Functional characterization of the presenilin homologue SPE-4 [Elektronische Ressource] / Aya Yamasaki-Meythaler

Aya Yamasaki-Meythaler

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München


Functional Characterization of
the Presenilin Homologue SPE-4




Aya Yamasaki-Meythaler
aus
Tokyo, Japan


2006 Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Herrn Prof. Dr. Christian Haass betreut und von Herrn Prof. Dr. Ralf-
Peter Jansen vor der Fakultät für Chemie und Pharmazie vertreten.




Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, am 2.11.2006



.....................................................
(Aya Yamasaki-Meythaler)


Dissertation eingereicht am 5.11.2006
1. Gutachter: Prof. Dr. Ralf-Peter Jansen
2. Gutachter: Prof. Dr. Christian Haass
Mündliche Prüfung am 5.12.2006
2








For my parents and Tobi,
who always believed in me.
3The results in this dissertation are partially presented in the following publication:

The GxGD Motif of Presenilin Contributes to Catalytic Function and Substrate
Identification of γ-Secretase
Aya Yamasaki, Stefan Eimer, Masayasu Okochi, Agata Smialowska, Christoph Kaether,
Ralf Baumeister, Christian Haass, and Harald Steiner
The Journal of Neuroscience, April 5, 2006 • 26(14):3821–3828 • 3821



This publication is enclosed in the end of this dissertation.
4 Abbreviations
Abbreviations

AD Alzheimer's Disease
Aβ Amyloid-β peptide
ADAM a disintegrin and metalloproteinase
APP intracellular domain AICD
APH-1 anterior pharynx-defective phenotype
APP like protein APLP
APP β-amyloid precursor protein
BACE β-site APP-cleaving enzyme
C. elgans Caenorhabditis elegans
Cluster of differentiation 44 CD44
CTF C-terminal fragment
egg laying defective Egl
FAD familial AD
Flag tagged NEXT F-NEXT
Flag tagged Nβ F-Nβ
germ-line proliferation defective GLP-1
HEK human embryonic kidney
homologue of PS HOP-1
LIN-12 abnormal cell lineage-12
mouse embryonic fibroblast MEF
Notch β peptide Nβ
Nicastrin NCT
NEXT Notch extracellular truncation
Notch intracellular domain NICD
NTF N-terminal fragment
PS enhancer-2 PEN-2
PS Presenilin
RNA interference RNAi
S1, S2, S3, S4 site 1, site 2, site 3, site 4
soluble APP sAPP
SEL-12 suppressors and/or enhancers of lin-12
spermatogenesis defective-4 SPE-4
SPP signal peptide peptidase
SPP like protein SPPL
sw Swedish mutant
TACE TNF-α convertase
Abbreviations
transmembrane domain TMD
TMIC transmembrane-intracellular
TNF-α tumor necrosis factor-α
wt wild type

6 Contents
Contents
Abbreviations .......................................................................................................................5
Contents ................................................................................................................................7
1. Introduction....................................................................................................................13
1.1 Histopathology of Alzheimer’s disease......................................................................13
1.2 Genetics of Alzheimer’s disease.................................................................................15
1.3 Molecular biology of Alzheimer’s disease.................................................................18
1.3.1 β-Amyloid precursor protein processing ............................................................18
1.3.2 γ-Secretase...........................................................................................................20
1.3.2.1 Notch and other substrates of γ-secretase ....................................................21
1.3.2.2 Presenilin......................................................................................................23
1.3.2.3 Nicastrin.......................................................................................................25
1.3.2.4 APH-1 and PEN-2........................................................................................26
1.3.2.5 Assembly of the γ-secretase complex ..........................................................27
1.3.2.6 Substrate recognition of γ-secretase.............................................................28
1.4 The presenilin homologues in C. elegans ..................................................................29
1.5 Aim of the study..........................................................................................................30
2. Materials and Methods..................................................................................................31
2.1 Machines, hardware and software.............................................................................31
2.1.1 Equipment and instrument ..................................................................................31
2.1.2 Recombinant DNA techniques............................................................................32
2.1.3 Cell culture..........................................................................................................32
2.1.4 Protein analysis ...................................................................................................32
2.1.5 Immunofluorescense microscopy .......................................................................33
2.2 Recombinant DNA techniques ...................................................................................34
2.2.1 Constructs and vectors ........................................................................................34
2.2.2 PCR .....................................................................................................................35
2.2.2.1 Primers and template cDNAs.......................................................................35
7Contents
2.2.2.2 Reaction mixture..........................................................................................36
2.2.2.3 PCR program ...............................................................................................37
2.2.2.4 Two-step PCR..............................................................................................37
2.2.3 Isolation and purification of PCR products ........................................................37
2.2.3.1 Materials ......................................................................................................37
2.2.3.2 Agarose gel electrophoresis .........................................................................38
2.2.3.3 Isolation and purification of PCR products from agarose gels....................38
2.2.4 Enzymatic modification of cDNA fragments .....................................................38
2.2.4.1 Enzymes.......................................................................................................38
2.2.4.2 Restriction enzyme treatment ......................................................................39
2.2.4.3 Alkaline phosphatase treatment ...................................................................39
2.2.4.4 Ligation of cDNA fragments .......................................................................39
2.2.5 Transformation of E. coli....................................................................................40
2.2.5.1 Materials ......................................................................................................40
2.2.5.2 Preparation of competent cells.....................................................................40
2.2.5.3 Transformation of E. coli.............................................................................40
2.2.6 Preparation of plasmid DNA from E. coli ..........................................................41
2.2.6.1 Materials41
2.2.6.2 Small-scale plasmid DNA preparation (mini-prep).....................................41
2.2.6.3 Mini-prep DNA analysis..............................................................................42
2.2.6.4 Large-scale plasmid DNA preparation (maxi-prep) ....................................42
2.2.7 DNA sequencing.................................................................................................42
2.3 Cell culture and cell lines ..........................................................................................43
2.3.1 Materials .............................................................................................................43
2.3.2 Cell lines and medium ........................................................................................43
2.3.3 Cell culture..........................................................................................................44
2.3.4 Transfection of mammalian cells........................................................................44
2.3.4.1 Materials ......................................................................................................44
2.3.4.2 Transfection mixture...................................