The hypothesis of this proposal is that CRALBP plays a fundamental role in vitamin A metabolism in the retina and retinal pigment epithelium (RPE). The unifying long range goals of the proposal are to establish a molecular basis for understanding the normal structure, function and regulation of CRALBP in order that questions concerning CRALBP-related visual disorders can be answered.
Specific aims focus on both the CRALBP protein and gene and include: Mapping the retinoid-binding pocket of rCRALBP by measuring retinoid binding properties and ligand associated conformational changes in mutant and wildtype proteins; Seeking direct protein-protein interaction between CRALBP and RPE 11- cis-retinol dehydrogenase; Developing an in vivo system for characterizing tissue-specific CRALBP gene expression; and characterize CRALBP knockout mice with regard to retinal vitamin A metabolism, electrophysiology and morphology. Methods will include recombinant protein production using bacterial and baculovirus expression systems, retinoid-binding analysis by UV-visible absorbance, fluorescence titrations and 13C and 19F NMR. Protein -protein interactions will be sought by chromatographic, electrophoretic and immunological methods and characterized by electrospray mass spectrometry, sequence analysis and by enzyme/substrate carrier activity. Standard molecular biology methods will be used to prepare and test mouse wildtype and PCE-1 mutant CRALBP reporter gene constructs in transgenic mice. Retinoid HPLC, Western analysis, ERG and ultra structural methods will be used to characterize CRALBP knockout mice. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. The proposed studies will lead to a better understanding of the structure, function and regulation of CRALBP and the visual disorders with which at may be associated.
| Marmorstein, Lihua Y; McLaughlin, Precious J; Stanton, J Brett et al. (2002) Bestrophin interacts physically and functionally with protein phosphatase 2A. J Biol Chem 277:30591-7 |
| Sakaguchi, Hirokazu; Miyagi, Masaru; Shadrach, Karen G et al. (2002) Clusterin is present in drusen in age-related macular degeneration. Exp Eye Res 74:547-9 |
| Pudota, B N; Miyagi, M; Hallgren, K W et al. (2000) Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation. Proc Natl Acad Sci U S A 97:13033-8 |
