Functional proteome analysis of pathophysiological variants of the AAA-ATPase Cdc48p, VCP [Elektronische Ressource] / Ralf Josef Braun

Ralf.Braun

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Institut für Humangenetik,
Technische Universität München und
GSF-Forschungszentrum für Umwelt und Gesundheit


Functional Proteome Analysis of
Pathophysiological Variants of the AAA-
ATPase Cdc48p/VCP

Dipl.-Biochem. Ralf Josef Braun

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Erwin Grill

Prüfer der Dissertation: 1. apl. Prof. Dr. Jerzy Adamski
2. Univ.-Prof. Dr. Bertold Hock, em.
3. Univ.-Prof. Dr. Frank Madeo,
Karl-Franzens-Universität Graz/Österreich

Die Dissertation wurde am 05.10.2006 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 21.12.2006 angenommen.


















Meinen Eltern,
meinem Bruder
und meiner Angelika










Table of Contents
Table of Contents
A. ABBREVIATIONS .......................................................................................................................................... 9
B. SUMMARY..................................................................................................................................................... 13
C. ZUSAMMENFASSUNG ............................................................................................................................... 15
D. INTRODUCTION.......................................................................................................................................... 17
1. PROTEOMICS AND FUNCTIONAL PROTEOMICS ............................................................................................. 17
1.1 Proteome and proteomics 17
1.1.1 Proteomics describes the dynamic protein expression pattern of a cell .......................................................... 17
1.1.2 Proteome analyses were enabled by the development of new techniques........................................................ 17
1.1.3 Combination of complementary techniques and reducing complexity of protein samples facilitate the full
description of a proteome......................................................................................................................................... 18
1.2 Functional proteomics.......................................................................................................................... 19
2. STRUCTURE AND CELLULAR ROLE OF THE AAA-ATPASE CDC48P/VCP..................................................... 21
2.1 Historical overview 21
2.2 Cdc48p/VCP is a ubiquitous highly conserved protein ....................................................................... 21
2.3 Cdc48p/VCP is a member of the AAA-ATPase family ........................................................................ 22
2.4 The Cdc48p/VCP-ATPase is controlled by ubiquitin .......................................................................... 24
2.5 Cdc48p/VCP participates in a variety of different cellular processes ................................................. 25
2.5.1 Protein degradation......................................................................................................................................... 25
2.5.2 ER-associated protein degradation (ERAD).................................................................................................... 25
2.5.3 Cell cycle regulation......... 28
2.5.4 Transcriptional control..... 28
2.5.5 Membrane fusion.............. 29
2.5.6 Chaperone activity............ 29
2.5.7 DNA repair....................... 30
3. ROLE OF CDC48P/VCP UNDER PATHOPHYSIOLOGICAL CONDITIONS........................................................... 31
3.1 Mutant VCP causes the rare dominant multisystem disorder IBMPFD ............................................ 31
3.2 Wild-type VCP and human protein deposit disorders.......................................................................... 31
3.3 Cdc48p/VCP and apoptosis: From humans to yeast ........................................................................... 33
4. YEAST AS A MODEL ....................................................................................................................................... 35
4.1 Yeast as a model for conserved cellular processes in eukaryotes........................................................ 35
4.2 Yeastdel for the analysis of evolutionary conserved mechanisms of apoptotic cell death .... 35
5. AIM OF THE STUDY........ 39
E. MATERIAL AND METHODS ..................................................................................................................... 41
1. MATERIAL...................... 41
1.1 Chemicals............... 41
1.2 General equipment.41
1.3 Protein chemistry... 41
1.3.1 Special equipment............. 41
1.3.2 Kits .................................................................................................................................................................. 42
1.4 Molecular biology.. 42
1.4.1 Special equipment............. 42
1.4.2 Kits 42
1.4.3 E. coli strains.................... 42
1.4.4 Enzymes............................ 42
1.4.5 Oligonucleotides............... 42
1.4.6 Plasmids and constructs... 43
1.4.6.1 Plasmids .................................................................................................................................................. 43
1.4.6.2 Constructs............................................................................................................. 44
1.5 Analysis of yeast and mammalian cell cultures................................................................................... 44
1.5.1 Special equipment............................................................................................................................................ 44
1.5.2 Kits................................... 44
1.5.3 Yeast strains and mammalian cell lines........................................................................................................... 45
1.5.3.1 Yeast strains ............................................................................................................................................ 45
1.5.3.2 Mammalian cell lines .............................................................................................................................. 45
1.5.4 Antibodies......................... 45
1.6 Software and databases ........................................................................................................................ 46
1.6.1 Software............................ 46
5 Ralf Braun, Dissertation

1.6.2 Databases ........................................................................................................................................................ 46
2. METHODS...................... 47
2.1 Protein chemistry... 47
2.1.1 Determination of protein concentration .......................................................................................................... 47
2.1.2 Protein precipitation......... 47
2.1.3 SDS-PAGE........................ 48
2.1.4 Staining of SDS gels......... 49
2.1.4.1 Silver staining.......................................................................................................................................... 49
2.1.4.2 Coomassie staining.................................................................................................................................. 50
2.1.4.3 Colloidal Coomassie staining .................................................................................................................. 50
2.1.4.4 Ruthenium-II-bathophenantroline disulfonate chelate (RuBP) staining .................................................. 51
2.1.4.5 Digitalizing and drying of SDS gels........................................................................................................ 52
2.1.5 Western blot analysis....................................................................................................................................... 52
2.1.6 Two-dimensional gel electrophoresis (2-DE) .................................................................................................. 54
2.1.6.1 Sample preparation............................. 54
2.1.6.2 Rehydration and sample loading ............................................................................................................. 55
2.1.6.3 Isoelectric focusing (IEF)........................................................................................................................ 56
2.1.6.4 Casting of the polyacrylamide gels for the second dimension................................................................. 56
2.1.6.5 Equilibration and transfer of the IPG strips............................................................................................. 57