Proteolysis of the receptor for advanced glycation end products by matrix metalloproteinases [Elektronische Ressource] / Ling Zhang

Qun

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2006
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Proteolysis of the Receptor for Advanced
Glycation End Products by Matrix Metalloproteinases





Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaften

Am Fachbereich Biologie
Der Johannes Gutenberg-Universität Mainz





Ling Zhang
geb. am 22-10-1972 in Wuhan, China



Mainz 2006





i Table of Content
Table of Contents

Table of Contents...................................................................................................................................ii
1. Introduction....................................................................................................................................... 1
1.1 The Aβ peptide................................................................................................................................. 1
1.1.1 Aβ peptide production and its pathologic role in Alzheimer’s disease (AD)........................... 1
1.1.2 Aβ peptide clearance from the brain .......................................................................................... 2
1.2 RAGE (receptor for advanced glycation end products) .............................................................. 4
1.2.1 Structure 4
1.2.2 Expression patterns...................................................................................................................... 5
1.2.3 Extracellular ligands and their pathophysiolologic functions.................................................. 5
1.2.4 Intracellular signaling.................................................................................................................. 7
1.2.5 RAGE-Aβ interaction 8
1.2.6 Isoforms of RAGE........................................................................................................................ 9
1.2.6.1 Full-length RAGE 9
1.2.6.2 C-terminal truncated RAGE .................................................................................................... 10
1.2.6.3 Soluble RAGE (sRAGE) .......................................................................................................... 10
1.2.6.4 N-truncated RAGE................................................................................................................... 12
1.3 Protein Ectodomain Shedding ..................................................................................................... 12
1.3.1 Ectodomain shedding................................................................................................................. 12
1.3.2.1 A disintegrin and metalloproteinases (ADAMs)...................................................................... 14
1.3.2.2. Matrix metalloproteinases (MMPs)........................................................................................ 14
2. Aim of the study .............................................................................................................................. 19
3. Materials and Methods 21
3.1 Materials ........................................................................................................................................ 21
3.1.1 Chemicals and Media 21
3.1.2 Enzyme and Kit systems............................................................................................................ 22
3.1.3 Laboratory instruments and accessories.................................................................................. 23
3.1.4 Solutions, buffers and media 24
3.1.5 Antibodies ................................................................................................................................... 26
3.1.6 Bacterial strains and cell lines................................................................................................... 28
3.1.7 Plasmids ...................................................................................................................................... 29
3.1.8 Oligonucleotides ......................................................................................................................... 32
3.1.9 Inhibitors and Activators .......................................................................................................... 33
3.2 Methods.......................................................................................................................................... 34
3.2.1 Molecular biology....................................................................................................................... 34
3.2.1.1 Maintenance of bacterial strains ............................................................................................. 34
3.2.1.2 Preparation of competent bacteria........................................................................................... 34
3.2.1.3 Transformation of E. coli......................................................................................................... 34
3.2.1.4 Plasmid Minipreparation 34
3.2.1.5 Plasmid Maxipreparation ........................................................................................................ 35
3.2.1.6 Enzymatic modification of DNA.............................................................................................. 36
3.2.1.7 DNA electrophoresis ................................................................................................................ 37
3.2.1.8 DNA purification...................................................................................................................... 38
3.2.1.9 DNA Sequencing 38
3.2.1.10 Polymerase Chain Reaction (PCR) ....................................................................................... 38
3.2.2 Protein biochemical methods .................................................................................................... 40
3.2.2.1 Determination of protein concentration (Bradford assay) ..................................................... 40
3.2.2.2 TCA precipitation of proteins .................................................................................................. 40
3.2.2.3 Chloroform-Methanol precipitation of proteins ..................................................................... 41
3.2.2.4 SDS-polyacrylamide gel electrophoresis ................................................................................. 41
ii Table of Content
3.2.2.5 Western Blot ............................................................................................................................. 42
3.2.2.6 Coomassie staining of polyacrylamide gels............................................................................. 43
3.2.2.7 Biotinylation of cell surface proteins....................................................................................... 43
3.2.2.8 Gelatin zymography assay........................................................................................................ 44
3.2.3 Cell biology methods.................................................................................................................. 44
3.2.3.1 Cell culture ............................................................................................................................... 44
3.2.3.2 Trypsinizing adhesive cells....................................................................................................... 44
3.2.3.3 Subculture adhesive cells......................................................................................................... 45
3.2.3.4 Freezing cells............................................................................................................................ 45
3.2.3.5 Thawing cells 45
3.2.3.6 Transfect cells with lipofectamine 2000 .................................................................................. 45
3.2.3.7 Transfect cell with DEAE-Dextran reagent............................................................................ 46
3.2.3.8 Selection of stable transfectants .............................................................................................. 46
3.2.4 Methods for analysis of ectodomain shedding......................................................................... 46
3.2.4.1 General procedures .................................................................................................................. 46
3.2.4.2 Handling of secretion medium ................................................................................................ 48
3.2.4.3 Handling of cells ...................................................................................................................... 48
3.3 Data analysis.................................................................................................................................. 48
4. Result................................................................................................................................................ 49
4.1 Overexpression of myc-RAGE or wild-type RAGE in Flp-In 293 cells as well as COS-7 cells
.............................................................................................................................................................. 49
4.2 Human soluble RAGE is released from the cell membrane...................................................... 50
4.2.1 Cleavage products of full-length RAGE.....................................................................