Exploring Crystallin Deamidation as a Causative Agent of Cataracts

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Project Summary Cataract is an opacity of the eye lens that can result in blindness. In 2010, cataract affected 15% of the population by age 60 and nearly 70% of the population by age 80. Despite the disease’s prevalence, surgery that replaces the lens with a synthetic implant remains the current standard of care. This strategy prevents access to treatment for people without adequate healthcare. Cataract is caused by protein aggregation in the lens, the majority of which is comprised of a class of proteins called crystallins. Understanding the mechanisms by which crystallins aggregate to form cataract is critical for developing therapeutics that can prevent their formation as an alternative to cataract surgery. Maturation of the lens is accompanied by loss of organelles and protein degradation mechanisms; therefore, the eye lens is metabolically inactive and crystallins by necessity are extremely long- lived proteins. Research on their biophysical properties have proven that crystallins are unusually soluble and stable. However, due to subjection to decades of damaging radiation as well as the loss of lens homeostasis mechanisms, crystallins accumulate post-translational modifications (PTMs) such as oxidation and deamidation. These PTMs are thought to alter their solubility and stability, thereby promoting cataract-related aggregation in the lens. Deamidation, the conversion of asparagine or glutamine to aspartic or glutamic acid, respectively, is the most common PTM. It has been shown that variants with one or two deamidation sites have increased susceptibility to aggregation and oxidation as well as decreased stability. The long-term goal of this project is investigate how deamidation promotes aggregation. This research will be performed on variants with 3, 5, 7, and 9 sites of deamidation and builds on previous insights developed by our lab. I hypothesize that the mechanism of aggregation of these variants will depend on the extent of deamidation. In variants with fewer sites of deamidation, I predict aggregation is formed by increased anion-p interactions. In highly deamidated variants, I propose aggregation increases hydrophobic exposure from altered protein dynamics. Here, I will investigate these possibilities with solution-state NMR and mass spectrometry dynamics experiments. These will be complimented with biophysical techniques that quantify the extent of aggregation, dimerization, and oxidation. I will then look structure of deamidation variant aggregates with solid-state NMR. Finally, preliminary evidence in our lens suggests the HgS could be performing a novel active role in the lens as a last resort redox buffer. I will investigate this with proteomics experiments that monitor the disulfide linkages that have been implicated in this role.