A Novel Method to Improve Protein Solubility and Elasticity in Aged Human Lenses

Abstract

Purpose : Presbyopia is a progressive, age-related visual impairment characterized by the lens's lack of ability to focus on nearby objects. During presbyopia, proteins in the lens become increasingly insoluble and aggregated, contributing to the loss of elasticity and accommodation. This study reports the development of a small molecule that improves protein solubility and elasticity in aged lenses.

Methods : Aggrelyte-2 (S,N-Diacetyl-L-cysteine methyl ester) or the control compound (N-Acetyl-L-cysteine methyl ester, aggrelyte-2C) was incubated (at 500 μM) with a suspension of 5 mg/ mL of water-insoluble protein (WI) of aged human lenses (65-75 years) for 24 or 48 h. The mixture was centrifuged, and the protein concentration in the supernatant was measured, N^ε-acetyllysine (AcK) in proteins was measured by western blotting and LC-MS/MS (in enzyme digested samples). The protein thiol content was estimated by a fluorescence method. Human (60-70 years) and mouse lenses (C57BL/6J, 5-8 months) were incubated with aggrelyte-2 at 2.5- or 2-mM concentration for 3 days to determine effects on the elasticity of lenses, using a coverslip method.

Results : Upon incubation of aggrelyte-2 with aged human lens WI, we measured 1.0-1.3 µg/µL of soluble proteins after 24 h and 1.0-1.5 µg/µL after 48 h. Incubation of aggrelyte-2C (that lacked the lysine acetylation capacity), we observed 0.7-0.9 µg/µL of soluble proteins after 24 h and 0.7-1.1 µg/µL after 48 h, which were ~1.35-fold lower efficient than aggrelyte-2. The AcK content in the solubilized proteins increased in the samples incubated with aggrelyte-2 but not with aggrelyte-2C. The LC-MS/MS results revealed AcK levels of 1.5–2.1 nmoles/mg protein in the aggrelyte-2 treated samples, compared to 0.6–0.9 nmoles/mg protein in the samples treated with aggrelyte-2C. Western blotting revealed a significant increase (p<0.01) in the αA-crystallin content in the solubilized protein. The protein-thiol content in the WI treated with aggrelyte-2 and aggrelyte-2C increased by 1.93- and 5.07-fold over untreated controls. The elasticity of lenses treated with aggreltyte-2 significantly improved (p<0.01 and p<0.001 for mouse, and human lenses, respectively) with a concomitant increase in the AcK levels.

Conclusions : Aggrelyte-2 solubilizes WI through acetylation and disulfide reduction and decreases stiffness in aged human lenses. It could be developed as a therapy against presbyopia.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.