Unconventional secretory pathways [Elektronische Ressource] : protein folding and quality control during FGF2 membrane translocation / presented by Rafael Backhaus

-

English
187 Pages
Read an excerpt
Gain access to the library to view online
Learn more

Description

Unconventional Secretory Pathways: Protein Folding and Quality Control During FGF2 Membrane Translocation Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biologe Rafael Backhaus born in Hildesheim Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biologe Rafael Backhaus born in Hildesheim Oral Examination: Unconventional Secretory Pathways: Protein Folding and Quality Control During FGF2 Membrane Translocation Referees: Prof. Dr. rer. nat. Walter Nickel Prof. Dr. rer. Nat. Michael Brunner List of Publications Backhaus, R., Zehe, C., Wegehingel, S., Kehlenbach, A., Schwappach, B. and Nickel, W. (2004). Unconventional protein secretion: membrane translocation of FGF-2 does not require protein unfolding. J. Cell Sci. 117, 1727-1736. Engling, A.*, Backhaus, R.*, Stegmayer, C., Zehe, C., Seelenmeyer, C., Kehlenbach, A., Schwappach, B., Wegehingel, S. and Nickel, W. (2002).

Subjects

Informations

Published by
Published 01 January 2006
Reads 16
Language English
Document size 9 MB
Report a problem





Unconventional Secretory Pathways:

Protein Folding and Quality Control During

FGF2 Membrane Translocation


Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences








presented by

Diplom-Biologe Rafael Backhaus

born in Hildesheim









Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto Carola University of Heidelberg, Germany
for the degree of

Doctor of Natural Sciences

















presented by

Diplom-Biologe Rafael Backhaus

born in Hildesheim


Oral Examination:







Unconventional Secretory Pathways:

Protein Folding and Quality Control During

FGF2 Membrane Translocation











Referees:
Prof. Dr. rer. nat. Walter Nickel
Prof. Dr. rer. Nat. Michael Brunner



List of Publications


Backhaus, R., Zehe, C., Wegehingel, S., Kehlenbach, A., Schwappach, B. and
Nickel, W. (2004). Unconventional protein secretion: membrane translocation of
FGF-2 does not require protein unfolding. J. Cell Sci. 117, 1727-1736.


Engling, A.*, Backhaus, R.*, Stegmayer, C., Zehe, C., Seelenmeyer, C.,
Kehlenbach, A., Schwappach, B., Wegehingel, S. and Nickel, W. (2002).
Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation
to the extracellular surface of CHO cells. J. Cell Sci. 115, 3619-3631.
(* these authors contributed equally to this work)



I

Table of Content

ZUSAMMENFASSUNG ..............................................................................................1
SUMMARY..................................................................................................................3
1 INTRODUCTION..................................................................................................5
1.1 Classical Protein Secretion 5
1.2 Unconventional Protein Secretion 10
1.2.1 Current Models of Unconventional Export Mechanisms..........................................................11
1.3 Non-classically Secreted Proteins 13
1.3.1 Fibroblast Growth Factors..........................................................................................................15
1.3.1.1 Fibroblast Growth Factor 1 .................................................................................................17
1.3.1.2 Fibroblast Growth Factor 2 .................................................................................................18
1.3.2 Galectins......................................................................................................................................23
1.3.3 Leishmania HASPB ....................................................................................................................25
1.3.4 Cytokines.....................................................................................................................................26
1.3.5 Viral Proteins...............................................................................................................................27
1.3.6 Homeodomain Containing Transcription Factors and Chromatin-binding Proteins...............28
1.4 Quality Control in Unconventional Secretion 29
1.5 Use of Dihydrofolate Reductase in Protein Folding Analysis 31
1.6 Aim of the Present Study 32
2 MATERIAL AND METHODS .............................................................................34
2.1 Material 34
2.1.1 Chemicals....................................................................................................................................34
2.1.2 Technical Devices.......................................................................................................................36
2.1.3 Plasmids ......................................................................................................................................37
2.1.4 Primers and Oligonucleotides ....................................................................................................37
2.1.5 DNA Modifying Enzymes............................................................................................................40
2.1.6 Bacteria and Bacterial Media .....................................................................................................40

II

2.1.7 Eukaryotic Cell Lines ..................................................................................................................41
2.1.8 Eukaryotic Cell Culture Media....................................................................................................42
2.1.9 Antibodies....................................................................................................................................42
2.2 Molecular Biological Methods 43
2.2.1 Bacterial Transformation ............................................................................................................43
2.2.2 Selection and Amplification of Plasmids ...................................................................................44
2.2.3 Plasmid Preparation ...................................................................................................................44
2.2.4 Determination of DNA Concentration ........................................................................................45
2.2.5 Agarose Gel Electrophoresis .....................................................................................................45
2.2.6 DNA Marker.................................................................................................................................46
2.2.7 Polymerase Chain Reaction.......................................................................................................46
2.2.8 PCR Purification..........................................................................................................................48
2.2.9 Gel Extraction of DNA Fragments .............................................................................................48
2.2.10 Restriction Digests......................................................................................................................48
2.2.11 DNA Dephosphorylation.............................................................................................................49
2.2.12 Ligation of DNA Fragments........................................................................................................49
2.2.13 DNA Sequencing ........................................................................................................................49
2.2.14 Cloning Strategy: cDNA Constructs of the DHFR Approach...................................................50
2.2.15 Cloning Strategy: cDNA Constructs of the Piggyback Approach............................................53
2.3 Eukaryotic Cell Culture Techniques 57
2.3.1 Maintaining Cell Lines ................................................................................................................57
2.3.2 Freezing of Eukaryotic Cells ......................................................................................................57
2.3.3 Thawing of Eukaryotic Cells.......................................................................................................58
2.3.4 Retroviral Transduction ..............................................................................................................58
2.3.5 Addition of Doxicycline ...............................................................................................................60
2.3.6 Addition of Aminopterin ..............................................................................................................60
2.4 Biochemical Methods 60
2.4.1 Preparation of Cell Lysates ........................................................................................................60
2.4.2 Preparation of Cell Free Supernatants......................................................................................61
2.4.3 Determination of Protein Concentration ....................................................................................61
2.4.4 Sample Preparation for SDS Polyacrylamide Gel Electrophoresis.........................................62
2.4.5 SDS Polyacrylamide Gel Electrophoresis.................................................................................62
2.4.6 SDS-PAGE Protein Molecular Weight Standards ....................................................................64
2.4.7 Western Blot Transfer.................................................................................................................64
2.4.7.1 Reversible Ponceau Staining of Proteins ..........................................................................65
2.4.7.2 Immunochemical Protein Detection Using the ECL System ............................................65
2.4.7.3 Immunochemical Protein Detection Using the LICOR System........................................66

III

2.4.8 Protease Protection Assay.........................................................................................................67
2.4.9 Immunoprecipitation of Proteins ................................................................................................68
2.4.10 Co-Immunoprecipitation of Proteins ..........................................................................................69
2.4.11 Gel Filtration Analysis.................................................................................................................69
2.4.12 Biotinylation of Cell Surface Proteins ........................................................................................70
2.5 Flow Cytometry 72
2.5.1 Sample Preparation for FACS Analysis Detaching Cells From Culture Dishes.....................72
2.5.2 Plate Labelling Technique..........................................................................................................73
2.5.3 FACS Sort ...................................................................................................................................74
2.6 Confocal Microscopy 74
2.6.1 Sample Preparation for Confocal Microscopy ..........................................................................74
2.6.2 Immunostaining of Cell Surface Proteins for Confocal Microscopy ........................................75
3 RESULTS...........................................................................................................76
3.1 Dihydrofolate Reductase Fusion Protein System to Study Protein Folding During
Membrane Translocation Events 76
3.1.1 Generation of Cell Lines.............................................................................................................77
3.1.2 Biochemical Analysis of Doxicycline Dependent Protein Expression .....................................80
3.1.3 Characterization of the Reporter Cell Lines by Confocal Microscopy.....................................82
3.1.4 Analysis of Doxicycline Dependent Protein Expression and Secretion by Flow Cytometry..83
3.1.5 Influence of Aminopterin on the Mitochondrial Import of the Reporter Construct MTS-GFP-
DHFR...........................................................................................................................................85
3.1.6 Biochemical Analysis of the Influence of Aminopterin on MTS-GFP-DHFR Import Into
Mitochondria................................................................................................................................86
3.1.7 Protease Protection Assay to Analyze the Stability of the DHFR Domain in the Absence or
presence of Aminopterin ............................................................................................................88
3.1.8 Rationale For the Analysis of the Aminopterin Effect on FGF2-GFP-DHFR Export..............90
3.1.9 Effect of Aminopterin on FGF2-GFP-DHFR Export As Analyzed by Confocal Microscopy ..91
3.1.10 Effect of Aminopterin on FGF2-GFP-DHFR Export As Analyzed by Flow Cytometry ...........92
3.1.11 Limitations of the DHFR Approach ............................................................................................95




IV

3.2 Molecular Piggyback Export Analysis System 97
3.2.1 Generation of Piggyback Reporter Cell Lines...........................................................................98
3.2.2 Biochemical Analysis of Doxicycline Dependent Protein Expression ...................................101
3.2.3 Analysis of Doxicycline Dependent Protein Expression Based on Flow Cytometry ............103
3.2.4 Rationale of the Piggyback System.........................................................................................105
3.2.5 Co-Immunoprecipitation Analysis of Piggyback Complex Formation ...................................107
3.2.6 Probing Complex Formation Employing Gel Filtration ...........................................................109
3.2.7 Export of Reporter Molecules as Analyzed by Flow Cytometry ............................................115
3.2.8 Biochemical Analysis of Piggyback Export .............................................................................120
3.2.9 Analysis of GFP-Protein A-NES Release into Culture Medium.............................................123
3.2.10 Summary of the Piggyback Export Approach.........................................................................125
4 DISCUSSION ...................................................................................................126
4.1 Analysis of the Need for Unfolding Employing a DHFR Fusion Protein System 129
4.1.1 Characterization of Model Cell Lines Expressing DHFR Fusion Proteins............................130
4.1.2 Analysis of Aminopterin Binding to DHFR Fusion Constructs...............................................132
4.1.3 Influence of Aminopterin on the Mitochondrial Import of MTS-GFP-DHFR..........................133
4.1.4 Analysis of FGF2-GFP-DHFR Membrane Translocation in the Presence of Aminopterin..134
4.2 Piggyback Export Analysis System 137
4.2.1 Characterization of Piggyback Model Cell Lines ....................................................................138
4.2.2 Verification of Complex Formation Employing Biochemical Methods...................................139
4.2.3 Analysis of the Folding State of FGF2 Employing Piggyback Export Analysis ....................142
4.3 Concluding Remarks 146
5 LITERATURE...................................................................................................150
6 ABBREVIATIONS............................................................................................174
7 ACKNOWLEDGEMENTS ................................................................................177

Zusammenfassung 1

Zusammenfassung

Fibroblast Growth Factor 2 (FGF2) ist ein proangiogener Wachstumsfaktor, der eine
entscheidende Rolle bei der Tumor-induzierten Angiogenese spielt. FGF2 wird über
einen bisher nicht bekannten Mechanismus von Säugetierzellen sezerniert, der auch
nach Blockierung des ER/Golgi Systems durch Brefeldin A vollständig funktionell
bleibt. Da FGF2 auch biomedizinisch ein sehr interessantes Targetprotein darstellt,
ist die molekulare Aufklärung des Sekretionsweges von ausserordentlich grosser
Bedeutung.

In dieser Arbeit wurde ein experimentelles Modellsystem etabliert, dass mittels
Durchflusszytometrie eine exakte Quantifizierung der FGF2 Expressionsrate unter
verschiedenen experimentellen Bedingungen erlaubt. Darüber hinaus wurden
Testsysteme entwickelt, die den Exportvorgang sowohl mittels konfokaler
Laserscanmikroskopie als auch durch biochemische Methoden wie die
Zelloberflächenbiotinylierung rekonstituieren.

Eine wesentliche Fragestellung bei der Aufklärung des molekularen Mechanismus
der FGF2 Sekretion bestand in der Analyse des Faltungszustandes von FGF2
während des Exportvorganges. Auf der Basis der oben beschriebenen
Modellsysteme wurde FGF2 als DHFR Fusionsprotein exprimiert, so dass der
Faltungszustand durch einen exogenen Liganden kontrolliert werden konnte. Es
konnte gezeigt werden, dass unter Bedingungen, die die Entfaltung des Moleküls
nicht erlauben, die FGF2 Sekretionsrate nicht beeinflusst wird. Darüber hinaus
konnten mit Hilfe eines sogenannten Piggyback Exportsystems Hinweise dafür
gewonnen werden, dass eine Interaktion eines zweiten Reportermoleküls mit FGF2
während des Exportvorganges erhalten bleibt. Jedoch war die Effizienz dieses
Piggyback Transportes gering, so dass diese Ergebnisse schwer interpretierbar
blieben. Dennoch sind diese Beobachtungen konsistent mit den Ergebnissen, die mit
Hilfe des DHFR Systems erhalten wurden. Weiterhin stimmen die in dieser Arbeit
geschilderten Experimente in überein mit neueren Befunden aus unserem Labor, die
auf eine Rolle von Heparansulfatproteoglykanen als Exportrezeptoren bei der
Sekretion von FGF2 hinweisen. Diese Daten deuten ebenfalls auf einen

Zusammenfassung 2

Exportvorgang hin, während dessen FGF2 vollständig gefaltet bleibt. Die Summe der
Daten hat wichtige Implikationen für den Mechanismus der FGF2 Sekretion, der nach
heutigem Kenntnisstand durch eine direkte Translokation über die Plasmamembran
erfolgt. Eine Verknüpfung des Exportvorgangs mit dem FGF2 Faltungszustand
könnte somit Qualitätskontrolle gewährleisten, die die Sekretion von nicht
funktionellen Molekülen ausschliesst.