Our long-term goal is to prevent human cataracts by understanding the molecular mechanisms involved. This project builds on our previous work of nearly 25 years on lens protein modifications in aging and cataractogenesis. Protein crosslinking is a major modification in aged and cataractous lenses. Ascorbate (ASC) is a major constituent of the lens and in humans is present at concentrations up to 2 mM. ASC is oxidized in aged and cataractous lenses, and its oxidation products react rapidly with lens proteins to form pigmented and cross-linked proteins through formation of advanced glycation end products (AGEs). Recent work suggests that ASC oxidation product-mediated reactions contribute significantly to protein crosslinking in cataractous lenses. Reduced glutathione (GSH) offers some protection against this process, but the decreased levels of GSH in aged and cataractous lenses favor ASC oxidation. Mechanisms for such oxidation are unclear. Although molecular oxygen-mediated oxidation is likely to occur in the cortex, it is unlikely to occur in the nearly anoxic nucleus. Despite this limitation, ASC oxidation, along with protein crosslinking, aggregation and AGE-modification of proteins are most prominent in the nucleus of cataractous lenses. Kynurenines are tryptophan oxidation products produced by the kynurenine pathway initiated by indoleamine 2, 3-dioxygenase. They are present in relatively high levels in human lenses. Kynurenines undergo spontaneous deamination and bind covalently to lens proteins. Our preliminary studies show that both protein-free and protein-bound kynurenines promote ASC oxidation. UVA light has been considered to be an important risk factor for cataractogenesis, although the mechanisms are still obscure. Our preliminary experiments suggest that kynurenine-mediated ASC oxidation is significantly accelerated by UVA light, and that such oxidation can occur both in the presence and absence of oxygen. Based on these observations, we hypothesize that kynurenine- mediated ASC oxidation followed by AGE-modification of proteins plays an important role in the etiology of senile cataracts. We will test this hypothesis with the following three aims.
In aim 1 we will determine kynurenine-mediated ASC oxidation in the presence and absence of oxygen and UVA light, conditions that emulate cortex and nucleus of the human lens.
In aim 2 we will determine the impact of kynurenine-mediated ASC oxidation on covalent crosslinking and aggregation of lens proteins, and in aim 3, we will test our newly developed prodrug compounds on Kyn/ASC-mediated protein modification and crosslinking, and evaluate their effects on cataract development. The proposed studies should unravel the interplay between kynurenines and ASC in lens protein modification in human cataracts, and the findings could lead to innovative therapies to prevent or delay cataracts in humans.
In this application; we will test our novel hypothesis that UVA light through kynurenines oxidizes vitamin C in the lens and causes protein pigmentation; crosslinking and aggregation during cataract development. We will employ sophisticated analytical methods and transgenic animal models to test this hypothesis. We will then develop targeted inhibitors to block this mechanism in the lens to prevent cataract development.
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