# Molecular mechanisms of protein crosslinking in the lens

> **NIH NIH R01** · UNIVERSITY OF COLORADO DENVER · 2021 · $339,378

## Abstract

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 α-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 α-crystallin-client protein
complexes under the conditions of the lens by employing fluorescence resonance energy transfer (FRET)-
based assays. We will then determine whether α-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 α-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 α-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.

## Key facts

- **NIH application ID:** 10072058
- **Project number:** 5R01EY023286-08
- **Recipient organization:** UNIVERSITY OF COLORADO DENVER
- **Principal Investigator:** Ram H Nagaraj
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $339,378
- **Award type:** 5
- **Project period:** 2013-09-01 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10072058

## Citation

> US National Institutes of Health, RePORTER application 10072058, Molecular mechanisms of protein crosslinking in the lens (5R01EY023286-08). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10072058. Licensed CC0.

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