Robert Molday


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Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Biochemical analysis of ABCA4 mutations responsible for Stargardt disease (2021)

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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The Role of Photoreceptor ABC Transporter ABCA4 in Retinal and Lipid Transport and Stargardt Disease (2014)

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-ATPase structure-funtion relationships: Mechanism and roles of ATP8A2-CDC50A in aminophospholipid transport, protein trafficking, and visual disorders (2013)

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 structure-function relationships : role in Stargardt disease and related retinal degenerative diseases (2009)

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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Purification and mass spectrometric characterization of human CXCR4 (2008)

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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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

The study of the P4-ATPase, ATP8A2, investigating disease-causing mutants and methods for high throughput production and purification (2019)

Human diseases have been linked to genetic mutations, causing the inability of proteins to function properly. Of these proteins, membrane proteins are crucial for the function of a cell; thus, a mutation affecting these classes of proteins causes extensive harm. This study was focused on the phospholipid transporter P₄-ATPase, ATP8A2, investigating the effects of mutations associated with human diseases. Six associated mutants have been studied: I376M, K429M, K429N, A544P, R625W, and W702R. The study allowed us to differentiate between two classes of mutants: proteins which fold in a native-like conformation allowing export from endoplasmic reticulum (ER), but lacking activity; and proteins that are highly misfolded, retained in the ER, and are rapidly degraded by proteasomes. The well-studied P2-ATPases transport small ion substrates, whereas P4-ATPases transport much larger substrates across the plasma membrane (PM). ATP8A2 was further studied with its β-subunit, CDC50A, to develop a high expression system for protein structural studies. The use of 20 mM CHAPS and 0.04% GDN was the best combination of detergents to solubilize HEK 293F cells with sonication and stirring at 4°C for 45 minutes for maximal purification. Samples analyzed under a transmission electron microscope (TEM) were monodispersed and non-aggregated; however, the same peptidisc-reconstituted sample was aggregated by cryo-electron microscopy (cryo-EM). Further studies are required to prevent aggregation during freezing of ATP8A2-CDC50A for analysis by cryo-EM.

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Characterization of members of type IV and type IIC of human P-type ATPase (2017)

P-type ATPases comprise a superfamily of proteins that play vital roles in the human body and can cause severe diseases if their functions are impaired. P₄-ATPases or type 4 of P-type ATPases are implicated in the ATP-dependent flipping of phospholipids across cell membranes. This generates and maintains transverse phospholipid asymmetry, a property important for biological processes including vesicle trafficking. ATP9A is a P₄-ATPase that remains poorly characterized despite its high expression in brain and testis. Interestingly, loss of Neo1p, the yeast ortholog of ATP9A, is lethal. The first part of this study investigates the functional properties and cellular localization of ATP9A. Human ATP9A was expressed in HEK293T cells and characterized using biochemical and cell-based approaches. ATP9A exhibited little if any phospholipid-dependent ATPase activity, but underwent hydroxylamine-sensitive phosphorylation, a characteristic feature of the P-type ATPase reaction cycle. A monoclonal antibody to ATP9A was generated for analysis of ATP9A in cells and brain tissues by western blotting and immunofluorescence microscopy. In transfected HEK293T cells ATP9A localized to perinuclear and peripheral punctate structures possibly related to the endocytic pathway. Our findings suggest that ATP9A undergoes autophosphorylation, but fails to dephosphorylate, possibly due to lack of an accessory protein or a specific substrate. Further studies on endogenous ATP9A should provide further insight into its physiological function and possible role in human disease.On the other hand, Na⁺/K⁺-ATPase (NKA) belongs to type 2C of P-type ATPases and establishes Na⁺ and K⁺ gradients across cell membranes. NKA has been shown to interact with retinoschisin (RS1), an adhesion protein essential for normal retinal structure and function. Mutations in the gene encoding RS1 cause a macular degeneration disorder called X-linked retinoschisis (XLRS). RS1 is thought to be anchored to the membranes of photoreceptor and bipolar cells through interaction with the α3 and β2 isoforms of NKA. The second part aims to characterize the RS1-NKA complex by generating monoclonal antibodies specific for the components. Indeed, immunoaffinity purification of NKAβ2 from bovine retinal membranes co-immunoprecipitated the α3 subunit and RS1. Tandem affinity purification of the native protein complexes should enhance understanding of the molecular and cellular mechanisms underlying XLRS.

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Characterization of hippocalcin-like protein 1 (HPCAL1), a neuronal calcium sensor protein in the retina (2015)

Hippocalcin-like protein 1 (HPCAL1) is a neuronal calcium sensor (NCS) protein found in the brain and in the retina. NCS proteins have important roles in signaling pathways including phototransduction; however, the role of HPCAL1 in the retina remains unresolved. The objective of this thesis is to characterize HPCAL1 and identify its potential interacting partners in the retina. The first part of the thesis examines the localization and characteristics of HPCAL1 using a variety of biochemical assays. The presence of HPCAL1 in the retina was confirmed with RT-PCR, Western blotting analysis, and immunofluorescence microscopy. Since NCS proteins respond to intracellular calcium level changes, HPCAL1 was expressed and isolated from both mammalian cells and E. coli to study its calcium binding properties and other characteristics. Results from a gel mobility shift assay and a fluorescence assay clearly indicated that HPCAL1 undergoes conformational changes upon calcium binding. Furthermore, membrane association assays confirmed that retinal HPCAL1 possesses the calcium-myristoyl switch mechanism which responds to the presence or absence of calcium. NCS proteins often interact with other proteins to perform their functions; therefore, the second part of the study involves the use of mass spectrometry in an attempt to identify calcium-dependent interacting partners of HPCAL1. Over 300 potential interacting partners were identified, and selected proteins were subjected to co-immunoprecipitation and Western blotting analysis or co-localization studies using immunofluorescence microscopy in order to confirm their interactions with HPCAL1. TorsinA has been identified as a calcium-dependent interacting partner to HPCAL1; however, further studies will have to be conducted to determine the significance of this interaction and to confirm other potential interacting partners that were identified in the mass spectrometry analysis. The results of this study provide important technical information on the biochemical characterization of HPCAL1 and the properties of HPCAL1 in the retina. Not only has the identification of potential interacting partners of HPCAL1 provided first insights at its possible role or function in the retina, it has also pointed out the potential for identifying interacting partners of other NCS proteins using mass spectrometry.

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Expression, purification, and characterization of RD3, the protein associated with Leber's congenital amaurosis type 12 (2013)

RD3 is a highly conserved 23 kDa soluble protein expressed in the retinal and testicular tissues required for the trafficking of GC1 (Guanylate Cyclase 1). A lack of expression of this protein, or of GC1, results in a rapid loss of photoreceptor cells after retinal development, indicating their importance in photoreceptor function and survival. Previous work has demonstrated that RD3 shows disperse and membrane-associated distribution in HEK293T cells. When expressed in E. coli, RD3 forms inclusion bodies exclusively. RD3 is non-amenable to vigorous centrifugation, resistant to purification by way of size exclusion, and binds to and inhibits GC1. The current investigation demonstrates that RD3 is highly conserved throughout sight capable vertebrates, forms large proteolipid macro-molecular structures of 10nm size or greater and that E. coli expressed protein conforms to the predicted secondary structure of RD3. Additionally, methods of isolating RD3 used in previous studies are carried out here and found to result in the formation of the expected structures. Multiple alignment and secondary structure prediction software, circular dichroism, dynamic light scattering, electron microscopy, and standard protein purification and visualization techniques are utilized in these endeavors.

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Identification and characterization of a novel retinal protein, ANKRD33, and its interacting partner HPCAL-1 (2010)

The outer segment is a specialized region of rod and cone photoreceptor cells located in vertebrate retina. It features stacks of membranous discs containing visual pigment molecules, and the membranous structures undergo continuous renewal process. In order to better understand the cellular mechanisms in the outer segment, Kwok et al. (2008) used tandem mass spectrometry on bovine rod outer segment preparations, and identified many proteins of unknown function, one of which was ankyrin repeat domain 33 (ankrd33). Ankrd33 belongs to the ankyrin repeat protein class, which has been described to be involved in a variety of functions, such as cell-cell signalling and cytoskeleton structure. However, the function and localization of Ankrd33 have not been previously investigated. In this project, ankrd33 was cloned from bovine retinal cDNA and RT-PCR experiments showed a retina specific gene expression of this protein. In addition, monoclonal antibodies were raised against N and C-termini of ankrd33. These antibodies were used to localize the protein in retina. In addition, they were used to identify the interacting partners of ankrd33 in photoreceptors. Ankrd33 was found to be exclusively expressed in the outer segments of photoreceptor cells and co-immunoprecipitation studies identified hippocalcin like 1 protein (HPCAL-1) as one of the interacting partners.HPCAL-1 belongs to the family of proteins called neuronal calcium sensors (NCS) which are EF-hand containing Ca2+-binding proteins and expressed in different neuronal cells. These proteins are involved in calcium modulation of numerous cellular activities based on the cell type and their interacting partners. As a first step in identifying the possible cellular pathways that ankrd33 and HPCAL-1 might be involved in, monoclonal antibodies were produced against the full length of HPCAL-1 and were used for immunofluorescence studies and in vitro confirmation of the interaction between the two proteins. Immunofluorescence studies showed labelling of ankr33 and HPCAL-1 in rod and cone outer segment with cone outer segment having a stronger signal. These results showed that ankrd33 and HPCAL-1 are both highly expressed in the cone outer segment and these proteins may be involved in calcium dependent-cone specific pathways.

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