Lens proteins undergo numerous physico-chemical changes during aging and cataract formation;pigmentation and crosslinking are the two major ones. Although several biochemical pathways participate in these changes, the actual mechanisms and their interrelationships remain unclear. Our project addresses this very issue. Reactions of dicarbonyl compounds with proteins appear to be major contributors to pigmentation and crosslinking in lens proteins. Recent studies, including our own, point to yet another mechanism for producing these changes, involving kynurenines, tryptophan oxidation products that react with lens proteins to generate pigments and crosslinking structures. Our preliminary data suggest that the dicarbonyl and kynurenine protein modification processes are related and interdependent. Although most reactions in these pathways are non-enzymatic, the reactive compounds formed can initiate further reactions that are regulated by enzymatic mechanisms. We will investigate these mechanisms in the context of lens physiology. Our proposed research is organized to achieve four specific aims. In the first aim, we will focus on methylglyoxal (MGO), a highly reactive dicarbonyl compound in the lens. We will determine how MGO is metabolized by glyoxalase I as well as the importance of glyoxalase I for survival of lens epithelial cells under conditions of hyperglycemic, oxidative or nitrosative stress.
In aim 2, we will establish the role of the dideoxyosone (DDO) pathway in lens protein modification. We will determine how this recently discovered pathway, through which sugars and ascorbic acid modify proteins, contributes to lens aging and cataract formation.
In aim 3, we will study kynurenine-mediated protein modification. We will focus mainly on indoleamine 2,3-dioxygenase (IDO), an enzyme that catalyzes metabolism of tryptophan to kynurenines. We will use transgenic and knockout mice to investigate the role of IDO in lens protein modification, and explore the effect of hyperglycemia and oxidative stress on IDO activity. Kynurenine-mediated damage to lens proteins will be assessed by chromatographic methods and immunological assays with highly specific monoclonal antibodies. Finally, in aim 4, we will determine the relationship among kynurenines, MGO and DDO in lens protein modifications. Cummulative information gained from these experiments will provide us with critical insights into the biochemical mechanisms that underlie lens aging and cataract formation.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
3R01EY009912-15S1
Application #
8428198
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Araj, Houmam H
Project Start
1994-09-01
Project End
2013-03-31
Budget Start
2010-04-01
Budget End
2013-03-31
Support Year
15
Fiscal Year
2012
Total Cost
$76,004
Indirect Cost
$27,594
Name
Case Western Reserve University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Nahomi, Rooban B; Oya-Ito, Tomoko; Nagaraj, Ram H (2013) The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin. Biochim Biophys Acta 1832:195-203
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Nagaraj, Ram H; Nahomi, Rooban B; Shanthakumar, Shilpa et al. (2012) Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function. Biochim Biophys Acta 1822:120-9
Linetsky, Mikhail; Kaid Johar, S R; Meltretter, Jasmin et al. (2011) Determination of dideoxyosone precursors of AGEs in human lens proteins. Arch Biochem Biophys 514:16-26
Mailankot, Maneesh; Nagaraj, Ram H (2010) Induction of indoleamine 2,3-dioxygenase by interferon-gamma in human lens epithelial cells: apoptosis through the formation of 3-hydroxykynurenine. Int J Biochem Cell Biol 42:1446-54
Mailankot, Maneesh; Howell, Scott; Nagaraj, Ram H (2010) Kynurenine inhibits fibroblast growth factor 2-mediated expression of crystallins and MIP26 in lens epithelial cells. Biochim Biophys Acta 1802:609-20
Pasupuleti, N; Matsuyama, S; Voss, O et al. (2010) The anti-apoptotic function of human αA-crystallin is directly related to its chaperone activity. Cell Death Dis 1:e31
Gangadhariah, Mahesha H; Wang, Benlian; Linetsky, Mikhail et al. (2010) Hydroimidazolone modification of human alphaA-crystallin: Effect on the chaperone function and protein refolding ability. Biochim Biophys Acta 1802:432-41
Pasupuleti, Nagarekha; Gangadhariah, Mahesha; Padmanabha, Smitha et al. (2010) The role of the cysteine residue in the chaperone and anti-apoptotic functions of human Hsp27. J Cell Biochem 110:408-19

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