Abstract
Purpose :
With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. Protein aggregation and insolubilization are the primary reasons for the increase in the stiffness of lenses during the onset of presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual properties, lysine acetylation and disulfide reduction.
Methods :
Water-insoluble protein (WI) of aged human lenses (58-75 years) (2 mg/ 0.4 mL) was incubated for 24 h with aggrelyte-1 or aggrelyte-1C, a control compound lacking the S-acetyl group, (N-acetyl glutathione diethyl ester). The solubilized protein was measured in the supernatant. Western blotting and LC-MS/MS (of the enzyme-digested protein samples) were employed to measure the Nε-acetyllysine (AcK) content. A fluorescent assay was used to estimate the thiol content. Human (44-66 years) and mouse lenses (C57BL/6J, 7 months) were incubated with aggrelyte-1 or -1C at 2000 µM concentration for 72 and 24 h, respectively, and the stiffness of the lenses was measured using a computer-controlled lens squeezer.
Results :
We tested the effect of aggrelyte concentration on the solubility of WI. The results showed that 2000 µM of aggrelyte-1 was able to improve solubility of WI significantly (p<0.0001). Aggrelyte-1 (2000 µM) treatment of WI from aged human lenses (58-75 years) resulted in 0.86 ± 0.10 mg (mean ± SD) of water-soluble protein, which was 2.6- and 1.2-fold higher than the untreated and aggrelyte-1C treated samples, respectively. The western blotting and LC/MS-MS results showed that aggrelyte-1-treated solubilized protein contained significantly greater amounts of AcK (p<0.0001 and p<0.01) than control samples. The free thiol content was 0.22 ± 0.06 µM in the control samples and 0.38 ± 0.03 µM and 0.40 ± 0.04 µM in the aggrelyte-1- and aggregate-1C-treated samples, respectively. Aged mouse and human lenses treated with aggregate-1 showed reduced stiffness compared to those treated with aggregate-1C or untreated controls (p<0.05), accompanied by higher free thiol and AcK levels.
Conclusions :
Aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.