Selective (nonlysosomal) proteolysis serves two vital functions in cells : (1) it controls steady-state levels of proteins whose concentrations and activities must be precisely regulated to maintain homeostasis and differentiation pathways; and (2) it protects cells from insults such as oxidative stress, viral infection and mutations by degrading potentially cytotoxic damaged or aberrant proteins. The importance of selective proteolysis to cell regulation and to stress and disease resistance is underscored by the fact that eukaryotic cells possess a highly conserved and exquisitely selective proteolytic pathway that recognizes and degrades both native regulatory proteins as well as aberrant proteins resulting from stress and disease. This pathway requires ubiquitin, a protein which is abundant in all cells; hence, the pathway is called the ubiquitin-dependent pathway (UDP). Studies from the applicant's laboratory indicate that the UDP functions in mammalian ROS and suggest that the visual transduction GTP-binding protein (G protein), transducin, is a UDP substrate. These observations suggest a novel mechanism by which photoreceptor proteins may be selectively regulated as well as the potential for modulation of the visual transduction cascade by the UDP. One long-term objective of the applicant's research program is to determine the function of the UDP in photoreceptors. In pursuit of this goal, the proposed research will (1) confirm the presence of the UDP protease in rat ROS using immunoelectron microscopy and (2) demonstrate UDP proteolytic function in cell-free preparations of gradient-purified bovine ROS (specific aim 1). The applicant will subsequently determine (3) if purified transducin is degraded by the UDP in ROS preparations and (4) the extent to which light-induced transducin dissociation alters its degradation by the ROS UDP (specific aim 2). In humans, protein damage in the normal retina resulting from (photo)oxidation is implicated in age-related macular degeneration (AMD), and expression of aberrant proteins in the diseased retina is causally associated with the pathogenesis of progressive retinal degenerations (retinitis pigmentosa). Thus, an additional long-term objective of the applicant's research is to determine how damaged and mutant proteins are removed from cells in the intact and diseased retina. In pursuit of this goal, experiments described under specific aim 3 will (1) use immunohistochemistry and western blotting to confirm a previous report of increased UDP-modified protein in retinas of Long-Evans rats exposed to photic stress (prolonged bright light) and to determine where within retinal cells these increases are manifest; (2) determine if increased protein flux through the UDP in response to photic stress reflects increased UDP efficacy and/or increased substrate availability; and (3) initiate studies designed to elucidate the molecular and biochemical mechanisms by which UDP efficacy is altered in response to photic stress.
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