Functional characterization of the interaction between Bex2 and torsinA, a protein involved in early-onset torsion dystonia [Elektronische Ressource] / von Susann Horn

Susi Rajah

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2007
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Functional characterization of the interaction between
Bex2 and torsinA, a protein involved in early-onset
torsion dystonia







der Fakultät für Biologie
der EBERHARD KARLS UNIVERSITÄT TÜBINGEN

zur Erlangung des Grades eines
Doktors der Naturwissenschaften

von

Susann Horn
aus Werdau
vorgelegte
Dissertation
2007



































Tag der mündlichen Prüfung: 25.07.2007

Dekan: Prof. Dr. F. Schöffl

1. Berichterstatter: Prof. Dr. T. Gasser
2. Berichterstatter: Prof. Dr. O. Rieß





Teile dieser Studie wurden bereits veröffentlicht:

Wissenschaftliche Publikationen:

Horn, S., Kullmann, S., Hewett, J. W., Gasser, T., and Kamm, C. (2007) The early-
onset torsion dystonia protein torsinA interacts with Bex2 at the nuclear
envelope. Manuscript submitted.

Grundmann K., Reischmann B., Vanhoutte, G., Huebner, J., Teismann, P., Hauser,
T. K., Bonin, M., Wilbertz, J., Horn, S., Nguyen, H. P., Kuhn, M., Chanarat, S.,
Wolburg, H., Van der Linden, A., and Riess, O. (2007) Overexpression of human
wildtype torsinA and human ΔGAG torsinA in a transgenic mouse model
causes phenotypic abnormalities. Neurobiol. Dis. doi:10.1016/j.nbd.2007.04.015



Tagungsbeiträge:

Poster

Horn, S., Kullmann, S., Hewett, J. W., Gasser, T., and Kamm, C. The early-onset
torsion dystonia protein torsinA interacts with Bex2. Society for Neuroscience
th36 Annual Meeting, Atlanta 2006.




























Meiner Familie gewidmet












Content
1 SUMMARY ..................................................................................1
2 INTRODUCTION .........................................................................3
2.1 Definition and classification of dystonia.................................................. 3
2.2 Primary dystonia ........................................................................................ 6
2.3 Predominantly generalized dystonia – EOTD .......................................... 7
2.4 TorsinA – structure and expression......................................................... 8
2.5 TorsinA - putative functions.................................................................... 12
2.5.1 TorsinA in the endoplasmic reticulum.............................................. 12
+2.5.2 TorsinA – an AAA protein with chaperone function ........................ 15
2.5.3 TorsinA at the nuclear envelope (NE) 16
2.5.4 TorsinA in dopaminergic neurons.................................................... 18
2.5.5 TorsinA, cell polarity and the cytoskeleton....................................... 19
2.6 Mouse models of early onset torsion dystonia ..................................... 20
2.7 TorsinA interactors .................................................................................. 22
2.7.1 TorsinB ............................................................................................ 22
2.7.2 Kinesin-I........................................................................................... 23
2.7.3 Vimentin........................................................................................... 24
2.7.4 LAP1 and LULL1 ............................................................................. 25
2.8 Objectives................................................................................................. 26
2.8.1 Preliminary work.............................................................................. 26
2.8.2 Goals of this study........................................................................... 27
3 RESULTS ..................................................................................29
3.1 Characterization of the polyclonal Bex2 antibody ................................ 29
3.2 Expression of Bex2 and torsinA in mammalian tissues and cell
lines........................................................................................................... 32
3.3 Bex2 and torsinA interact in in vitro affinity precipitation assays....... 35
3.4 Bex2 and torsinA co-localize at the NE and in processes of
neuron-like cells....................................................................................... 39
3.5 Bex2 and torsinA accumulation in the membranous fraction of
mouse neuroblastoma cells.................................................................... 42
3.6 Bex2 and torsinA are associated with synaptosomal membranes
in the mammalian brain ........................................................................... 45
3.7 Bex2, torsinA and kinesin-I co-localize in primary cortical rat
neurons..................................................................................................... 47
3.8 Characterization of hBex2 deletion variants.......................................... 51
3.9 Bex2 and torsinA are similarly distributed throughout adult rat
brain .......................................................................................................... 55
3.10 TorsinA and Bex2 co-localization in neurons of adult rat brain .......... 59
i Content
4 DISCUSSION.............................................................................61
4.1 The polyclonal Bex2 antibody specifically recognizes Bex2 ............... 61
4.2 Bex2 interacts with wildtype and EOTD-causing torsinA in vitro........ 64
4.3 Bex2 and torsinA co-localize at the NE of neuronl-like cells ............... 66
4.4 Bex2 and TorsinA are associated with synaptosomal membranes..... 68
4.5 Expression of Bex2 and torsinA in neurons of the adult rat brain ...... 70
4.6 hBex2 deletions........................................................................................ 71
4.7 The role of Bex1 in neurotrophin signaling, the cell cycle and
neuronal differentiation – putative functions for Bex2?....................... 73
4.8 Orientation of Bex2 and torsinA at cellular membranes 76
4.9 Conclusion................................................................................................ 78
4.10 Outlook...................................................................................................... 79
5 MATERIALS AND METHODS ..................................................83
5.1 Equipment and consumables ................................................................. 83
5.1.1 Molecular biology............................................................................. 83
5.1.2 Protein biochemistry ........................................................................ 83
5.1.3 Cell biology...................................................................................... 84
5.2 Chemicals and solutions......................................................................... 84
5.2.1 Molecular biology 87
5.2.2 Protein biochemistry 87
5.2.3 Cell biology 89
5.3 Antibodies................................................................................................. 90
5.4 Expression and cloning vectors ............................................................. 92
5.5 Oligonucleotides ...................................................................................... 93
5.6 Methods in molecular biology................................................................. 94
5.6.1 Polymerase chain reaction (PCR) ................................................... 94
5.6.2 Reverse transcriptase PCR (RT-PCR) ............................................ 94
5.6.3 Quantitative Real time PCR (qRT-PCR).......................................... 95
5.6.4 Agarose gel electrophoresis ............................................................ 97
5.6.5 Isolation and purification of DNA-fragments .................................... 97
5.6.6 Enzymatic digestion of DNA 97
5.6.7 Ligation of DNA ............................................................................... 98
5.6.8 Transformation of DNA into One Shot® TOP10Electrocomp™
E. coli .............................................................................................. 98
5.6.9 Production of bacterial glycerol stocks ............................................ 98
5.6.10 Production of electrocompetent cells............................................... 98
5.6.11 Preparation and purification of plasmid DNA................................... 99
5.7 Methods in protein biochemistry.......................................................... 100
5.7.1 Preparation of protein extracts....................................................... 100
ii Content
5.7.2 Protein quantification by Bradford and Bicinchoninic Acid Assay
(BCA)