Translocation proteomics [Elektronische Ressource] : a novel proteomic approach for the identification of signalling intermediates / by Milošević, Jadranka
102 Pages
English
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Translocation proteomics [Elektronische Ressource] : a novel proteomic approach for the identification of signalling intermediates / by Milošević, Jadranka

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
102 Pages
English

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“TRANSLOCATION PROTEOMICS: A NOVEL PROTEOMIC APPROACH FOR THE IDENTIFICATION OF SIGNALLING INTERMEDIATES” Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen by Miloševi ć, Jadranka of Virovitica, Croatia Giessen 2008 From the Department of Internal Medicine Chairman: Prof. Dr. Werner Seeger of the Faculty of Medicine of the Justus Liebig University Giessen First Supervisor and Committee Member: Dr. Oliver Eickelberg Second Supervisor and Committee Member: Prof. Dr. Martin Diener Committee Members: .Prof. Dr. Andreas Günther Date of Doctoral Defence: 04.04.2008 I declare that I have completed this dissertation single-handedly without the unauthorized help of a second party and only with the assistance acknowledged therein. I have appropriately acknowledged and referenced all text passages that are derived literally from or are based on the content of published or unpublished work of others, and all information that relates to verbal communications.

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Published 01 January 2008
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“TRANSLOCATION PROTEOMICS: A NOVEL PROTEOMIC
APPROACH FOR THE IDENTIFICATION OF SIGNALLING
INTERMEDIATES”








Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen









by
Miloševi ć, Jadranka

of
Virovitica,
Croatia


Giessen 2008
















From the Department of Internal Medicine
Chairman: Prof. Dr. Werner Seeger
of the Faculty of Medicine of the Justus Liebig University Giessen


















First Supervisor and Committee Member: Dr. Oliver Eickelberg
Second Supervisor and Committee Member: Prof. Dr. Martin Diener
Committee Members: .Prof. Dr. Andreas Günther







Date of Doctoral Defence:
04.04.2008




















I declare that I have completed this dissertation single-handedly without the
unauthorized help of a second party and only with the assistance
acknowledged therein. I have appropriately acknowledged and referenced all
text passages that are derived literally from or are based on the content of
published or unpublished work of others, and all information that relates to
verbal communications. I have abided by the principles of good scientific
conduct laid down in the charter of the Justus Liebig University of Giessen
in carrying out the investigations described in the dissertation.


























I TABLE OF CONTENTS
I TABLE OF CONTENTS I
II LIST OF FIGURES V
III LIST OF TABLES VII
IV LIST OF ABBREVIATIONS VIII
SUMMARY XII
ZUSAMMENFASSUNG XIV
1 INTRODUCTION
1.1 TGF-β SIGNAL TRANSDUCTION 3
1.1.1 THE TGF- β SUPERFAMILY OF LIGANDS
1.1.2 TGF- β SIGNALLING VIA SERINE/THREONINE KINASE RECEPTORS 4
1.1.3 INTRACELLULAR SIGNALLING MOLECULES 5
1.1.3.1 Smads 5
1.1.3.2 Smad phosphorylation and dephosphorylation 7
1.1.3.2.1 Smad phosphorylation by other pathways 8
1.1.3.3 Nucleocytoplasmic Smad Shuttling 9
1.2 NEGATIVE REGULATION OF TGF- β SIGNALLING 11
1.2.1 INHIBITOR SMADS 11
1.2.2 SMAD UBIQUITINATION 12
1.3 SMAD-INDEPENDENT SIGNALING CASCADES ACTIVATED BY TGF- β
FAMILY MEMBERS
1.4 TGF- β SIGNALLING IN HUMAN DISORDERS 13
1.4.1 IDIOPATHIC PULMONARY FIBROSIS
1.4.2 OTHER DISORDERS 14
1.5 PROTEOMICS 15
1.5.1 SUBCELLULAR PROTEOMICS 16
1.5.2 PHOSPHOPROTEOMICS
1.5.2.1 Immobilized metal-ion affinity chromatography: enrichment of
phosphoproteins 17
1.5.3 TWO DIMENSIONAL GEL ELECTROPHORESIS 18
1.5.4 PROTEIN DETECTION AND QUANTITATION
1.5.5 PROTEIN IDENTIFICATION AND CHARACTERIZATION 20
1.5.5.1 Mass spectrometry and protein analysis 20
1.5.5.2 Matrix assisted laser desorption/ionization 20
2 AIM OF THE STUDY
3 MATERIALS AND METHODS
3.1 MATERIALS 23
3.1.1 EQUIPMENTS 23
3.1.2 GENERAL CHEMICALS 24
3.1.3 CELL CULTURE REAGENTS AND OTHER LABORATORY CHEMICALS 25
3.1.4 ANTIBODIES 26
3.1.5 CELL LINES AND MEDIA
3.1.6 SAMPLES FROM PATIENTS 27
3.2 METHODS
3.2.1 WESTERN BLOTTING
3.2.2 TWO-DIMENSIONAL GEL ELECTROPHORESIS 29
3.2.2.1 Sample preparation and protein solubilisation 29
3.2.2.2 First dimension: Isoelectric focusing 30
3.2.2.3 Second dimension: Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) 31
3.2.2.4 Gel analysis 32
3.2.3 TRYPTIC IN-GEL DIGEST AND MALDI TOF MS ANALYSIS 32
3.2.3.1 Protein identification by peptide mass fingerprinting 32
3.2.3.2identification by sequencing of peptides and peptide mass fingerprinting 33
3.2.3.3 Phosphoprotein enrichment by phosphate metal affinity chromatography 34
3.2.3.4 Concentrating and desalting the phosphoprotein fraction 34
3.2.3.5 Separation of enriched phosphoproteins by two-dimensional electrophoresis 35
3.2.4 IMMUNOFLUORESCENCE 35
4 RESULTS
4.1 SUBCELLULAR FRACTIONATION 37
4.2 TWO-DIMENSIONAL SEPARATION OF CYTOPLASMIC AND NUCLEAR
PROTEIN FRACTIONS FROM A549 CELL LINE 39
4.2.1 TWO-DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF CYTOSOLIC ENRICHED
FRACTION 40
4.2.2 VALIDATION OF THE TRANSLOCATION DATA 45
4.2.3 TWO-DIMENSIONAL GEL ELECTROPHORESIS ANALYSIS OF NUCLEAR ENRICHED
FRACTION 47
4.3 NEWLY IDENTIFIED PHOSPHOPROTEINS IN TGF- β SIGNALLING PATHWAY 50
4.3.1 PURIFICATION OF PHOSPHOPROTEINS 50
4.3.2 ANALYSIS OF TOTAL PHOSPHOPROTEIN USING TWO-DIMENSIONAL
ELECTROPHORESIS 52
4.3.3 IDENTIFICATION OF DIFFERENTIAL PHOSPHOPROTEINS TRIGGERED BY TGF- β1 54
4.3.4 PREDICTION OF PHOSPHORYLATRION SITES 55
5 DISCUSSION
5.1 TRANSLOCATION IN RESPONSE TO TGF- β STIMULATION 59
5.1.1 ANALYSIS OF CYTOPLASMIC FRACTION 59
5.1.2 ANALYSIS OF NUCLEAR FRACTION 61
5.2 IDENTIFICATION OF NOVEL PHOSPHORYLATED PROTEINS IN TGF- β
SIGNALLING PATHWAY 63
6 ACKNOWLEDGEMENTS
7 DECLARATION
8 REFERENCES
9 CURRICULUM VITAE LIST OF FIGURES V
II LIST OF FIGURES
Figure 1.1 The canonical TGF- β Smad Pathway
Figure 1.2 Structure of R-Smads, Smad4 and I-Smads (Smad6 and
Smad7)
Figure 1.3 Model of nucleocytoplasmic shuttling of Smads
Figure 1.4 General proteome analysis using two-dimensional gel
electrophoresis and mass spectrometry
Figure 4.1 Schematic illustration of a strategy to study translocation
events
Figure 4.2 Subcellular fractionation of A549 cells
Figure 4.3 Control of subcellular fractionation of A549 cells after 120
minutes TGF- β stimulation
Figure 4.4 Two-dimensional separation of cytoplasmic and nuclear
fractions
Figure 4.5 Alterations in protein profiles between cytosolic fractions
from stimulated cells and unstimulated controls
Figure 4.6 Translocation of KHSRP, FUBP1, hnRNP H, hnRNP L in
A549 cells after TGF- β1 stimulation
Figure 4.7 The TGF- β1-dependent translocation of hnRNP L, FUBP1,
KHSRP
Figure 4.8 Phosphoprotein enrichment procedure
Figure 4.9 Complete separation of non-phosphorylated and
phosphorylated proteins
Figure 4.10 Phosphoproteome of A549 cells after elution of
phosphoproteins from phosphate metal affinity LIST OF FIGURES VI
chromatography (PMAC) separated by one dimensional gel
electrophoresis
Figure 4.11 Two-dimensional electrophoretic analysis of A549 cell
fractions after selective phosphoprotein enrichment
Figure 4.12 Two-dimensional electrophoretic analysis and alignment of
phosphorylated proteins after stimulation with TGF- β for
60 minutes
Figure 4.13. Two-dimensional electrophoretic analysis of proteins in
TGF- β1 stimulated cells compared with the non-stimulated
control
LIST OF TABLES VII
III LIST OF TABLES
Table 1 A network controlling a transforming growth factor - β
signalling pathway
Table 2 Laboratory equipments
Table 3 General laboratory chemicals
Table 4 Cell culture reagents
Table 5 Primary antibodies
Table 6 Secondary antibodies
Table 7 Composition of 10% resolving gels (40 ml)
Table 8 Composition of 4% stacking gels (20 ml)
Table 9 Composition of 12% gels (40 ml)
Table 10 Identified proteins from cytoplasmic fraction
Table 11 Identified proteins from nuclear fraction
Table 12 Phosphorylated proteins identified by MALDI-TOF-MS
Table 13 Classification of identified phosphoproteins divided into
functional categories LIST OF ABBREVIATIONS VIII
IV LIST OF ABBREVIATIONS
2-DE Two dimensional gel electrophoresis
ACN Acetonitirile
ACVRII Activin type II receptor
ACVRII-B e II-B receptor
ALK1 Activin receptor like kinase 1
ALK2 Activin receptor like kinase 2
ALK3 Activin receptor like kinase 3
ALK4 Activin receptor like kinase 4
ALK5 Activin receptor like kinase 5
ALK6 Activin receptor like kinase 6
ALK7 Activin receptor like kinase 7
ALK8 Activin receptor like kinase 8
AMH Anti-Müllerian hormone
AMHRII Anti-Müllerian hormone type II receptor
APS Ammonium persulfate
BAMBI BMP and activin membrane-bound inhibitor
BMP Bone morphogenetic protein
BMPRII Bone morphogenetic receptor 2
CA Carrier ampholitcs
CE Capillary electrophoresis
CF Cytosolic fraction
CE-MS Capillary electrophoresis-Mass spectrometry
CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-
propanesulfonate
cDNA Complementary deoxyribonucleic acid
Co-Smad Common Smad
CRAC Cultured rat articular chondrocytes
DAPI 4´, 6´-diamidino-2-phenylindole
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
ECD Extracellular domain