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The Role of Photoreceptor ABC Transporter ABCA4 in Retinal And THE ROLE OF PHOTORECEPTOR ABC TRANSPORTER ABCA4 IN RETINAL AND LIPID TRANSPORT AND STARGARDT MACULAR DEGENERATION by Faraz Quazi A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2013 © Faraz Quazi, 2013 Abstract ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters implicated in the clearance of retinoids from photoreceptor outer segments and associated with Stargardt macular degeneration. Stargardt patients display lipofuscin deposits and photoreceptor degeneration that invariably lead to the loss of central vision. In biochemical and knockout mice studies, ABCA4 has been implicated in the transport of N-retinylidene- PE, the Schiff base conjugate formed from all-trans retinal and phosphatidylethanolamine (PE). The principal challenge in assessing direction and transport has been impeded by the unstable hydrophobic nature of the proposed substrate. As part of this study, a novel biochemical transport assay was developed and used to show that ABCA4 actively flips N- retinylidene-PE from the lumen to the cytosolic side of membranes. This is the first mammalian ABC transporter shown to function as an importer. 11-cis retinal delivered in excess to photoreceptors is a defining cause of lipofuscin/A2E formation. Purified ABCA4 transports 11-cis retinylidene-PE. By HPLC analysis, 11-cis retinal also showed isomerization to all-trans retinal with PE present in discs. Thus, ABCA4 insures the complete removal of N-retinylidene-PE conjugates from the disc- lumen, thereby preventing accumulation of lipofuscin precursors including A2PE. Stargardt mutants which showed reduced functionality were rescued by allosteric enhancement of ATPase with dronedarone, an anti-arrhythmic drug, acting in concert with an increase in N- retinylidene-PE transport. Additionally, protein misfolding was selectively restored by 4- phenylbutyrate, a chemical chaperone. ii Genetic mutations in several ABCA subfamily transporters cause severe-inherited lipid disorders. The structure-function relationships underlying substrate specificity remain unclear. ABCA1 linked to Tangier disease has been implicated in the export of cholesterol and phospholipids to the apolipoproteinA-I acceptor by cell-based assays. In this study, biochemical analysis indicated that purified and reconstituted ABCA1 actively flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the lumenal side of membranes, while ABCA7 preferred phosphatidylserine. In contrast, ABCA4 transported PE in the reverse direction. Tangier and Stargardt mutants showed reduced lipid transport activities. This thesis demonstrates the importance of ABCA4 as a unique ABC importer, reports the first reconstitution of phospholipid flippase activity for ABCA transporters, and identifies several compounds as potential therapeutic drugs for Stargardt disease. iii Preface A version of chapter 2 has been published. [Quazi, F.], Lenevich. S., Molday, R.S., 2012. The ABC Transporter ABCA4 Linked to Stargardt Disease Functions as an N-retinylidene- phosphatidylethanolamine and Phosphatidylethanolamine Importer. Nat Comm. 3:925. Stepan Lenevich synthesized the N-retinyl PE analog. Mice retina were dissected by Laurie Molday and Hidayat Djajadi. I performed the experiments and wrote the original manuscript. The final manuscript was edited by Molday, R.S. A version of chapter 4 has been accepted. [Quazi, F.], Molday R.S. 2013. Differential Phospholipid Substrates and Directional Transport by ATP Binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants. J. Biol. Chem. In press. I performed the experimental work and wrote the original manuscript. The final manuscript version was edited by Molday, R.S. iv Table of Contents Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iv Table of Contents .................................................................................................................... v List of Tables ......................................................................................................................... xii List of Figures ....................................................................................................................... xiii Acknowledgements ............................................................................................................... xv Abbreviations ...................................................................................................................... xvii Chapter 1: Introduction .......................................................................................................... 1 1.1 Casting Light ............................................................................................................. 1 1.1.1 Anatomy of the Eye .............................................................................................. 1 1.1.2 The Retina ............................................................................................................. 3 1.1.3 Photoreceptors and Retinal Chromophore ............................................................ 4 1.1.4 Retinal Pigment Epithelium .................................................................................. 7 1.1.5 Phototransduction Cascade ................................................................................... 8 1.1.6 Visual Cycle .......................................................................................................... 9 1.2 Retinoid Fluxes and Lipofuscin .............................................................................. 10 1.3 Retinal Disorders .................................................................................................... 13 1.3.1 Stargardt Disease ................................................................................................ 15 1.4 The ABC Transporter Superfamily ......................................................................... 16 1.4.1 Role of ABC Proteins in Phospholipid Transport ............................................... 19 1.4.2 General Mechanisms ........................................................................................... 21 1.4.3 ABCA Transporters ............................................................................................ 24 v 1.4.3.1 ABCA1 ....................................................................................................... 25 1.4.3.2 ABCA7 ....................................................................................................... 26 1.4.3.3 ABCA4 ....................................................................................................... 27 1.4.3.3.1 ABCA4 Knockout Mice ........................................................................ 29 1.4.3.3.2 ABCA4 and the Visual Cycle ............................................................... 29 1.4.3.3.3 ABCA4 and Implication in Stargardt Disease ...................................... 30 1.5 Thesis Investigation ................................................................................................ 34 Chapter 2: The ABC Transporter ABCA4 Linked to Stargardt Disease Functions as an N- retinylidene-phosphatidylethanolamine and Phosphatidylethanolamine Importer* ............... 39 2.1 Introduction ............................................................................................................. 39 2.2 Experimental Procedures ........................................................................................ 41 2.2.1 Materials ............................................................................................................. 41 2.2.2 Isolation of Retina and Outer Segment Membranes ........................................... 42 2.2.3 Generation of ABCA4 Mutant Constructs .......................................................... 42 2.2.4 Expression and Purification of ABCA4-1D4 from HEK293T Cells .................. 42 2.2.5 Purification of ABCA4 ....................................................................................... 43 2.2.6 Preparation of Donor Proteoliposomes and Acceptor Liposomes ...................... 43 2.2.7 Measurement of ATP-Dependent [3H]-All-Trans Retinal (ATR) Transfer ....... 44 2.2.8 Preparation of Rod Outer Segment (ROS) Disc Vesicles ................................... 45 2.2.9 Retinoid Transfer Assay in Bovine/Mouse Outer Segment Disc Vesicles ......... 46 2.2.10 Fl-Labeled Phospholipid Flipping Assay ........................................................ 47 2.2.11 Analysis of Retinoid Binding ......................................................................... 47 2.2.12 Preparation and Purification of N-retinyl PE .................................................. 48 vi 2.2.13 ATPase Assay ................................................................................................. 49 2.2.14 SDS-PAGE and Western Blots ....................................................................... 50 2.3 Results ..................................................................................................................... 51 2.3.1 ATP-Dependent Transfer
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