PROJECT 4 - POLYPHENOLS AND DAMAGE IN THE EYE

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The proteins in the eye perform a vital role in sustaining human quality of life, particularly with respect to aging. Several of them, such as the lens proteins, are synthesized one time and preserved for the rest of life. Since they are exposed to light > 320 nm, they are subject to chronic sunlight-induced damage. This damage includes several post-translational modifications and cross-linking of the lens proteins, the extent of which is poorly defined. The latter lead to precipitation of proteins in the lens and ultimately to cataract formation. An important chemical species that is formed in the eye by the action of the ultraviolet portion of sunlight is singlet oxygen. It reacts with several protein amino acid side chains (Cys, His, Met, Trp, Tyr) introducing oxygen atoms. It also causes cleavage of the peptide backbone at limited sites. The local chemical environment in the aqueous of the eye, an ultrafiltrate of plasma that bathes the lens, includes important antioxidant species such a vitamin C and glutathione. Our hypothesis is that polyphenols and their metabolites derived from botanical dietary supplements enter the aqueous of the eye and thereby synergistically enhance its anti-oxidant environment. This is proposed to prevent UV light-induced post-translational protein modification and polymerization and therefore deter age-related degeneration of eye function. In this proposal, we will examine the following: (1) to determine in a rat model the effect of selected botanical polyphenol preparations on their composition in the lens and aqueous humor of the eye, (2) to determine the effects of polyphenols on the levels of the endogenous antioxidant glutathione and the exogenous antioxidants vitamins C and E and carotenoids in specific regions of the eye, (3) to determine the effects of dietary polyphenols on the extent of post-translational modifications and cross-linking of the lens acrystallin (a heat shock protein with chaperone activity), and (4) use specifically mutated forms of a model protein in vitro and lens aB-crystallin to examine the mechanism of singlet oxygen-induced protein cleavage and posttranslational modifications. These experiments will utilize techniques and expertise in microfluidics analysis and protein mass spectrometry that have been developed in the Purdue-UAB Botanicals Center in the previous grant period. They will use a combination of in vivo experiments in animal models and in vitro experiments to examine the mechanisms of singlet oxygen-induced protein damage and the role of specific polyphenols and their metabolites in combination with physiological antioxidants.