The lens contains three major types of structural protein: alpha-, beta-, and gamma-crystallins. Previous biochemical and spectroscopic studies indicate the possibility of interactions between alpha-crystallin and beta- or gamma-crystallin. Our recent studies using a mammalian two-hybrid system confirm that there are interactions among crystallins and that these interactions are altered by some congenital cataract crystallin mutations. The two-hybrid system can detect not only inter-molecular interactions but also intra-molecular interactions and is especially useful in studying alpha- (alphaA- and alphaB) crystallin. Mapping interface domains is one of the major applications of the two-hybrid system. alphaA- and alphaB-crystallins are oligomers whose three-dimensional structures have not been determined because of their inability to be crystallized for x-ray diffraction study. In this proposal, it is hypothesized that inter-subunit reaction domains can be mapped by the two-hybrid system assay with the aid of site-specific mutation, and that interface domains consist mainly of beta-strands and can be targeted for mutations. Once the interface beta-strands are identified, small peptides mimicking the beta-strands can be designed and synthesized. The reactions between peptides and alphaA- or alphaB-crystallin will prevent formation of dimers and oligomers, and native monomers will be available for structural studies. The first specific aim will be preliminary studies with known structural beta-and gamma-crystallins; the second specific aim will be mapping interface domains of alphaA- and alphaB-crystallins, followed by studying peptide-protein interactions. The methodology includes cloning, subcloning, site-specific mutagenesis, cell culture, protein expression and purification, small peptide design and synthesis, and two-hybrid system assay. The long-term objective is to understand the mechanisms of dimerization and oligomerization, and the results may help us to understand the function of alpha-crystallin in normal lens and in cataract formation.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY013968-02
Application #
6838768
Study Section
Special Emphasis Panel (ZRG1-VISA (01))
Program Officer
Liberman, Ellen S
Project Start
2004-01-01
Project End
2008-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
2
Fiscal Year
2005
Total Cost
$432,500
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
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Liu, Bing-Fen; Song, Shuhua; Hanson, Mark et al. (2008) Protein-protein interactions involving congenital cataract T5P gammaC-crystallin mutant: a confocal fluorescence microscopy study. Exp Eye Res 87:515-20
Song, Shuhua; Hanson, Mark J; Liu, Bing-Fen et al. (2008) Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching. Mol Vis 14:1282-7
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Liu, Bing-Fen; Anbarasu, Kumarasamy; Liang, Jack J-N (2007) Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells. Mol Vis 13:854-61
Liu, Bing-Fen; Liang, Jack J-N (2007) Protein-protein interactions among human lens acidic and basic beta-crystallins. FEBS Lett 581:3936-42
Liu, Yizhi; Zhang, Xinyu; Luo, Lixia et al. (2006) A novel alphaB-crystallin mutation associated with autosomal dominant congenital lamellar cataract. Invest Ophthalmol Vis Sci 47:1069-75
Zetterberg, Madeleine; Zhang, Xinyu; Taylor, Allen et al. (2006) Glutathiolation enhances the degradation of gammaC-crystallin in lens and reticulocyte lysates, partially via the ubiquitin-proteasome pathway. Invest Ophthalmol Vis Sci 47:3467-73
Liu, Bing-Fen; Liang, Jack J-N (2006) Domain interaction sites of human lens betaB2-crystallin. J Biol Chem 281:2624-30
Liang, Jack J; Liu, Bing-Fen (2006) Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants. Protein Sci 15:1619-27

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