Biochemical characterization of the Sec14-PH module from the neurofibromatosis type I protein [Elektronische Ressource] / presented by Stefan Welti

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 89
• Langue: English
• Poids de l'ouvrage: 22 Mo

Extrait

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
Stefan Welti, Diplom-Biologe
born in: Heidelberg
Oral exam:Biochemical Characterization
of the Sec14-PH Module
from the Neurofibromatosis Type I Protein
Referees: PD Dr. Klaus Scheffzek
Prof.Dr. Michael BrunnerCONTENTS
Contents
1 Abstracts 8
1.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2 Zusammenfassung . . . . . . . . . . . . . . . . . . . . . . 9
2 Abbreviations 10
3 Introduction 13
3.1 Neurofibromatosis Type I . . . . . . . . . . . . . . . . . 13
3.2 The NF1 gene . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3 Animal models of NFI . . . . . . . . . . . . . . . . . . . 15
3.3.1 Mouse . . . . . . . . . . . . . . . . . . . . . . . . 15
3.3.2 Drosophila melanogaster . . . . . . . . . . . . . . 16
3.4 The NF1 gene product Neurofibromin . . . . . . . . . . . 17
3.4.1 The GAP related domain and Ras. . . . . . . . . 20
3.4.2 The tubulin binding region . . . . . . . . . . . . . 23
3.4.3 The syndecan binding region . . . . . . . . . . . . 25
3.4.4 The CSRD domain . . . . . . . . . . . . . . . . . 26
3.4.5 The Sec14 homology - PH like module . . . . . . 27
3.5 Current situation and goals . . . . . . . . . . . . . . . . 29
4 Results and discussion 31
4.1 Improved overlay assays and PIP binding . . . . . . . . . 31
4.1.1 New tools: the αNF1-SecPH antibody . . . . . . 31
4.1.2 Overlay assays and the PIP binding Site . . . . . 32
4.2 Assessment of typical PH- and Sec14-domain activities . 39
4.2.1 NF1-SecPH does not bind phosphotyrosine . . . . 39
4.2.2 Localization studies in life cells . . . . . . . . . . 41
4.3 Structural investigation of lipid bound NF1-SecPH . . . 45
4.3.1 Improved purification procedure for NF1-SecPH . 45
4.3.2 Crystallization of detergent free NF1-SecPH . . . 47
4.3.3 Structure of glycerophospholipid bound NF1-SecPH 49
4.4 Properties of the lipid binding cage . . . . . . . . . . . . 52
3CONTENTS
4.4.1 Identification of the bound ligands as PtdEtn and
PtdGro . . . . . . . . . . . . . . . . . . . . . . . 52
4.4.2 NF1-SecPH has lipid exchange activity . . . . . . 56
4.5 Patient derived mutations of NF1-SecPH . . . . . . . . . 58
4.5.1 PurificationandcharacterizationofNF1-SecPHmu-
tants . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.5.2 Structure of the ΔK1750 mutant . . . . . . . . . 62
4.5.3 of the TD mutant . . . . . . . . . . . . 66
4.6 Access to the lipid binding cage can be inhibited with PIPs 69
4.7 Conclusions and outlook . . . . . . . . . . . . . . . . . . 71
5 Materials and methods 77
5.1 Common Methods . . . . . . . . . . . . . . . . . . . . . 77
5.2 Expression and purification of NF1-SecPH . . . . . . . . 78
5.3 Protein - lipid overlay assays . . . . . . . . . . . . . . . . 79
5.3.1 Generation of αNF1-SecPH antibodies . . . . . . 79
5.3.2 Protein lipid overlay assays . . . . . . . . . . . . 80
5.3.3 Site directed mutagenesis . . . . . . . . . . . . . . 81
5.4 Crystallographic techniques . . . . . . . . . . . . . . . . 82
5.4.1 Crystallization . . . . . . . . . . . . . . . . . . . 82
5.4.2 X-ray data collection . . . . . . . . . . . . . . . . 83
5.4.3 Structure determination by molecular replacement 85
5.5 AnalysisoftheNF1-SecPH-glycerophospholipidinteraction 86
5.5.1 Lipid extraction . . . . . . . . . . . . . . . . . . . 86
5.5.2 Preparation of liposomes . . . . . . . . . . . . . . 87
5.5.3 Lipid exchange reactions and inhibition . . . . . . 88
5.5.4 Mass spectrometry analysis . . . . . . . . . . . . 88
5.6 Protein characterization . . . . . . . . . . . . . . . . . . 90
5.6.1 Analytical size exclusion chromatography . . . . . 90
5.6.2 Circular dicroism spectroscopy . . . . . . . . . . . 90
5.6.3 Isothermal titration calorimetry . . . . . . . . . . 91
5.7 Mammalian cell culture and microscopy. . . . . . . . . . 92
4CONTENTS
6 Appendix 94
6.1 Publication list . . . . . . . . . . . . . . . . . . . . . . . 94
6.2 Abbreviations used in Fig. 3-4 (p.24) . . . . . . . . . . . 95
6.3 Key to the lipid arrays . . . . . . . . . . . . . . . . . . . 97
6.4 Units, amino acids and prefixes . . . . . . . . . . . . . . 98
References 99
5Acknowledgement
Foremost, I want to thank my supervisor Klaus Scheffzek for arous-
ing my fascination for structural biology, introducing me to X-ray
crystallography and for offering me the opportunity to elaborate this
thesis. ForfurthervaluableinputanddiscussionsIwouldliketothank
the members of my Thesis Advisory Committee Philippe Bastiaens,
Michael Brunner, Luis Serrano and Jochen Wittbrodt. In addition, I
would like to thank Thomas Rausch for participating in the disputa-
tion.
Thisthesiswouldnothavebeenpossiblewithoutthehelpofmanypeo-
ple - I want to thank especially Igor D’Angelo for uncounted help and
advice,SvenFratermanandMatthiasWilmforagreatandfruitfulcol-
laboration as well as Massimiliano Mazza, Ivan Yudushkin, Wolfram
Antonin and Fabien Bonneau for patience and showing me new tech-
niques. For years of friendship, help and a good working atmosphere
I am further grateful to Annabel Parret, Esther Lenherr, Jeanette
Seiler, Vladimir Pena, Vadim Sidorovitch, Kanchan Anand, Ulrike
Laabs, Michael Hothorn, Stefan Reinelt, Hus¨ seyin Besir, Ann-Marie
Lawrence, Ines Racke and Uli Steuerwald. A big thank also to Sonja
Kuhn¨ who gave me the chance to guide her to her Diploma and who
managed to advance two projects at once, as well as to Uli Karst, a
summer student I had the privilege to work with. For additional valu-
able help I also want to thank Oriol Galego, Stefanie Kandels-Lewis,
Simone Prinz, Stephanie Kronenberg, Christopher Roome and Frank
Thommen.
Furhtermore, I am grateful to my parents, Friederike and my brother
Wilfried for their support and I want to address a very special thank
to my wife Henriette for her continuous help, encouragement and un-
derstanding; without you, this thesis would not have been possible.
6fur¨ Henriette und
meine Eltern1 - Abstracts
1 Abstracts
1.1 Abstract
Neurofibromatosis type I (NF1) is an inherited neurocutaneous disor-
der with a high incidence of 1 in 3500 newborns. Clinical manifesta-
tions include pigment anomalies, Lisch nodules and the formation of
differenttumorslikeneurofibroma. NF1iscausedbyalterationsofthe
NF1 gene, encoding the Ras specific GTPase activating protein Neu-
rofibromin, which participates in several major signaling pathways.
A structural proteomics approach recently led to the discovery of an
unpredicted pleckstrin homology (PH)- and a Sec14-like domain.
In this thesis I have investigated the biochemical properties of the
NF1-SecPH module. NF1-SecPH can bind glycerophospholipids with
a preference for phosphatidylethanolamine and -glycerol (PtdEtn,
-Gro), of which PtdEtn is abundant in Neurofibromin containing cells
and thus a likely physiological ligand. It was furthermore possible to
crystallize NF1-SecPH in complex with glycerophospholipids which is
the first structure of a CRAL Trio domain bound to such ligands and
shows that PtdEtn binds to the interior of the Nf1-Sec portion. Lipid
exchange experiments revealed that PtdEtn and PtdGro are readily
exchanged, but phosphatidylcholine, -serine and -inositol (PtdCho,
-Ser,-Ins)areonlyincorporatedtoaminordegree. Thelipidexchange
activity can be modulated by soluble headgroups of phosphorylated
PtdIns derivatives (PIPs), which is consistent with a regulatory in-
teraction between Nf1-Sec and NF1-PH. While some patient derived
mutants show significant structural alterations compared to the cellu-
lar NF1-SecPH module, their properties with respect to lipid content
and PIP binding are only affected slightly. Localization studies in the
presence and absence of stimuli did not reveal a specific compartment
association compared to other PH domain containing proteins.
Taken together, PtdEtn is probably a physiological ligand of NF1-
SecPH, which seems able to incorporate membrane derived lipids in a
regulated fashion.
81 - Abstracts
1.2 Zusammenfassung
NeurofibromatoseTypI(NF1)isteineneurokutaneErbkrankheit, die
bei einem von 3500 Neugeborenen auftritt. H¨aufige Symptome sind
Pigmentanomalien, Lisch-Knoten und verschiedene Tumore, wie Neu-
rofibrome. DieErkrankungwirddurchMutationenimNF1Genverur-
sacht, das mit dem Ras spezifischen GTPase aktivierenden Protein
Neurofibromin einen Regulator zentraler Signalwege kodiert. Durch
strukturbezogene Proteomik wurde kurzlic¨ h eine unerwartete Pleck-
strin Homologie- (PH) und eine Sec14-¨ahnliche Dom¨ane entdeckt.
IndervorliegendenArbeituntersuchteichdiebiochemischenEigen-
schaften des NF1-SecPH Moduls. NF1-SecPH bindet Glycerophos-
pholipide – im Besonderen Phosphatidylethanolamin und -glycerol
(PtdEtn, -Gro) – was zusammen mit seiner H¨aufigkeit in Neurofi-
bromin enthaltenden Zellen fur¨ PtdEtn als wahrscheinlichen physi-
ologischen Liganden spricht. Zudem konnte NF1-SecPH im Komplex
mitGlycerophospholipidenkristallisiertwerden,wasdieersteStruktur
einer CRAL Trio Dom¨ane mit solchen Liganden darstellt und zeigt,
dass PtdEtn im Inneren von NF1-Sec