Robert S. Molday - US grants

Molecular properties and structural organization of proteins in retinal rod outer segment (ROS) disk and plasma membranes will be studied to elucidate their functional role in visual excitation and cell-cell recognition and regulation in normal and retinal degenerative disease states. We will study the molecular properties of the high Mr proteins of disk membranes and define their possible role as structural and functional elements in disk-disk and disk-plasma membrane interactions. Monoclonal antibodies will be used with solid-phase radioimmune assays, immunoaffinity chromatography, immunospecific cell membrane labeling and biochemical analysis; i.e. electrophoresis, HPLC peptide mapping, amino acid analysis etc. to purify, quantify and characterize ROS membrane proteins. Their molecular properties and interaction with other proteins including actin and calmodulin will be studied and correlated with the molecular properties of potentially-related cytoskeletal proteins (myosin and spectrin) of other cell types. The effect of limited proteolytic digestion, divalent cations, ionic strength and addition of specific ROS cytoplasmic proteins on the association-dissociation behavior of isolated disks will be studied and correlated with changes in the properties of high Mr proteins and other proteins of disk membranes. Monoclonal antibodies against specific regions of rhodopsin will be characterized with the aim of using these reagents to probe structural organization, conformational changes and functional domains of rhodopsin in disk membranes. Identification and characterization of antigenic sites on rhodopsin will be determined by limited proteolytic digestion of rhodopsin and synthetic peptides in conjunction with RIA assays, HPLC separations, amino acid analysis and protein binding and ion transport assays. ROS plasma membrane will be isolated for analysis of its structural and functional properties. Lectins and monoclonal antibodies will be used in conjunction with newly developed ferromagnetic iron dextran and gold-dextran microspheres to specifically label the outer surface of ROS plasma membranes and separate these membranes from disk membranes by affinity magnetic chromatography and/or affinity density perturbation. Purify and sideness of the ROS plasma membranes will be studied by RIA and lectin binding assays and factors such as Ca++, cGMP, ROS cytoplasmic proteins, etc. affecting Na efflux from these vesicles will be studied. Protein components of Ros plama membranes will also be studied by immunochemical, biochemical and microscopic techniques and compared with those present in disk membranes.

Over the previous grant period, the rod outer segment (ROS) plasma membrane has been isolated from disk membranes and shown to contain a number of major proteins not present in disk membranes. These include the cGMP-gated cation channel, Na+Ca++ exchange protein, a 240kD spectrin-like protein, and a 38 kD membrane associated protein. In this proposal the molecular properties of these and other plasma membrane proteins will be studied in detail in order to elucidate their role in visual transduction process and other important metabolic and structural functions in rod and cone outer segments. Monoclonal antibodies (Mabs) recently produced in this laboratory will be used with biochemical, immunochemical, microscopic, molecular biology and reconstitution techniques to define the structural, functional and antigenic domains of these proteins and to compare their properties with corresponding proteins in cone photoreceptor cells and other cell types. Proposed areas of research are as follows: (1) The antigenic binding sites for anti cGMP-gated channel Mabs (PMcIDI,PMc 2GII, others) will be determined by recombinant DNA techniques, radioimmune assays (RIA) using synthetic peptides and immunocytochemical methods to determine the topography of the 63kD channel in ROS plasma membranes. Site-directed antibodies against known sequences will also be prepared and their binding cites localized by immunogold-labeling studies. The effect of these antibodies on the cGMP-gated channel activity will be studies reconstituted vesicle systems. (2) The 230KD NaCa exchange protein will be studied using a number of anti-230KD monoclonal antibodies we have recently generated. (3) A 240KD spectrin-like protein associated with the cGMP gated channel will be partially sequenced for comparison to other spectrin cytoskeletal proteins and the morphological features of the isolated 240 KD protein will be studied by electron microscopy. (4) A major plasma membrane associated 38 KD protein will be isolated and its molecular properties and its interaction with other plasma membrane proteins will be studied. (5) Rhodopsin from plasma membrane and disk membranes will be purified by immunoaffinity chromatography. The C-terminal, N-terminal segments, and tryptic peptide maps of these rhodopsins will be compared in order to determine if plasma membrane and disk membrane rhodopsin are identical or if they undergo differential post-translational modification as part of the sorting process.

DESCRIPTION (Adapted from applicant's abstract): The outer segment of rod photoreceptor cells consists of a plasma membrane that surrounds a highly organized stack of discs. The long term goal of this research program is to identify and characterize rod outer segment (ROS) plasma membrane proteins and define their role in phototransduction, outer segment structure and stability, metabolic and regulatory and retinal degenerative disease including retinitis pigmentosa. The specific aims are: (1) to study the structure-function relationships of the rod cGMP-gated channel: studies will include (a) analysis of the subunit composition and subunit-subunit interactions of the channel complex; (b) defining the role of the glutamic rich region (GARP) of the beta-subunit in outer segment structure; and (c) elucidating mechanisms that regulate the activity of the channel as part of the phototransduction and adaptational processes; (2) to study the structure, function and regulation of the rod Na+/Ca2+-K+ exchanger: studies will be directed toward determining the role of the large intracellular and extracellular domains of the exchanger in: (a) cation transport function; (b) Ca2+-mediated regulation of exchange activity; and (c) protein-protein interactions that may serve to stabilize the outer segment structure; and (3) to identify and characterize two calmodulin-binding proteins of 67 kDa and 160 kDa in ROS as a first step in determining their role in the Ca2+-dependent regulation of outer segment processes. A variety of current biochemical, molecular biological, cell biological, immunochemical and biophysical methods will be employed along with a unique panel of highly specific monoclonal and polyclonal antibodies and cDNAs to outer segment proteins. The results of these studies should provide new insight into the structure, function and regulation of the cGMP-gated channel, the Na+/Ca2+-K+ exchanger and calmodulin-binding proteins of rod segments and define their role in retinal degenerative diseases.

DESCRIPTION (Adapted from applicant's abstract): Rod and cone outer segments are unique compartments of photoreceptor cells where the process of phototransduction occurs. The overall goal of this research program is to identify and characterize outer segment membrane proteins and elucidate their role in i) the visual process, ii) outer segment morphogenesis, structure, and renewal, and iii) inherited retinal degenerative diseases. The specific aims of the current grant period are as follows: 1) To study the structural, functional, and regulatory properties of ABCR, the photoreceptor ATP binding cassette (ABC) transporter of photoreceptors, and determine its cellular and subcellular distribution in rod and cone photoreceptors. 2) To develop a heterologous cell (COS-1) system to express wild-type and mutant forms of ABCR for structure-function analyses. This system will be used to determine how missense mutations in ABCR implicated in Stargardt disease alter the structure of ABCR and its possible function as a transporter. 3) To identify, clone, characterize and localize novel low abundant rod outer segment membrane proteins as a first step in determining their role in outer segment structure, function and renewal and their possible involvement in inherited retinal degenerative diseases. For these studies, a variety of current biochemical, molecular biology, cell biology and biophysical methods will be employed along with a unique panel of highly specific monoclonal antibodies and cDNAs to ABCR and novel, undefined outer segment proteins. The results of these studies should lead to new information about the morphogenesis, structure and function of rod and cone outer segments and provide insight into the role of ABCR in the visual cycle and retinal degenerative diseases including Stargardt macular dystrophy and age-related macular degeneration.

DESCRIPTION (provided by applicant): Outer segments are specialized compartments of rod and cone photoreceptors that mediate the primary events of vision. The rod outer segment (ROS) consists of a highly ordered stack of disks surrounded by a plasma membrane. Filaments bridge adjacent disks together and the disk rim to the plasma membrane, however, the proteins that constitute these structural elements have not been identified. Since disorganization and loss in ROS is known to cause photoreceptor degeneration, it is important to define the molecular basis for ROS morphogenesis and structure. An overall objective of this research is to catalogue all the proteins in ROS with the goal of identifying a subset of "novel" proteins critical for the structure, stabilization and formation of ROS and proteins likely to be associated to retinal degenerative diseases. Another key objective is to define the molecular basis for selected inherited retinal degenerative diseases that cause significant loss in vision. These objectives will be achieved in the following specific aims. 1) To determine the complete proteosome of the ROS using current and emerging mass spectrometry-based approaches together with subcellular fractionation procedures. Proteins predicted to play a role in ROS structure and morphogenesis will be characterized using an array of biochemical, immunochemical, molecular and cell biology techniques. The role of these proteins in ROS structure and photoreceptor degeneration will be assessed;2) To evaluate the role of protein-protein interactions between the peripherin-2:rom-1 complex in the disk rim and the channel:exchanger complex in the plasma membrane in stabilizing the structure of ROS. Immuno-affinity techniques, site-directed mutagenesis, heterologous cell expression, membrane reconstitution and knockout mice will be used in the study;and 3) to examine the molecular mechanisms underlying selected retinal diseases. The function of RS1 (retinoschisin) as a retinal cell adhesion protein will be examined in order to understand how defects in this protein cause X-linked Juvenile Retinoschisis. The possible function of ELOVL4 in the biosynthesis of very long chain fatty acids and in autosomal dominant Stargardt disease will also be studied. This research will provide new insight into the molecular mechanisms underlying ROS structure and inherited retinal dystrophies and serve as a basis for developing novel treatments for this set of diseases that cause significant visual loss.

DESCRIPTION (provided by applicant): Inherited retinal degenerative diseases (RDDs) are a heterogeneous group of disorders that constitute a major cause of vision loss in the world population. In most cases, they are caused by mutations in genes that encode proteins essential for photoreceptor cell structure, function and survival. Although significant progress has been made in the genetics of RDDs, mechanisms by which various genetic defects cause photoreceptor degeneration and a loss in vision are not well understood. The objective of this application is to increase our understanding of the molecular and cellular mechanisms underlying two severe early onset disorders, Stargardt macular degeneration associated with mutations in the retinoid transporter ABCA4 and Leber Congenital Amaurosis Type 12 (LCA12) linked to mutations in RD3, a novel protein essential for guanylate cyclase expression in photoreceptor cells. This information will be used to test the hypothesis that novel therapeutic treatments can be developed in animal models for these diseases as an essential step for translation into human clinical trials. Three specific aims of this application are: 1) To examine the structural, functional and regulatory properties of ABCA4 and identify novel nontoxic chemical compounds that enhance the retinoid transport activities of WT and Stargardt disease-causing mutants of ABCA4 and hence serve as potential therapeutic drugs; 2) to further evaluate the therapeutic potential of selected drug compounds in animal models for Stargardt disease; and 3) to investigate the mechanisms underlying the function of RD3 in guanylate cyclase expression and trafficking in photoreceptors and evaluate adeno-associated viral (AAV) vectors for the delivery and expression of RD3 and the rescue of rod and cone function and survival in the rd3 mouse model for LCA12. These aims will be achieved by using newly developed in vitro retinoid transport assays, established and newly generated animal models, in vivo and in vitro imaging methods, protein expression and purification, novel viral gene delivery vectors, and high throughput drug screening methods. The results of this research will yield novel insight into the structure and function of ABCA4 and RD3 in rod and cone photoreceptor cells and further define how mutations in these proteins cause photoreceptor degeneration in Stargardt disease, LCA12, and related diseases. Importantly, this work will provide a 'proof of concept' that novel drug and gene based treatments can be developed for translation into future clinical trials for these diseases. These studies will also have a strong impact in other biomedical research. The knowledge gained from our studies of ABCA4 and Stargardt disease will enhance our understanding and treatment of other diseases (cystic fibrosis, Tangiers disease, adrenoleukodystrophy, etc.) linked to genetic defects in related ABC transporters and the studies on RD3 and LCA12 will extend our understanding of protein expression and trafficking and AAV-mediated gene therapy for the treatment of various diseases.