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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY019665-05
Application #
8518333
Study Section
Biology and Diseases of the Posterior Eye Study Section (BDPE)
Program Officer
Neuhold, Lisa
Project Start
2009-09-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
5
Fiscal Year
2013
Total Cost
$297,612
Indirect Cost
$94,464
Name
West Virginia University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
191510239
City
Morgantown
State
WV
Country
United States
Zip Code
26506
Sinha, Satyabrata; Belcastro, Marycharmain; Datta, Poppy et al. (2014) Essential role of the chaperonin CCT in rod outer segment biogenesis. Invest Ophthalmol Vis Sci 55:3775-85
Sinha, Satyabrata; Majumder, Anurima; Belcastro, Marycharmain et al. (2013) Expression and subcellular distribution of UNC119a, a protein partner of transducin * subunit in rod photoreceptors. Cell Signal 25:341-8
Gao, Xueli; Sinha, Satyabrata; Belcastro, Marycharmain et al. (2013) Splice isoforms of phosducin-like protein control the expression of heterotrimeric G proteins. J Biol Chem 288:25760-8
Belcastro, Marycharmain; Song, Hongman; Sinha, Satyabrata et al. (2012) Phosphorylation of phosducin accelerates rod recovery from transducin translocation. Invest Ophthalmol Vis Sci 53:3084-91
Yang, Jun; Wang, Le; Song, Hongman et al. (2012) Current understanding of usher syndrome type II. Front Biosci (Landmark Ed) 17:1165-83
Posokhova, Ekaterina; Song, Hongman; Belcastro, Marycharmain et al. (2011) Disruption of the chaperonin containing TCP-1 function affects protein networks essential for rod outer segment morphogenesis and survival. Mol Cell Proteomics 10:M110.000570