The transparency of the eye lens depends on maintaining the native tertiary structures and solubility of the lens crystallin proteins over a lifetime. Cataract, the leading cause of blindness worldwide, is caused by protein aggregation within the protected lens environment. With age, covalent protein damage accumulates through pathways thought to include UV radiation, oxidation, deamidation, and truncations. Experiments suggest that the resulting protein destabilization leads to partially unfolded, aggregation-prone intermediates and the formation of insoluble, light-scattering protein aggregates. These aggregates either include or overwhelm the protein chaperone content of the lens. Here we review the causes of cataracts and non-surgical methods being investigated to inhibit or delay cataract development, including natural product-based therapies, modulators of oxidation, and protein aggregation inhibitors.

The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human γD-crystallin (HγD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key β-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the γ-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of γ-crystallins, mouse mutant substitutions were introduced into HγD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HγD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.

BackgroundThe transparency of the eye lens depends upon maintenance of the native state of the γ- and β-crystallins, which is aided by the abundant chaperones αA- and αB-crystallin. Mature onset cataract, the leading cause of blindness worldwide, involves the polymerization of covalently damaged or partially unfolded crystallins into light-scattering aggregates. A number of single amino acid substitutions and truncations of γ-crystallins result in congenital cataract in both humans and mice, though in many cases the coupling between the protein alterations and the accumulation of aggregates is poorly defined.Methodology/Principal FindingsWe have studied the aggregation properties and chaperone interactions of human γD-crystallin carrying substitutions of two buried core mutants, I90F and V75D, which cause congenital cataract in mice. The in vitro aggregation pathway competing with productive refolding was not altered by either substitution. Furthermore, this aggregation pathway for both mutant proteins–originating from a partially folded intermediate–was efficiently suppressed by αB-crystallin. Thus the cataract pathology was unlikely to be associated with a direct folding defect. The native state of wild-type human γD-crystallin exhibited no tendency to aggregate under physiological conditions. However both I90F and V75D native-like proteins exhibited slow (days) aggregation to high molecular weight aggregates under physiological conditions. The perturbed conformation of I90F was recognized and bound by both αA and αB chaperones. In contrast, the aggregation derived from the perturbed state of V75D was not suppressed by either chaperone, and the aggregating species were not bound by the chaperone.Conclusions/SignificanceThe cataract phenotype of I90F in mice may be due to premature saturation of the finite α- crystallin pool. The V75D aggregation pathway and its escape from chaperone surveillance and aggregation suppression can account for the congenital cataract pathology of this mutant. Failure of chaperone recognition may be an important source of pathology for many other protein folding defects.

The eye lens is composed of fiber cells packed with crystallins up to several hundred mg/ml. α‐Crystallin, the chaperone system of the lens, is a small heat shock protein composed of αA and αB (HαB) subunits. γD‐Crystallin (HγD) is a monomeric, two‐domain protein found in the lens nucleus. It is synthesized in utero and must maintain its native fold for a lifetime. WT HγD exhibited a three‐state unfolding pathway with the N‐terminal domain (N‐td) unfolding first, followed by the C‐terminal domain (C‐td). The intermediate populated on this pathway had a folded C‐td and unfolded N‐td. Upon dilution from denaturant, aggregation competed with productive refolding. The HγD mutants V75D and I90F were destabilized in equilibrium unfolding/refolding experiments and this destabilization was dependent on the location of the mutation. Like WT, both mutants aggregated upon dilution from denaturant. HαB recognized and bound an intermediate species, suppressing WT HγD aggregation. HαB suppressed aggregation of both mutants and formed stable complexes with them. This demonstrated that HαB recognized and bound aggregation‐prone species of both WT and mutant HγD proteins. Because precursors to aggregation and cataract formation are not accessible in the lens, these results provide insights into the conformation of aggregation‐prone crystallin intermediates. This work was supported by NEI grant EY015834 to JK.

The aggregation of damaged or misfolded proteins is associated with a number of human diseases, including Alzheimer's disease, Huntington's disease, and cataract. In this study, we investigate the ability of the Group II chaperonin from Methanococcus marapaludis, Mm‐Cpn, a homolog of the eukaryotic chaperonin TRiC, to bind and refold human γD crystallins. Crystallins are a family of structural proteins found in the lens of the human eye, and aggregation of these proteins is thought to be the cause of cataract. Mm‐Cpn interactions with both wild type HγD‐Crys, and damage and disease model mutant HγD‐Crys were evaluated. Solution turbidity studies indicate that Mm‐Cpn suppresses aggregation of both wild type and disease model mutant HγD‐Crys, and exhibits a greater affinity for the destabilized mutant HγD‐Crys. In addition, size exclusion chromatography and fluorescence spectroscopy show that Mm‐Cpn can refold HγD to a native like state, as well as form a long‐lived Mm‐Cpn/HγD complex with both the wild type and mutant HγD crystallins. This long‐lived complex may be ideal for imaging of the chaperonin/substrate complex by cryo‐EM or x‐ray crystallography. These data suggest that the Mm‐Cpn/HγD interaction may serve as a model for general chaperonin/substrate interaction, as well as provide a better understanding of the mechanism of binding and refolding by the Group II chaperonins.