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Hassane S. Mchaourab, Sanjay Mishra, Ricahrd A. Stein; Cataract-Linked D-crystallin Mutants Escape Detection by α-Crystallin. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1301.
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The chaperone activity of the lens chaperone, α-crystallin, is hypothesized to play a central role in lens transparency by suppressing the aggregation of damaged or modified proteins prone to unfolding. However, despite a wealth of studies describing its interactions with model substrates, the understanding of α-crystallin binding to lens β- and γ-crystallin is limited. The discovery of γ-crystallin mutants that cause congenital cataract in human and mouse models provides a unique opportunity to explore their recognition by α-crystallin and ensuing chaperone interactions.
For this purpose, we characterized the unfolding equilibrium of two cataract-linked γ-crystallin mutants, I4F and V76D, and investigated their interaction with αA- and αB-crystallin subunits. Binding to α-crystallins and the phosphorylation mimic of αB-crystallin was detected by changes in the fluorescence intensity and anisotropy of a bimane probe site-specifically attached to γD-crystallin.
Located in the core of the N-terminal domain, the substitutions stabilize an intermediate state of γD-crystallin with an unfolded N-terminus. Binding to αA-crystallin, detected by fluorescence of a bimane probe attached to γD-crystallin mutants, was marginal even at high concentrations and large excess of chaperones. This result implies that the folding intermediate has low affinity to α-crystallin and that high affinity binding requires global unfolding of γD-crystallin. Consonant with this conclusion, detectable binding to αA-crystallin and the phosphorylation mimic of αB-crystallin requires further reduction of γD-crystallin stability which was achieved by combination of the two mutations.
The data suggest that a subclass of γ-crystallin destabilized mutants escape quality control by the resident lens chaperones, a result which rationalizes the observation that these mutants form large aggregates in the lens. Our findings predict that γ-crystallins destabilized by age-related modifications of residues in N-terminal domain may not interact with α-crystallin allowing them to nucleate aggregation and scatter light.
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