The overall goal of this project has been to provide a biochemical mechanism for formation of selenite cataract in young rats. Our previous work has shown that partial proteolysis of Beta-crystallins by calcium- activated protease, calpain II, plays a central role in the mechanism of selenite cataract. The current proposal is focused on providing a biochemical mechanism on how partially truncated B-crystallin polypeptides become insoluble in selenite cataract and other models of cataract using older rat lenses.
Aim #1 will use an in vitro model of crystallin insolubilization to determine why removal of the N-termini on B-crystallin subunits by calpain causes insolubilization.
Aim #2 will directly compare results of the in vitro studies of proteolytic insolubilization to insolubilization occurring in the in vivo model of selenite cataract and in other models of cataract using older rat lenses.
Aim #3 will determine the role of a-crystallin chaperon activity in proteolytic insolubilization of B-crystallins. Techniques to be used include: two dimensional electrophoresis followed by N-terminal protein sequencing of spots, fast atom mass spectroscopy, laser light scattering, UV circular dichroism, protein-protein cross- linking, and turbidity measurements. The results will extend our on- going studies concerning how crystallins become insoluble in cataracts and how crystallins interact in normal lens. This knowledge may be used in the future to develop drugs for prevention of cataract by maintaining the solubility of lens proteins.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY003600-15
Application #
2414985
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1981-09-30
Project End
1998-12-31
Budget Start
1997-05-01
Budget End
1998-12-31
Support Year
15
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Dentistry
Type
Schools of Dentistry
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Azuma, Mitsuyoshi; Tamada, Yoshiyuki; Kanaami, Sayaka et al. (2003) Differential influence of proteolysis by calpain 2 and Lp82 on in vitro precipitation of mouse lens crystallins. Biochem Biophys Res Commun 307:558-63
Lampi, Kirsten J; Kim, Yung H; Bachinger, Hans Peter et al. (2002) Decreased heat stability and increased chaperone requirement of modified human betaB1-crystallins. Mol Vis 8:359-66
Kim, Yung Hae; Kapfer, Deborah M; Boekhorst, Jos et al. (2002) Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin. Biochemistry 41:14076-84
Lampi, Kirsten J; Shih, Marjorie; Ueda, Yoji et al. (2002) Lens proteomics: analysis of rat crystallin sequences and two-dimensional electrophoresis map. Invest Ophthalmol Vis Sci 43:216-24
Nakajima, Takeshi; Nakajima, Emi; Fukiage, Chiho et al. (2002) Differential gene expression in the lens epithelial cells from selenite injected rats. Exp Eye Res 74:231-6
Tamada, Y; Fukiage, C; Mizutani, K et al. (2001) Calpain inhibitor, SJA6017, reduces the rate of formation of selenite cataract in rats. Curr Eye Res 22:280-5
Shih, M; David, L L; Lampi, K J et al. (2001) Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses. Curr Eye Res 22:458-69
Nakamura, Y; Fukiage, C; Azuma, M et al. (2001) Calpain-induced light scattering in young rat lenses is enhanced by UV-B. J Ocul Pharmacol Ther 17:47-58
Lampi, K J; Oxford, J T; Bachinger, H P et al. (2001) Deamidation of human beta B1 alters the elongated structure of the dimer. Exp Eye Res 72:279-88
Shearer, T R; Ma, H; Shih, M et al. (2000) Calpains in the lens and cataractogenesis. Methods Mol Biol 144:277-85

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