With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for 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 property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58–75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44–66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that 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.

Advanced glycation end products (AGEs) are protein modifications resulting from the chemical reaction between lysine and arginine residues in proteins, and carbonyl compounds, including glyoxal (GO) and methylglyoxal (MGO). N^ε-carboxymethyllysine (CML) and N^ε-carboxyethyllysine (CEL), formed by glycation from GO and MGO, are among the major AGEs in tissue proteins. Incubation with GO or MGO resulted in higher CML and CEL formation in the two cysteine residues containing αA-crystallin (αAC) than in the cysteine lacking αB-crystallin (αBC). Mass spectrometric data showed K70 and K166 to be heavily modified with CML and CEL in GO- and MGO-modified αAC. In silico analysis of the structure of αAC showed K70 and K166 to be proximal to C142. Mutation or reductive alkylation of cysteine residues in αAC significantly reduced CML and CEL formation. The addition of GSH or N-acetylcysteine enhanced CML and CEL formation in αBC. The introduction of a cysteine residue proximal to a lysine residue in αBC increased the CML and CEL accumulation. Our data showed that CML and CEL formation occurs through a hemithioacetal intermediate formed from the reaction between thiols and GO or MGO. Together, these results highlight a mechanism by which thiols influence protein AGE levels. In addition, CML and CEL are ligands for RAGE, a receptor for AGEs, which has been implicated in several aging and diabetes-associated diseases. Therefore, further understanding of the biosynthesis of CML and CEL could lead to the development of new therapies against those diseases.

Mutations in Duchenne muscular dystrophy (DMD) gene that affect the expression of dystrophin protein lead to a number of disorders collectively called dystrophinopathies characterized by muscle degeneration and loss. Dystrophin is a large protein (427 KDa) made up of N-terminal calponin homology (CH) domains, 24 spectrin repeats, cystein rich (CR) domain and C-terminal (CT) domain.

Mutations in Duchenne muscular dystrophy (DMD) gene that affect the expression of dystrophin protein lead to a number of disorders collectively called dystrophinopathies characterized by muscle degeneration and loss. Dystrophin is a large protein (427 KDa) made up of N-terminal calponin homology (CH) domains, 24 spectrin repeats, cystein rich (CR) domain and C-terminal (CT) domain.