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, which in the human lens 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 crosslinked proteins through formation of advanced glycation end products (AGEs). Reduced glutathione (GSH) offers some protection against this process, but the decreased levels of GSH in aged and cataractous lenses favor ASC oxidation. Recent work suggests that much of the protein crosslinking in cataractous lenses are ASC oxidation product-mediated. We know that ASC is oxidized in aging and cataractous lenses, but we do not know the mechanisms for such oxidation. Although molecular oxygen- mediated oxidation is likely to occur in the cortex, it is unlikely to occur in the near anoxic nucleus. Despite this limitation, protein crosslinking and aggregation through AGE formation is 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 as 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 protein modification 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. Together, the proposed studies will 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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