Identification of a BACE dimer and characterization of its biochemical and enzymatic properties [Elektronische Ressource] / vorgelegt von Gil Gregor Westmeyer

Gil Gregor Westmeyer

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem
Adlolf-Butenandt-Institut
Lehrstuhl für Stoffwechselbiochemie
der
Universität München
Vorstand: Prof. Dr. rer. nat. Christian Haass




Identification of a BACE dimer and characterization of its
biochemical and enzymatic properties




Dissertation
zum Erwerb des Doktorgrades der Medizin
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität München





vorgelegt von
Gil Gregor Westmeyer
aus Essen
2006

Mit Genehmigung der Medizinischen Fakultät
der Universität München





Berichterstatter: Prof. Dr. C. Haass
2. Berichterstatter: Prof. Dr. Th. Heinzeller



Mitberichterstatter: Prof. Dr. Dr. h.c. Th. Brandt
Mitberichterstatter: Prof. Dr. H. A. Kretschmar




Mitbetreuung durch den
promovierten Mitarbeiter: Dr. Michael Willem



Dekan: Prof. Dr. med. D. Reinhardt




Tag der mündlichen Prüfung: 20.7.2006


2 C ONTENTS
1 Contents
1 CONTENTS......................................................................................................... 3
1.1 Index of Figures................................................................................................. 8
2 ZUSAMMENFASSUNG...................................................................................... 9
3 SUMMARY........................................................................................................ 10
4 LIST OF ABBREVIATIONS.............................................................................. 11
5 INTRODUCTION............................................................................................... 14
5.1 Epidemiology of Dementia and Alzheimer´s Disease ................................. 14
5.2 Definition of Alzheimer`s Dementia............................................................... 14
5.3 Morphology...................................................................................................... 15
5.4 Pathogenesis................................................................................................... 17
5.4.1 β-Amyloid Precursor Protein (APP) .................................................................... 17
5.4.2 Proteolytic Processing of APP ............................................................................ 17
5.4.2.1 Amyloid- β as the necessary pathogen of AD............................................ 18
5.4.2.2 Toxicity of Amyloid- β ................................................................................. 18
5.4.3 The Tau pathology as a consequence of A β formation ...................................... 20
5.5 Risk factors...................................................................................................... 21
5.5.1 Environmental risk Factors.................................................................................. 21
5.5.2 Genetic risk factors ............................................................................................. 21
5.5.2.1 Missense mutations in APP and overexpression of APP.......................... 22
5.5.2.2 the Presenilins ...................................................... 23
5.5.2.3 Apolipoprotein E4 Allele ............................................................................ 23
5.6 Proteolytic processing of APP....................................................................... 23
5.6.1 The α-Secretase.................................................................................................. 23
5.6.2 BACE2 has a non-amyloidogenic effect ............................................................. 24
5.6.2.1 Sequence Structure of BACE2.................................................................. 25
5.6.2.2 Tissue Expression of BACE2 .................................................................... 26
5.6.2.3 Localization of BACE2............................................................................... 26
5.6.2.4 Cleavage Site .......................................................................... 26
5.6.2.5 3D Structure of BACE2 ............................................................................. 27
5.7 The amyloidogenic pathway 27
5.7.1 The Presenilin Complex................................................................................ 27
3 C ONTENTS
5.7.1.1 Role of the Presenilin complex in signal transduction............................... 29
5.7.2 The β-Secretase.................................................................................................. 30
5.7.2.1 Identification of β-Secretase (BACE) ....................................................... 30
5.7.2.2 Domain Structure of BACE........................................................................ 31
5.7.2.3 Mouse Models studying BACE.................................................................. 32
5.7.2.3.1 BACE Knock-out Mice 32
5.7.2.3.2 Transgenic Mice overexpressing BACE ............................................. 32
5.7.2.4 Posttranslational Maturation of BACE...................................................... 33
5.7.2.4.1 The role of the pro-domain of BACE................................................... 33
5.7.2.4.2 Posttranslational Modifications of BACE ............................................ 33
5.7.2.4.3 Dissulfide Bonds in BACE 34
5.7.2.5 Localization and Trafficking of BACE........................................................ 34
5.7.2.5.1.1 Cellular localization of BACE ....................................................... 34
5.7.2.5.1.2 Cellular localization of β-secretase activity .................................. 34
5.7.2.5.1.3 Trafficking through the Exocytotic Pathway................................. 34
5.7.2.5.1.4 Role of the TM domain of BACE.................................................. 36
5.7.2.5.1.5 Polarized Sorting of BACE........................................................... 36
5.7.2.5.1.6 Lipid Rafts and BACE .................................................................. 36
5.7.2.6 Tissue Expression and Tissue localization of β-secretase activity ........... 38
5.7.2.6.1 BACE mRNA – distribution................................................................. 38
5.7.2.6.2 Tissue Expression of the BACE protein.............................................. 38
5.7.2.6.3 Altered protein expression of BACE in AD patients............................ 39
5.7.2.6.4 Mechanisms regulating BACE expression.......................................... 39
5.7.2.7 Crystallographic Structure of BACE .......................................................... 40
5.7.2.8 Subsite Specificity of BACE ...................................................................... 41
5.7.2.8.1 Cleavage at Asp 1 and the “swedish mutation” .................................. 41
5.7.2.8.2 Alternative cleavage at Glu11 by BACE ............................................. 42
5.7.2.8.3 Sequence of alternative cleavages of BACE...................................... 43
5.7.2.8.4 The ideal substrate sequence of BACE.............................................. 44
5.7.2.9 Known substrates for BACE 45
5.7.2.10 Enzyme Kinetics of BACE ..................................................................... 47
5.7.2.10.1 Comparison of BACE ectodomain and full length BACE.................. 47
5.7.2.10.2 Kinetic parameters of the pro-domain............................................... 47
5.7.3 Interaction partners of BACE .............................................................................. 47
6 GOALS OF THE CURRENT WORK ................................................................ 50
7 MATERIAL AND METHODS............................................................................ 52
7.1 Cloning............................................................................................................. 52
7.1.1 Existing constructs used ..................................................................................... 52
7.1.2 Cloning Vectors................................................................................................... 52
7.1.2.1 Maps of the Vectors .................................................................................. 52
7.1.3 Summary of Cloning of BACE expression constructs......................................... 52
7.1.4 Generation of myc-tagged BACE variants .......................................................... 53
4 C ONTENTS
7.1.4.1 Primers used for cloning............................................................................ 53
7.1.4.1.1 BACE-pA:............................................................................................ 53
7.1.4.1.2 BACE-GPI........................................................................................... 53
7.1.4.1.3 BACE-KKXX ....................................................................................... 53 <