Novel anti-inflammatory targets and mechanisms of boswellic acids and celecoxib [Elektronische Ressource] / von Lars Tausch

Tausch

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 36
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Novel anti-inflammatory targets and mechanisms
of boswellic acids and celecoxib




Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften






vorgelegt beim Fachbereich
Chemie, Biochemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main








von
Lars Tausch
aus Bad Nauheim




Frankfurt am Main (2008)
(D30)
















vom Fachbereich Chemie, Biochemie und Pharmazie der Johann Wolfgang Goethe –
Universität als Dissertation angenommen.

























Dekan: Prof. Dr. Harald Schwalbe

Gutachter: Prof. Dr. Dieter Steinhilber

Prof. Dr. Oliver Werz

Datum der Disputation: 29.08.2008





















Meiner Familie!





















Lieber einsam mit den Freien fallen, als mit den Massen im Triumphe ziehen! 4
Table of contents

1 Abbreviations and reagents ................................................................................................ 7
1.1 Abbreviations ............................................................................................................. 7
1.2 Reagents ................................................................................................................... 11
2 Introduction ...................................................................................................................... 13
2.1 Boswellic acids in traditional medicine and treatment of chronic diseases ............. 13
2.2 Cathepsin G; a neutrophil serine protease and its role in disease ............................ 15
2.3 DNA-dependent protein kinase and Akt .................................................................. 17
2+2.4 Cellular signalling: Ca -homeostasis and MAP kinases......................................... 19
2.5 COX and LOs: Key enzymes in eicosanoid biosynthesis........................................ 21
2.6 Aim of this work ...................................................................................................... 25
3 Methods............................................................................................................................ 27
3.1 Cells and cell culture................................................................................................ 27
3.1.1 Cell culture ....................................................................................................... 27
3.1.2 Mono Mac 6 cells............................................................................................. 27
3.1.3 LNCaP / HL-60 27
3.1.4 MCF-7 .............................................................................................................. 27
3.1.5 RBL-1............................................................................................................... 28
3.2 Isolation of human PMNL (polymorphonuclear leukocytes) from venous blood ... 28
3.3 an platelets from venous blood ...................................................... 28
3.4 Human in vitro whole blood assay........................................................................... 28
3.5 Expression and purification of 5-LO from Escherichia coli .................................... 29
3.6 Expression and purification of his-tagged platelet-type 12-LO from Escherichia coli
.................................................................................................................................. 30
3.7 Determination of 5-LO product formation in intact cells ........................................ 30
3.8 ination of 5-LO product formation in cell-free systems............................... 31
3.9 Determination of 12-LO product formation............................................................. 31
33.10 [ H]-Arachidonic acid release .................................................................................. 32
3.11 Immobilization of boswellic acids and protein pull-down assays............................ 32
3.12 SDS-PAGE............................................................................................................... 33
3.13 Western Blot............................................................................................................. 33
3.14 Colloidal Coomassie staining................................................................................... 34
3.15 Silver staining........................................................................................................... 34
3.16 In-gel digestion......................................................................................................... 34
3.17 MALDI-TOF-MS..................................................................................................... 34
3.18 In vitro kinase assay for DNA-PK ........................................................................... 35
3.19 In vivo phosphorylation ........................................................................................... 35
3.20 Akt activity assay 36
3.21 In vitro kinase assay of PKC .................................................................................... 36
3.22 Farnesyl pyrophosphate synthase activity assay ...................................................... 36
3.23 Release of TNF α in MM6........................................................................................ 37
3.24 Protease activity assays ............................................................................................ 37
3.25 Docking experiments................................................................................................ 38
3.26 Measurement of platelet aggregation (turbidimetric)............................................... 39
3.27 ent of platelet activation markers CD62 and PAC-1 by flow cytometry 39
3.28 Endogenous thrombin potential ............................................................................... 40
2+3.29 Intracellular Ca measurement................................................................................ 41
3.30 PMNL chemoinvasion assay 41
3.31 Purification of CatG from PMNL ............................................................................ 41
3.32 Determination of protein concentration ................................................................... 42 5
3.32.1 Lowry ............................................................................................................... 42
3.32.2 Bradford ........................................................................................................... 42
3.33 Crystallography and X-ray determination................................................................ 43
3.34 Statistics ................................................................................................................... 43
4 Results .............................................................................................................................. 44
4.1 Target identification of BAs using a protein-fishing strategy.................................. 44
4.1.1 Pulldown experiments...................................................................................... 44
4.1.2 Separation of precipitated proteins by SDS-PAGE.......................................... 45
4.1.3 Analysis of proteins by mass spectrometry...................................................... 46
4.1.4 Immunodetection of proteins ........................................................................... 48
4.1.5 FPPs activity..................................................................................................... 49
4.1.6 PKC activity 50
4.1.7 Rap1b ............................................................................................................... 51
4.2 Cathepsin G.............................................................................................................. 52
4.2.1 Protein fishing with immobilized boswellic acids selectively precipitates
cathepsin G ....................................................................................................... 52
4.2.2 Boswellic acids inhibit the proteolytic activity of cathepsin G........................ 54
4.2.3 Effects of boswellic acids on related serine proteases ..................................... 56
4.2.4 Docking of boswellic acids to cathepsin G ...................................................... 57
4.2.5 Boswellic acids inhibit (cathepsin G-mediated) PMNL chemoinvasion ......... 58
2+4.2.6 cibit cathepsin G-mediated Ca mobilisation in human
platelets............................................................................................................. 59
4.2.7 Inhibition of cathepsin G by Boswellia serrata extracts ex vivo...................... 60
4.2.8 Crystallography of CatG and A β-BA..............................