The highly concentrated and ordered proteins of the human lens are under continuous oxidative and photooxidative stress. Moreover, there is little turnover or repair of the lens proteins and change in protein structure accumulate with age. Fortunately, the lens ages slowly, with major malfunction (cataracts) occurring in the fifth or sixth decade. The long term objective of this work is to understand the etiology of cataracts on the molecular level. Our hypothesis is that postranslational modifications of lens proteins resulting from oxidative and other processes result in loss of transparency of the lens and that the design of methods for preventing these changes can only begin after i) understanding the primary covalent structure of the major lens proteins, ii) determining the structures of these proteins in diseased or senile tissue, and iii) correlating the structures with the effects on protein function. Few attempts have been made to investigate the specific molecular details on the peptide or protein level or to relate those molecular changes with functional alterations. A multidisciplinary approach is advanced in this proposal in which sate-of-the-art mass spectrometric methods will be employed to characterize structural modifications of lens proteins in native states, after insult, and during aging and cataractogenesis. In addition, functional studies will be carried out to establish structure/function relationships in modified proteins.
Specific aims i nclude: 1) to determine the primary covalent structure of modifications to human alpha crystallin and MP26, 2) to determine the nature and extent of modifications to bovine alpha crystallin and Mp26 upon exposure to photolysis and oxidation, 3) to determine the structure of alpha crystallin and MP26 in aged and cataractous human lenses, 4) to correlate structural modifications with the effect of age and cataract on lens protein function. Although numerous studies have been performed on the broad general aspects of photolytic and oxidative mechanisms in aging and cataractogenesis, the results of this work are expected to provide new detailed information on the structure and function of modified lens proteins, a critical need in the fundamental understanding of aging and cataract formation.
| Roberts, J E; Finley, E L; Patat, S A et al. (2001) Photooxidation of lens proteins with xanthurenic acid: a putative chromophore for cataractogenesis. Photochem Photobiol 74:740-4 |
| Schey, K L; Little, M; Fowler, J G et al. (2000) Characterization of human lens major intrinsic protein structure. Invest Ophthalmol Vis Sci 41:175-82 |
| Schey, K L; Finley, E L (2000) Identification of peptide oxidation by tandem mass spectrometry. Acc Chem Res 33:299-306 |
| Schey, K L; Patat, S; Chignell, C F et al. (2000) Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John's Wort). Photochem Photobiol 72:200-3 |
| Schey, K L; Fowler, J G; Shearer, T R et al. (1999) Modifications to rat lens major intrinsic protein in selenite-induced cataract. Invest Ophthalmol Vis Sci 40:657-67 |
| Finley, E L; Dillon, J; Crouch, R K et al. (1998) Radiolysis-induced oxidation of bovine alpha-crystallin. Photochem Photobiol 68:9-15 |
| Finley, E L; Dillon, J; Crouch, R K et al. (1998) Identification of tryptophan oxidation products in bovine alpha-crystallin. Protein Sci 7:2391-7 |
| Finley, E L; Busman, M; Dillon, J et al. (1997) Identification of photooxidation sites in bovine alpha-crystallin. Photochem Photobiol 66:635-41 |
| Schey, K L; Fowler, J G; Schwartz, J C et al. (1997) Complete map and identification of the phosphorylation site of bovine lens major intrinsic protein. Invest Ophthalmol Vis Sci 38:2508-15 |
| Swamy-Mruthinti, S; Schey, K L (1997) Mass spectroscopic identification of in vitro glycated sites of MIP. Curr Eye Res 16:936-41 |
