Impact of Mdm2-p53 on the proteasome assembly and disassembly Role of the ubiquitination of some 19S subunits [Elektronische Ressource] / Justine Letienne. Betreuer: Christine Blattner

Justine Letienne

Description

"Impact of Mdm2-p53 on the proteasome assembly and disassemblyRole of the ubiquitination of some 19S subunits"Zur Erlangung des akademischen Gradeseines Doktors der Naturwissenschaften von der Fakultät für Chemie und Biowissenschaften desKarlsruher Instituts für Technologie (KIT)genehmigte DissertationvonJustine Letienneaus Lescar, FranceHauptreferent: PD Dr. Christine BlattnerKorreferent: Prof. Dr. Jörg KämperTag der mündlichen Prüfung: 18.10.2011TABLE OF CONTENTSZUSAMMENFASSUNG .................................................................................... 5 ABSTRACT .........................................................................................................7 LIST OF FIGURES AND TABLES.................................................................. 8 ABBREVIATIONS ............................................................................................. 91. INTRODUCTION.........................................................................................111.1 The Ubiquitin Proteasome System “UPS”.................................................... 12 1.1.1 The ubiquitin conjugation pathway................................................................................. 12 1.1.1.1 Ubiquitination of substrates........................................................................................................12 1.1.1.2 E3 ubiquitin ligases .....................................................................................

Sujets

• Biologie

Informations

• Publié par: karlsruher_institut_fur_technologie
• Publié le: 01 janvier 2011
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

ome proteas on the 3"Impact of Mdm2-p5assembly and disassembly
Role of the ubiquitination of some 19S subunits"

Zur Erlangung des akademischen Grades

eines Doktors der Naturwissenschaften

von der Fakultät für Chemie und Biowissenschaften des

Karlsruher Instituts für Technologie (KIT)

genehmigtetation Disser

nvo

tienne LeJustine

aus Lescar, France

Hauptreferent: PD Dr. Christine Blattner
f. Dr. rent: ProKorrefe KämperJörgTag der mündlichen Prüfung: 18.10.2011

CONTENTSTABLE OF

ZUSAMMENFASSUNG....................................................................................5

7.........................................................................................................ABSTRACT

LIST OF FIGURES AND TABLES..................................................................8

ABBREVIATIONS.............................................................................................9

1. INTRODUCTION.........................................................................................11

1.1 The Ubiquitin Proteasome System “UPS”....................................................12
1.1.1 The ubiquitin conjugation pathway.................................................................................12
1.1.1.1 Ubiquitination of substrates........................................................................................................12
1.1.1.2 E3 ubiquitin ligases....................................................................................................................14
1.1.1.4 Ubiquitin and ubiquitin like modifications.................................................................................15
1.1.1.4.1 Ubiquitin chains.....................................................................................................................................15
1.1.1.4.2 Ubiquitin-like “UBLs” modifications....................................................................................................17
1.1.2 Degradation of targeted substrates by the 26S proteasome.............................................17
1.1.2.1 The 26S proteasome...................................................................................................................17
1.1.2.1.1 The 20 S proteasome..............................................................................................................................19
1.1.2.1.1.2.2 S1.2 iTghne 19aling of S proteasprotomeien degradation...............................................................................................................................................................................................................................2019
1.1.2.1.1.2.2.2 Deu2.1 Recogbiqunitionitin ofation s,u ubstnfroateslding, translocation into the 20S ............................................................................................and degradation of substrates....................................................2122
1.1.3 Deubiquitinases “DUBs”.................................................................................................23
1.1.4 Other roles of proteasomal subunits in cells...................................................................23

1.2 Mdm2 and its role in p53 regulation.............................................................24
1.2.1 The “RING” E3 ubiquitin ligase Mdm2.........................................................................24
1.2.1.1 Generalities.................................................................................................................................24
1.2.1.2 Structure of Mdm2.....................................................................................................................25
1.2.1.3 Post-translational modifications of Mdm2.................................................................................26
1.2.2 P53 ”guardian of the genome”........................................................................................26
1.2.3 The Mdm2-p53 pathway.................................................................................................27
1.2.3.1 In normal conditions, p53 is one of the major substrate of Mdm2.............................................27
1.2.3.2 The p53-Mdm2 pathway upon stress condition.........................................................................28
1.2.3.3 Monoubiquitination versus polyubiquitination of p53...............................................................29
1.2.3.4 The feedback loop......................................................................................................................29
1.2.4The ternary complex “p53-Mdm2-19S subunits”...........................................................30

1.3 Aim of the study............................................................................................31

2

2. MATERIALS AND METHODS.................................................................32

2.1Materials........................................................................................................32
2.1.1 Chemicals and consumables............................................................................................32
2.1.2 Kits2.1.3 Standards...........................................................................................................................................................................................................................................................3333
2.1.4 Binding matrices.............................................................................................................33
2.1.5 Oligonucleotides..............................................................................................................34
2.1.6 Enzymes..........................................................................................................................34
34........................................................................................................................2.1.7 Cells lines2.1.8 Bacteria............................................................................................................................35
35..........................................................................................................................2.1.9 Plasmids36.....................................................................................................................2.1.10 Antibodies

2.2 Methods.........................................................................................................38
38..................................................................................................................2.2.1 DNA methods2.2.1.1 Agarose gel electrophoresis........................................................................................................38
2.2.1.2 Transformation of DNA into bacteria........................................................................................38
2.2.1.3 Small-scale purification of DNA................................................................................................38
2.2.1.4 High-scale purification of DNA.................................................................................................39
2.2.1.5 Quantification of plasmid DNA.................................................................................................39
2.2.2 Cell culture and transfection methods.............................................................................40
2.2.2.1 Maintenance of mammalian cell lines........................................................................................40
2.2.2.2 Transfection with Calcium Phosphate reagent (Chen and Okayama, 1987)..............................40
2.2.2.3 Transfection using Lipofectamine 2000.....................................................................................41
41...............................................................................................................2.2.3 Protein methods2.2.3.1 Preparation of protein lysates from cells....................................................................................41
2.2.3.2 Quantification of protein extracts (Bradford Assay)..................................................................41
2.2.3.3 Separation of proteins by SDS-PAGE (polyacrylamide gel electrophoresis)............................42
2.2.3.4 Western blotting and immunodetection......................................................................................42
2.2.3.5 Production of the enzyme USP2-core........................................................................................43
2.2.3.6 Binding assay.............................................................................................................................44
2.2.5.7 Production of the MLG affinity matrix and purification of ubiquitinated conjugates................44
2.2.3.8 Staining with Coomassie® blue.................................................................................................45
2.2.3.9 Ubiquitination Assay..................................................................................................................46
2.2.3.10 Sucrose gradient.......................................................................................................................46
2.2.3.11 Immunoprecipitation................................................................................................................47

3. RESULTS...........