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

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