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.
|Nagaraj, Ram H; Nahomi, Rooban B; Mueller, Niklaus H et al. (2016) Therapeutic potential of Î±-crystallin. Biochim Biophys Acta 1860:252-7|
|Raghavan, Cibin T; Smuda, Mareen; Smith, Andrew J O et al. (2016) AGEs in human lens capsule promote the TGFÎ²2-mediated EMT of lens epithelial cells: implications for age-associated fibrosis. Aging Cell 15:465-76|
|Panda, Alok Kumar; Nandi, Sandip Kumar; Chakraborty, Ayon et al. (2016) Differential role of arginine mutations on the structure and functions of Î±-crystallin. Biochim Biophys Acta 1860:199-210|
|Raghavan, Cibin T; Nagaraj, Ram H (2016) AGE-RAGE interaction in the TGFÎ²2-mediated epithelial to mesenchymal transition of human lens epithelial cells. Glycoconj J 33:631-43|
|Rakete, Stefan; Nagaraj, Ram H (2016) Identification of Kynoxazine, a Novel Fluorescent Product of the Reaction between 3-Hydroxykynurenine and Erythrulose in the Human Lens, and Its Role in Protein Modification. J Biol Chem 291:9596-609|
|Nahomi, Rooban B; Pantcheva, Mina B; Nagaraj, Ram H (2016) Î±B-crystallin is essential for the TGF-Î²2-mediated epithelial to mesenchymal transition of lens epithelial cells. Biochem J 473:1455-69|
|Nahomi, Rooban B; DiMauro, Michael A; Wang, Benlian et al. (2015) Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities. Biochem J 465:115-25|
|Smuda, Mareen; Henning, Christian; Raghavan, Cibin T et al. (2015) Comprehensive analysis of maillard protein modifications in human lenses: effect of age and cataract. Biochemistry 54:2500-7|
|DiMauro, Michael A; Nandi, Sandip K; Raghavan, Cibin T et al. (2014) Acetylation of Gly1 and Lys2 promotes aggregation of human Î³D-crystallin. Biochemistry 53:7269-82|
|Linetsky, Mikhail; Raghavan, Cibin T; Johar, Kaid et al. (2014) UVA light-excited kynurenines oxidize ascorbate and modify lens proteins through the formation of advanced glycation end products: implications for human lens aging and cataract formation. J Biol Chem 289:17111-23|
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