Lens proteins undergo disulfide and non-disulfide crosslinking during aging. Such crosslinkings are associated with protein aggregation, insolubilization, light scattering and loss of lens accommodation. While disulfide crosslinking is well studied, the biochemical nature and the mechanism of formation of non-disulfide crosslinking are poorly understood. We propose to test a novel hypothesis that non-disulfide covalent crosslinking of proteins in the lens could arise from glycation-mediated crosslinking of the complexes that are formed between ?-crystallin and its chaperoned proteins, which leads to the formation of high molecular weight proteins and protein insolubilization during lens aging. Our preliminary studies strongly support this hypothesis. In the proposed project, we will systematically investigate this hypothesis via three specific aims.
In Aim 1, we will perform experiments to establish the long-term stability of ?-crystallin-client protein complexes under the conditions of the lens by employing fluorescence resonance energy transfer (FRET)- based assays. We will then determine whether ?-crystallin-client protein complexes undergo more covalent crosslinking by glycation than their individual protein components by quantifying protein-crosslinking advanced glycation end products (AGEs).
In Aim 2, we will extend our studies to intact human and mouse lenses to determine whether oxidative or thermal stress (to promote ?-crystallin-client protein complex formation) would promote glycation-mediated protein crosslinking in the lens. We will then determine the collective effects of stress and glycation on light transmittance and stiffness (resilience) in lenses.
In Aim 3, we will first determine whether crosslinking by ?-crystallin-client protein glycation has a direct relationship with non-disulfide crosslinked high-molecular-weight proteins in aging lenses; we will then use a novel inhibitor that we developed during the previous funding period to inhibit protein crosslinking in human lenses. Finally, we will determine whether the inhibitor prevents losses in light transmittance and losses in resilience due to AGE-mediated protein crosslinking. Together, the three aims will test an innovative concept of protein crosslinking in the lens and test a novel chemical inhibitor against such crosslinking. The findings in this study could lead to innovative therapies against presbyopia and cataracts.

Public Health Relevance

Presbyopia and cataracts are two major lens-related complications that can result in poor or total loss of vision. Although removal of the lens and transplantation of an intraocular lens would remedy both problems, the cost associated with such surgeries is enormous and not affordable in many developing and under- developed countries. Moreover, cataract surgeries can lead to complications and may require additional treatments. Thus, understanding mechanisms at the biochemical and molecular levels would enable us to develop therapies against both presbyopia and cataracts. In this application, we have proposed to test a novel hypothesis regarding how lens proteins aggregate to cause presbyopia. We have also proposed to use an innovative chemical that we have developed to block protein aggregation. Together, if successful, the results of this proposal will be the first proof of a biochemical mechanism for presbyopia and the first demonstration of an inhibitor that prevents it.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY023286-06A1
Application #
9658846
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Araj, Houmam H
Project Start
2013-09-01
Project End
2022-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
6
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Fl├╝ckiger, Rudolf; Cocuzzi, Enzo; Nagaraj, Ram H et al. (2018) DAF in diabetic patients is subject to glycation/inactivation at its active site residues. Mol Immunol 93:246-252
Rakete, Stefan; Nagaraj, Ram H (2017) UVA Light-mediated Ascorbate Oxidation in Human Lenses. Photochem Photobiol 93:1091-1095
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
Panda, Alok Kumar; Nandi, Sandip Kumar; Chakraborty, Ayon et al. (2016) Differential role of arginine mutations on the structure and functions of ?-crystallin. Biochim Biophys Acta 1860:199-210
Nahomi, Rooban B; Pantcheva, Mina B; Nagaraj, Ram H (2016) ?B-crystallin is essential for the TGF-?2-mediated epithelial to mesenchymal transition of lens epithelial cells. Biochem J 473:1455-69
Raghavan, Cibin T; Smuda, Mareen; Smith, Andrew J O et al. (2016) AGEs in human lens capsule promote the TGF?2-mediated EMT of lens epithelial cells: implications for age-associated fibrosis. Aging Cell 15:465-76
Nagaraj, Ram H; Nahomi, Rooban B; Mueller, Niklaus H et al. (2016) Therapeutic potential of ?-crystallin. Biochim Biophys Acta 1860:252-7
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
Holm, Thomas; Raghavan, Cibin T; Nahomi, Rooban et al. (2015) Effects of photobleaching on selected advanced glycation end products in the human lens. BMC Res Notes 8:5
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

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