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Identification and Characterization of
ABCA1-Interactive Proteins and Their
Relevance to Atherosclerosis



Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)
an der Fakultät für Chemie und Pharmazie
der Universität Regensburg




vorgelegt von
Salim Maa Bared
Regensburg im April 2005
This work was performed at the institute of Clinical Chemistry and Laboratory
Medicine at the University of Regensburg between August 2000 and December
2004 under the supervision of Prof. Dr. Gerd Schmitz.














Date of colloquium: 17. 10. 2005
Board of examiners: Chairman: Prof.Dr. Sigurd Elz
First Examiner: Prof. Dr. Armin Buschauer Second Examiner: Prof. Dr. Gerd Schmitz
Third Examiner: Prof. Dr. Jörg Heilmann




























„When you make the finding yourself, . . . even if you're the last person on earth to
see the light, you'll never forget it. “

Carl Sagan
American astronomer and science writer (1934-1996)
Acknowledgement

I would like to express my gratitude to Prof. Dr. Gerd Schmitz for supporting me in
performing my PhD at his fine institute and who was such a great help in correcting my
thesis always pushing it with new ideas and interesting views.
Further, my thanks go to PD. Dr. Christa Buechler, my group leader, for sharing her
knowledge with me. Special thanks go to Prof. Dr. Armin Buschauer, my supervisor at the
Faculty of Pharmacy at Regensburg University as well as to my colleagues and friends:
Prof. Dr. Charalampos Aslanidis, Dr. Alfred Boettcher and Dr. Wolfgang Kaminski, all
specialists in their fields.
My friends: Margot Grandl, Alex Sigruener, Mirko Ritter and many others.
Last, but definitely not least, for their technical assistance: Nadine, Connie, Andrea and
Sylvia.
I thank my Parents who spared no efforts and always care so much for me.
On top of them all, I thank the crown of my life, Rania. Without her, many things would
have come in another unexpected way.



Table of contents
I. INTRODUCTION............................................................................................................................. 1
1. PATHOGENESIS OF ATHEROSCLEROSIS ............................................................................... 2
1.1. RESPONSE TO INJURY HYPOTHESIS ........................................................................................... 2
1.2. RESPONSE TO RETENTION HYPOTHESIS..................................................................................... 4
2. METABOLISM OF APOB CONTAINING LIPOPROTEINS.......................................................... 5
2.1. CHYLOMICRONS ........................................................................................................................ 5
2.2. VERY LOW DENSITY LIPOPROTEIN (VLDL) ................................................................................. 6
2.3. LOW DENSITY LIPOPROTEIN (LDL)............................................................................................. 6
2.4. LDL-RECEPTOR 6
2.5. NIEMANN-PICK DISEASE TYPE C, A CHOLESTEROL TRAFFIC DISORDER....................................... 7
3. MODIFIED LOW DENSITY LIPOPROTEINS................................................................................ 8
3.1. ENZYMATICALLY MODIFIED LOW DENSITY LIPOPROTEIN (E-LDL)................................................. 9
3.2. OXIDIZED LOW DENSITY LIPOPROTEIN (OX-LDL)........................................................................ 9
4. CHOLESTEROL INFLUX PATHWAYS ...................................................................................... 10
4.1. CLATHRIN-DEPENDENT ENDOCYTOSIS ..................................................................................... 10
4.2. CLATHRIN-INDEPENDENT ENDOCYTOSIS................................................................................... 12
4.2.1. Phagocytosis ................................................................................................................. 13
4.2.2. Pinocytosis and Macropinocytosis ................................................................................ 13
4.2.3. Caveolin-Dependent Endocytosis ................................................................................. 14
4.2.4. Novel Uptake Mechanisms............................................................................................ 15
4.2.4.1 Surface Connected Compartments (SSC) .............................................................................15
4.2.4.2 Endocytosis Through Compartments Involving CD14............................................................15
4.2.4.3. Deep Tubular Invaginations......16
4.2.4.4. Continuous Cellular Membrane System................................................................................17
5. METABOLISM OF APO-AI CONTAINING HIGH DENSITY LIPOPROTEIN (HDL) .................................... 18
5.1. CHOLESTEROL EFFLUX FROM MACROPHAGES .............................................................. 19
5.2. MULTIPLE PATHWAYS FOR CELLULAR CHOLESTEROL EFFLUX.................................................... 20
5.2.1. Passive Diffusion ........................................................................................................... 20
5.2.2. SR-BI-Facilitated Diffusion of Cholesterol to HDL......................................................... 21
5.2.3. ABCA1-Mediated Active Efflux...................................................................................... 22
5.3. ATP-BINDING CASSETTE TRANSPORTER.................................................................................. 23
5.3.1. ABCA Subfamily ............................................................................................................ 24
5.3.2. ABCA1 and Familial HDL-Deficiency ............................................................................ 24
5.4. TANGIER DISEASE................................................................................................................... 25
5.5. ATP-SYNTHASE, A NEW CONCEPT IN THE DUAL REGULATION OF ENDOCYTOSIS AND APOAI-
MEDIATED CHOLESTEROL EFFLUX .................................................................................................. 25
6. VESICLE FORMATION IN THE GOLGI-DEPENDENT ABCA1 SECRETORY PATHWAY...... 27
6.1. COATED VESICLE ASSEMBLY ................................................................................................... 32
6.2. SYNTAXINS AS CONSTITUENTS OF THE SNARE FAMILY ............................................................ 35
6.3. THE RAB PATHWAY AND ITS INVOLVEMENT IN VESICULAR TRANSPORT....................................... 36
7. GENE ARRAYS........................................................................................................................... 37
8. THE YEAST TWO-HYBRID SYSTEM......................................................................................... 39
II. AIM OF THE THESIS................................................................................................................... 41
III. MATERIALS.............. 43
1. CHEMICALS................................................................................................................................44
2. STANDARDS AND KITS................................................................................................................. 44
3. RADIOACTIVE MATERIALS ............................................................................................................ 45
4. ENZYMES.... 45
5. HUMAN PRIMARY CELLS AND CELL LINES ...................................................................................... 46
5.1. Monocytes....... 46
5.2. Fibroblasts ........................................................................................................................ 46
5.3. Human cell lines ............................................................................................................... 46
6. BACTERIA (E.COLI) ..................................................................................................................... 46
7. PLASMIDS .................................................................................................................................. 46
8. MEDIA AND BUFFERS .................................................................................................................. 47
9. MICROARRAYS ........................................................................................................................... 47
10. TECHNICAL EQUIPMENTS 47
11. SILENCING RNA....................................................................................................................... 49
12. GENE BANKS............................................................................................................................ 49
13. FILMS AND MEMBRANES ............................................................................................................ 49
14. ANTIBODIES ............................................................................................................................. 49
15. PREPARATION OF SOLUTIONS ................................................................................................... 50
IV. METHODS .................................................................................................................................. 53
1. PRIMARY CELLS AND CELL LINES.................................................................................................. 54
1.1. Elutriation of human monocytes ....................................................................................... 54
1.2. Cultivation and differentiation of human monocytes ........................................................ 55
1.3. Cultivation of human tumor cell lines................................................................................ 55
1.4. Transfection of cell lines................................................................................................... 55
2. LIPOPROTEINS............................................................................................................................ 56
2.1. Isolation of Lipoproteins.................................................................................................... 56
2.2. Enzymatic and Oxidative Modification of LDL.................................................................. 56
3. PROTEIN METHODS .................................................................................................................... 56
3.1. Isolation of proteins .......................................................................................................... 56
3.2. Protein concentration Determination ................................................................................ 56
3.3. SDS-PAGE and western blotting...................................................................................... 57
3.4. Isolation of phagosomes with phagobeads ...................................................................... 57
3.5. Sucrose gradient centrifugation and isolation of rafts ...................................................... 58
3.6. Co-Immunoprecipitation ................................................................................................... 59
3.7. Immunofluorescent staining and microscopy ................................................................... 59
3.8. Flow cytometry ................................................................................................................. 59
4. RADIOACTIVE LIPID EFFLUX ......................................................................................................... 60
5. CULTIVATION OF ESCHERICHIA COLI............................................................................................ 60
6. NUCLEIC ACID METHODS ............................................................................................................. 60
6.1. Restriction enzyme digestion of DNA............................................................................... 60
6.2. DNA gel electrophoresis and DNA extraction from agarose gels .................................... 61
6.3. Cloning of DNA fragments................................................................................................ 61
6.4. Small interference RNA (siRNA) ...................................................................................... 62
6.5. RNA isolation....... 62
6.6. Quality assessment and quantification of RNA (Agilent).................................................. 63
6.7. RNA gel electrophoresis................................................................................................... 63
6.8. Reverse Transcription PCR (RT-PCR)............................................................................. 64
6.9. Real-Time PCR ................................................................................................................ 64
6.9.1. Gene Expression Monitoring With SYBR-Green-I Dye (LightCycler) ........................... 64
6.9.2. Gene Expression Monitoring With Hydrolysis Probes (Taqman).................................. 65
7. AFFYMETRIX® MICROARRAYS..................................................................................................... 66
8. YEAST-TWO-HYBRID SYSTEM 68
9. MASS SPECTROMETRY................................................................................................................ 68
V. RESULTS .................................................................................................................................... 69
1. IDENTIFICATION OF ABCA1 INTERACTIVE PROTEINS BY YEAST-TWO-HYBRID SYSTEM ....................... 70
2. CHARACTERIZATION OF THE ABCA1-FADD COMPLEX ................................................................. 71
3. CON OF THE ABCA1 - 2-SYNTROPHIN COMPLEX................................................. 76
4. PROTEIN-PROTEIN INTERACTIONS OF SYNTAXIN 13, ABCA1 AND FLOTILLIN-1 .............................. 79
5. LIPOPROTEIN ANALYSIS OF THE PALLIDIN KO MICE....................................................................... 87
6. IDENTIFICATION OF MACROPHAGE-SPECIFIC GENES INVOLVED IN LIPID TRAFFICKING USING GENE-
CHIPS............................................................................................................................................ 89
6.1. E-LDL mediated cholesterol flux ...................................................................................... 93
6.2. Ox-LDL mediated cholesterol flux .................................................................................... 96
6.3. Biochemical Pathways and Candidate Genes With Relevance to Lipid Traffic in
Macrophages........................................................................................................................... 97
6.3.1. Regulation of Genes in the Endocytic and Phagocytic Pathway in Macrophages....................98
6.3.2. Regulation of Genes in the Coated Vesicle Pathway in Macrophages.....................................99
6.3.3. Regulation of Genes in the Vesicular Transport Pathway in Macrophages............................100
6.3.4. Regulation of Genes in the COP Machinery in Macrophages ................................................101
6.3.5. Regulation of Genes in the Calcium Signalling Pathway in Macrophages .............................102
6.3.6. Regulation of ABCA1-Related Genes in the Exocytosis Pathway in Macrophages ...............104
VI. DISCUSSION............................................................................................................................ 106
VII. REFERENCES ........................................................................................................................ 121
VIII. SUMMARY ............................................................................................................................. 142
PUBLICATION LIST...................................................................................................................... 145








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