 63 
4.2.1  Expression and characterization of PDE5 NAAIRS-CyaB1 AC....................... 63 
4.3  hPDE10 and hPDE5 α-helical linker in tandem GAF signaling ............................... 66 
4.3.1  Expression and characterization of PDE10, PDE5 (G311-S346)-CyaB1 AC
chimeras (No.1-16)........................................................................................................... 67 
4.3.2  Expression and characterization of chimeras No.17 and 18 .............................. 75 
4.3.3 on and chartion of chimeras No.19-21..................................... 78 
4.3.4  Expression and characterization of chimeras No.22 and 23 .............................. 81 
4.4  Tandem CaM-binding domains in signaling: hPDE1-CyaB1 AC chimeras ............. 85 
4.4.1  Expression and characterization of PDE1A3- and PDE1B1-CyaB1 AC chimeras
.............................................................................................................................86 
4.5  Tandem UCR domains in signaling: hPDE4-CyaB1 AC chimeras .......................... 91 
4.5.1  Expression and characterization of hPDE4D3-CyaB1 AC chimeras... 92 
4.5.2 on and charDE4A4-CyaB1 AC chimeras................. 95 
4.5.3  Expression and characterization of hPDE4B1-CyaB1 AC chimeras............... 100 
5  Discussion ...................................................................................................................... 103 
5.1  Role of hPDE5 N-terminus in tandem GAF signaling.................... 104 
5.2  R