Robert Molday

ABCA4 is an ABC transporter encoded by the ABCA4 gene. This transporter is predominantly expressed in the disc membranes of photoreceptor cells in the retina where it plays a crucial role. ABCA4 transports N-retinylidene-phosphatidylethanolamine (N-ret-PE), the Schiff-base adduct formed by all-trans-retinal or 11-cis-retinal with phosphatidylethanolamine, across the disc membranes of photoreceptors. In doing so, ABCA4 acts as an importer that helps to prevent the formation and accumulation of harmful bisretinoids in the disc membranes of photoreceptors. To date, more than 1000 point-mutations in the ABCA4 gene have been linked to the development of Stargardt disease (STGD1) and other retinopathies. Although significant progress has been made in identifying disease-causing mutations in ABCA4, understanding genotype-phenotype relationships remains challenging because most patients are compound heterozygous for disease mutations in ABCA4 and phenotypic variations have been found in individuals with the same genotype. Furthermore, with some exceptions, analysis of STGD1 has relied on clinical and genetic data while lacking any biochemical characterization of disease-causing variants. As a result, it remains to be determined the extent to which the molecular properties of ABCA4 disease variants contribute to the etiology of STGD1 in relation to other factors, such as age, lifestyle, and environmental factors. To address this issue, a major aim of this thesis is to understand how the protein solubilization levels and residual activity of ABCA4 variants influence the disease outcome of STGD1 patients harboring these mutations. Furthermore, to help us delineate the different molecular mechanisms that underpin STGD1, and to help us understand the transport mechanism of ABC transporters in general, this thesis will characterize disease variants found in the transmembrane and the nucleotide binding domains of ABCA4. An in-depth functional analysis of the transmembrane domains (TMDs) will help us identify residues essential for the binding and translocation of N-ret-PE. Likewise, an investigation of common disease variants localized to the Walker-A motif of the nucleotide-binding-domains (NBDs) could help understand how ABCA4 couples binding and hydrolysis of ATP with N-ret-PE transport. Taken together, this information will be useful to develop a full understanding of the mechanism of transport of ABCA4 and similar transporters.

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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. 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.

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P₄-ATPases are a family of membrane transporters which have been implicated in the energy-dependent transport of aminophospholipids from the exocytoplasmic to cytoplasmic surface of biological membranes. This thesis investigation examined the structure-function relationships of ATP8A2, a novel member of the P₄-ATPase family initially discovered in a proteomic study of photoreceptor outer segments. Photoreceptor outer segments are organelles which consist of stacks of membraneous discs containing visual pigment molecules. ATP8A2 is shown to be present in photoreceptor outer segment discs and preferentially transports phosphatidylserine towards the cytosolic leaflet, providing the first direct demonstration of lipid transport by a purified mammalian P₄-ATPase. CDC50A, the β-subunit of ATP8A2 was discovered using mass spectrometry and Western blotting. Subunit interactions are mediated through the extracellular and membrane domains of CDC50A. The N-terminal domain of CDC50A appears to play a role in pump modulation. ATP8A2 forms a phosphoenzyme intermediate at Asp⁴¹⁶ and phosphatidylserine appears to be transported in a similar manner to that of other cation-transporting P-type ATPases. The phosphoenzyme exists in two distinct conformations: E₁P and E₂P. The E₂P form interacts with aminophospholipids. Lys⁸⁷³ in transmembrane segment M5 located in a region known to be important for cation binding for P-type ATPases is critical for phosphatidylserine binding. Glu¹⁹⁸ in the DGET motif is essential for E₂P dephosphorylation. ATP8A2 gene expression was disrupted in mice using a neo cassette. Knockout mice develop short outer segments and visual function is impaired. Modification of transbilayer asymmetry and composition appear to be responsible for reduced visual function rather than structural defects. The decrease in outer segment length suggests that ATP8A2 is involved in vesicular trafficking of proteins to the outer segment by regulating the step of vesicle budding possibly from the trans-Golgi network. We speculate that ATP8A2 plays a similar role in other neuronal cells and thus provide insight into the phenotype of human disorders caused by mutations in ATP8A2. In summary, this study has identified for the first time the transported substrate of a mammalian P₄-ATPase, discovered protein-protein interactions regulating function, elucidated the mechanism of lipid transport, and illuminated the function of ATP8A2 in photoreceptor and neuronal biology.

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ABCA4, also known as ABCR or the rim protein, is a member of the family of ATP binding cassette (ABC) proteins expressed in rod and cone photoreceptors. Mutations in ABCA4 have been linked to Stargardt macular degeneration and related retinal degenerative diseases and implicated in the transport of retinoid compounds across the outer segment disk membrane.This dissertation investigation describes various aspects of the structure and function relationships for ABCA4 and examines the mechanisms by which mutations in ABCA4 lead to various retinal degenerative diseases. A pull-down was employed to identify the retinoid substrate that interacts with ABCA4. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, ~1 mol of N-retinylidene-phosphatidylethanolamine was bound per mol of ABCA4 with an apparent Kd of 5.4 μM. These results provided the first direct biochemical evidence for the identity of the retinoid substrate for ABCA4. To determine the role of that C-terminus of ABCA4 plays in structure and function, a series of deletion and chimera mutants of ABCA4 was expressed, purified by immunoaffinity chromatography, and their biochemical properties analyzed. Removal of the C-terminal 30 amino acids including a conserved VFVNFA motif or substitution of the VFVNFA motif with alanines resulted in the complete loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal stimulated ATPase activity and caused retention of ABCA4 in the endoplasmic reticulum. In contrast mutants lacking the C-terminal 8, 16 or 24 amino acids but retaining the VFVNFA motif were active. These studies indicated that the VFVNFA motif in ABCA4 is required for proper folding of ABCA4 into a functionally active protein. These results provide a molecular rationale for the disease phenotype displayed by individuals with mutations in the C-terminus of ABCA4. Co-IP studies coupled to mass spectrometry were performed to identify novel protein interactors of ABCA4. Rhodopsin and arrestin (including a splice variant of arrestin, p⁴⁴) were identified and confirmed by western blotting. All-trans-retinal was found as a regulator of this interaction.This study for the first time has identified the retinoid substrate for ABCA4, demonstrated a role for the C-terminus and has found protein partners of ABCA4.

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G protein- coupled receptors (GPCR5) are seven transmembrane receptors that comprise thelargest superfamily of proteins in the body that are involved in a variety of fundamentalprocesses including sight, smell, tactile as well as nervous responses. Tightly coordinated,control of these receptors is critical for normal physiology. Emphasis has been placed inpharmaceuticals to find ways to inhibit or accelerate these processes in GPCR implicateddiseases. Although tertiary structural information would be beneficial for rational drug design,little is known about the structure of GPCRs despite extensive research in the field. Only twohigh resolution structures exist for any mammalian GPCR. Structural studies are challenged bythe intrinsic difficulty associated with purifying these receptors in high quality and quantity. Toovercome these challenges, we propose a mass spectrometric, sequence-based approach tocharacterize GPCRs allowing for the rapid characterization of an ectopically as well asendogenously expressed receptor employing a receptor tag. The sensitivity of the approachallows for the implementation of a mammalian expression system with the physiologicallyrelevant post-translational modifications (PTMs). The sites of N-linked glycosylation has beenmapped and receptor isoforms were identified for a model GPCR, CXCR4 and a related receptor,CCR5. This approach is versatile and applicable to other membrane proteins such as ATPbinding cassette (ABC) transporters and has been adapted into an antibody screening platform.As proof- of- principle, we have developed a monoclonal anti-human CXCR4 antibody withbroad applications and potential for clinical diagnostics.In view of the challenges preventing high resolution three-dimensional structure determination,photoaffinity crosslinking was combined with our sequence-based strategy for receptor bindingsite footprinting. CXCR4 ligand analogs containing a non-natural amino acid photocrosslinker,benzophenylalanine and a biotin tag for complex isolation were chemically synthesized andcrosslinked to CXCR4 expressed on intact cells. Individual components of the receptor-ligandcomplex were identified by western blotting and tandem mass spectrometry. Regions on thebound receptor that were protected from protease treatment and consequently tandem MS/MSsequencing were identified as potential sites of ligand-receptor contact. These regions wereidentified as the receptor N-terminus and/or the first extracellular loop for ligandbinding/docking.

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