Functional characterization of early growth response transcription factor 2 (EGR-2) [Elektronische Ressource] / von Katarina Ludajic

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• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2005
• Nombre de lectures: 33
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

Functional characterization of Early Growth
Response Transcription Factor 2 (EGR-2)
DISSERTATION
zur Erlangung des akademischen Grades
Doctor rerum naturalium (Dr. rer. nat.)
vorgelegt dem Rat der
Biologisch-Pharmazeutischen Fakultät der
Friedrich-Schiller-Universität Jena
von
Katarina Ludajic
geboren am 7. April 1975
in BelgradGutachter:
1. Prof. Dr. Peter Zipfel………………………..................................……….
2. Prof. Dr. Frank Große…………………..................................……………….
3. PD Dr. Hermann Eibel……………..................................…………………….
Tag des Rigorosums: 22.06.2005………………………………….
Tag der öffentlichen Verteidigung: 18.07.2005.……………………………….Table of Contents
Table of Contents
Table of contents I
Abbreviations V
Zusammenfassung VII
Summary IX
1 Introduction .................................................................................................................... 1
1.1 The Immune System................................................................................................... 1
1.2 Inflammation................. 2
1.2.1 Mediators of inflammation.................................................................................. 4
1.2.2 The role of transcription factors in inflammation............. 6
1.3 The ‘Immediate early’ genes...................... 8
1.4 The ‘Early growth response’ protein family ............................................................. 8
1.4.1 The structure of EGR proteins........................................... 9
1.4.2 Regulation of EGR protein expression...........................11
1.4.3 Function of EGR proteins.................................................14
1.5 The EGR-2 protein....................................................................17
1.6 The NF-kB proteins...................................18
1.7 The NAB proteins......20
1.8 Project aim..................................................................................................................23
2 Materials.........................24
2.1 Established cell lines and bacterial strains............................................................24
2.2 Cell culture reagents .................................................................24
2.3 Buffers and media.....................................24
2.4 Oligonucleotides........25
2.5 Plasmids .....................................................................................................................25
2.6 Antibodies...................28
2.7 Other reagents and materials..................29
3 Methods.........................................................................................................................30
3.1 DNA and RNA techniques........................30
ITable of Contents
3.1.1 RNA isolation and RT- PCR............................................................................30
3.1.2 Polymerase chain reaction (PCR)..30
3.1.3 Restriction and ligation of DNA.......31
3.1.4 Preparation and transformation of competent bacteria...............................32
3.1.5 Plasmid preparation ..........................................................................................33
3.1.6 DNA sequencing................................34
3.2 Cell culture methods.34
3.2.1 Cultivating of mammalian and insect cells ....................................................34
3.2.2 Transient transfections and Luciferase reporter gene assay.....................35
3.3 Baculovirus expression system...............................................35
3.3.1 Generating recombinant Baculoviruses by co-transfection........................35
3.3.2 Plaque assay and virus amplification.............................................................36
3.4 Expression of proteins in eucaryotic and procaryotic systems ..........................37
3.4.1 Expression of endogenous proteins...............................37
3.4.2 Expression of recombinant EGR proteins .....................................................37
3.4.3 Extraction of proteins expressed in eucaryotic cells ....................................37
3.4.4 Affinity chromatography purification of recombinant EGR proteins...........38
3.4.5 Expression and extraction of E. coli expressed recombinant proteins .....38
3.4.6 Affinity chromatography purification of E. coli expressed proteins ............39
3.5 Confocal Laser-Scanning Microscopy and Fluorescence Resonance Energy
Transfer (FRET).........................................................................................................39
3.6 Protein chemical methods........................................................................................40
3.6.1 SDS-PAGE (Sodiumdodecylsulfate-polyacrylamid gel electrophoresis)..40
3.6.2 Silver staining .....................................................................................................41
3.6.3 Protein transfer and Western blot...41
3.6.4 In vitro protein binding studies.........42
3.6.5 Immunoprecipitation..........................................................................................43
4 Results...........................................................44
4.1 Expression of native and recombinant EGR-2 and NF-kB proteins ..................44
4.1.1 Expression of native and recombinant EGR-2 protein................................44
4.1.2 Expression of native and recombinant NF-kB proteins ...............................45
4.2 Purification of recombinant EGR-2 and NF-kB proteins.....46
IITable of Contents
4.3 Cellular distribution of EGR-2 protein.....................................................................47
4.4 Characterization of EGR-2/NF-kB interactions.....................................................50
4.4.1 Interaction of NF-kB with immobilized EGR-2 protein.51
4.4.2 In vivo interaction of EGR-2 and NF-kB proteins .........................................52
4.4.3 Functional interaction of EGR-2 and NF-kB proteins in regulation of TNFa
and ICAM-1 promoters.....................................................................................54
4.4.4 Functional interaction of EGR-1/p65, EGR-3/p65 and EGR-4/p65
heterodimers in regulation of TNFa and ICAM-1 promoters......................55
4.5 Expression and purification of NAB-2 protein.......................................................56
4.6 Characterization of EGR-2/NAB interaction..........................57
4.6.1 Interaction of EGR-2 with immobilized NAB-2 protein.................................57
4.6.2 Co-immunoprecipitation of EGR-2 and NAB-2 proteins..............................58
4.6.3 In vivo interaction of EGR-2 and NAB-2 proteins .........................................59
4.6.4 Functional interaction of EGR-2 and NAB proteins in regulation of TNFa
and ICAM-1 promoters.....................................................................................60
4.6.5 Functional interaction of EGR-1, EGR-3, EGR-4 and NAB proteins in
regulation of TNFa and ICAM-1 promoters...................61
4.6.6 Localization of the interaction domains in the EGR-4 protein....................62
4.7 Interaction of EGR-1, EGR-3 and EGR-4 proteins with NAB.............................63
4.7.1 Interaction of recombinant EGR-1, EGR-3, EGR-4 proteins with
immobilized NAB-2........................................................................................................64
4.7.2 In vivo interaction of EGR-4 and NAB-2 proteins.........65
4.8 Interaction of NAB-2 and p65 proteins...65
4.9 EGR-EGR interactions..............................................................................................67
4.9.1 Interactions of EGR-2, EGR-3 and EGR-4 with immobilized EGR-1
protein...................................................................................................................67
4.9.2 In vivo interaction of EGR-EGR proteins.......................69
4.9.3 Functional interaction of EGR-EGR heterodimers in regulation of TNFa
and ICAM-1 promoters .................................................................................................70
5 Discussion....................................................................................................................71
5.1 Expression and localization of EGR-2 protein......................71
5.2 Interaction of EGR-2 with NF-kB proteins .............................................................73
5.2.1 Expression and localization of NF-kB proteins .............................................74
IIITable of Contents
5.2.2 Interaction of EGR-2 with NF-kB ....................................................................74
5.3 Interaction of EGR- and NAB proteins...78
5.3.1 Interaction of EGR-4 with NAB proteins ........................................................79
5.3.2 Interaction of NAB and p65 proteins..............................81
5.4 EGR-EGR complexes...............................................................82
6 References....................................................................................84
Acknowledgments i
Publications and scientific posters ii
Selbstständigkeitserklärung iii
IVAbbreviations
Abbreviations
Amp Ampicillin
AP-1 Activator protein 1
APS Ammoniumpersulfate
BCR B cell receptor
b-FGF Basic Fibroblast Growth Factor
BSA Bovine Serum A