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. ? ? ?
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