Cataract-Causing Defect of a Mutant γ-Crystallin Proceeds through an Aggregation Pathway Which Bypasses Recognition by the α-Crystallin Chaperone

Abstract

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.

Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability

“…Although consistent with in vitro studies (32,33), the lack of rescue for lens phenotypes by ␣A-crystallin is somewhat counterintuitive with the abundant expression of this chaperone in mammalian lenses. DECEMBER 2, 2016 • VOLUME 291 • NUMBER 49…”

“…We interpret these findings as revealing the presence of a chaperone network (35) capable of "sensing" the thermodynamic stability and aggregation propensity of proteins. Consistent with in vitro studies on destabilized ␥D-crystallin mutants (32,33), as well as on other modified crystallin proteins (36,37) demonstrating that ␣A-crystallin does not interact strongly with these aggregation-prone mutant proteins, the buffering capacity of ␣A-crystallin in the lens is not limiting, suggesting the contribution of other chaperones to lens proteostasis.…”

“…Induced by ␥D-crystallin I4F/V76D-In vitro biochemical studies showed that cataractlinked ␥D-crystallin mutants could evade quality control by ␣-crystallins (32,33), consistent with their ability to induce cataracts in mouse models (30,31). We set to probe the interactions between ␥D-crystallin and ␣-crystallins in vivo using our transgenic zebrafish model.…”

Aggregation-Prone Near-Native Intermediate Formation during Unfolding of a Structurally Similar Nonlenticular βγ-Crystallin Domain

“…4,44 The two-domain lens γD-crystallin follows a three-state transition during unfolding; 23 however, the tendency of intermediate species to aggregate is observed in some of those mutant forms of lens-specific crystallins that are implicated in cataract. [14][15][16]18,22 Such a tendency to aggregate or a decreased solubility of γ-crystallin is attributed to the alteration of surface properties. 19 However, it is not known if an individual domain of these lens γ-crystallin mutants would form such aggregation-prone intermediates.…”

Dynamics of βB2-Crystallin Motion Based on Principal Component Analysis and Normal Mode Analysis

“…Cataractogenesis is a downstream process emerging from loss of solubility and/or stability of crystallins [44][45][46]. However, the cataract pathology was unlikely to be associated with a direct folding defect [47]. Collective motions suggest that flexibility and internal motions are important for many protein functions [48][49][50].…”