Our long-term research objective is to understand the roles of molecular chaperones in vertebrate photoreceptors. In this research proposal, we address the role of a large chaperonin complex, CCT, and its co-chaperone, phosducin-like protein 1 (PhLP1), in the folding and assembly of the visual heterotrimeric G protein, transducin. Our working hypothesis is that CCT and PhLP1 are required for the folding of the beta subunit of transducin into its characteristic seven-blade beta-propeller structure. We also propose that a close homolog of PhLP1, phosducin, acts in concert with PhLP1 during folding of transducin-beta, and regulates trafficking of transducin in the photoreceptor cell. To test our hypotheses, we generated several transgenic mouse models, including those with the suppressed PhLP1/CCT function in photoreceptors, and mice expressing mutant phosducin. These animal models provide a unique opportunity to study the functions of CCT, PhLP1 and phosducin in vivo.
In Specific Aim 1, we will use mice with the suppressed PhLP1/CCT function to explore the physiological significance of these chaperones in photoreceptors and to determine the molecular mechanism of the retinal degeneration caused by suppression of their function. Studies within Specific Aim 2 will address the mechanism of phosducin and PhLP1 chaperone synergy, and demonstrate the feasibility of manipulating chaperone activity in photoreceptors, with a purpose to counteract retinal degeneration due to the aberrant protein folding.
In Specific Aim 3, we will determine the physiological roles of the serine 54 and serine 71 phosphorylation sites on phosducin that regulate its interaction with transducin in a light-dependent manner. Using mice expressing phosducin without these phosphorylation sites, we will determine in vivo the significance of each site in regulating trafficking of transducin to the rod outer segments. The proposed studies will reveal the function of the eukaryotic chaperonin, CCT, in vertebrate photoreceptors, and shed light on the mechanism of folding and assembly of essential visual signaling proteins. The knowledge of this mechanism is important for developing strategies against neurodegenerative blinding diseases caused by molecular chaperone malfunction and aberrant protein folding. We expect that our results will also significantly improve our understanding of G protein signaling and its cellular regulation.
The proposed studies are designed to elucidate the roles of molecular chaperones - proteins that help the newly synthesized polypeptides to assume proper secondary structure - in the folding of the visual heterotrimeric G protein, transducin, in the photoreceptor neurons of the retina. Aberrant folding of proteins in neurons is an established cause of neurodegenerative diseases;therefore, the proposed studies will advance our understanding of the mechanisms of neurodegenerative diseases, including those caused by aberrant folding of transducin. We expect that our results will also significantly improve our understanding of G protein-mediated signaling and its cellular regulation.
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