For many years, a major goal of lens protein chemists has been the identification and quantitation of posttranslational modifications occurring in all major proteins of the aging and cataractous human lens. Accomplishment of this goal will provide the basis for understanding, on a molecular level, the nature of biochemical processes that may contribute towards the opacification process. As a first step towards the accomplishment of this objective total alpha-A and alpha-B crystallins will be purified from total cataractous and normal human lens proteins. both these proteins have been implicated in possible etiologies of human cataractogenesis, through decreases in their molecular chaperon properties and through their involvement in protein aggregation. Following endoprotease digestion of the purified crystallins, the mixture of peptides will be screened for the presence of posttranslationally modified sequences, using various procedures that take advantage of recent advances in mass spectrometry and reverse phase HPLC. After purification of the modified peptide, the exact chemical nature of the modification will be identified by mass spectrometry, and the quantitative extent o the modification will be determined by reverse phase HPLC. The above mentioned experimental approach will then be used to identify and quantitate the extent of the same modifications in lens of animal cataract model systems, to determine whether the time course of these modifications precedes the development of lens opacification. Together, the studies outlined in this proposal will identify and quantitate the major posttranslational modification occurring in alpha-A and alpha-B crystallins during human senile cataractogenesis, and will provide the basis for understanding the temporal sequence of these modifications during development of lens opacifications in animal model systems.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY002932-23
Application #
6384303
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Liberman, Ellen S
Project Start
1979-06-01
Project End
2002-05-31
Budget Start
2001-06-01
Budget End
2002-05-31
Support Year
23
Fiscal Year
2001
Total Cost
$894,435
Indirect Cost
Name
Kansas State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Manhattan
State
KS
Country
United States
Zip Code
66506
Takemoto, Larry; Sorensen, Christopher M (2008) Protein-protein interactions and lens transparency. Exp Eye Res 87:496-501
Sabah, Judith R; Schultz, Bruce D; Brown, Zach W et al. (2007) Transcytotic passage of albumin through lens epithelial cells. Invest Ophthalmol Vis Sci 48:1237-44
Brown, Zachery; Ponce, Aldo; Lampi, Kirsten et al. (2007) Differential binding of mutant (R116C) and wildtype alphaA crystallin to actin. Curr Eye Res 32:1051-4
Takemoto, Larry J; Ponce, Aldo A (2006) Decreased association of aged alpha crystallins with gamma crystallins. Exp Eye Res 83:793-7
Lin, Dingbo; Barnett, Micheal; Grauer, Laura et al. (2005) Expression of superoxide dismutase in whole lens prevents cataract formation. Mol Vis 11:853-8
Ponce, Aldo; Takemoto, Larry (2005) Screening of crystallin-crystallin interactions using microequilibrium dialysis. Mol Vis 11:752-7
Peterson, James J; Young, Malin M; Takemoto, Larry J (2004) Probing alpha-crystallin structure using chemical cross-linkers and mass spectrometry. Mol Vis 10:857-66
Nguyen, Thu Annelise; Takemoto, Larry J; Takemoto, Dolores J (2004) Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites. J Biol Chem 279:52714-25
Boyle, Daniel L; Takemoto, Larry; Brady, James P et al. (2003) Morphological characterization of the Alpha A- and Alpha B-crystallin double knockout mouse lens. BMC Ophthalmol 3:3
Reddy, V N; Giblin, F J; Lin, L R et al. (2001) Glutathione peroxidase-1 deficiency leads to increased nuclear light scattering, membrane damage, and cataract formation in gene-knockout mice. Invest Ophthalmol Vis Sci 42:3247-55

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