Client-Induced Elongation, Expansion, and Co-Aggregation of the Lens Alpha-Crystallins

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Biophysical Journal

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The long-lived nature of the eye lens presents unique challenges to the maintenance of protein stability and function. Age-related accumulation of chemical modifications to proteins in the lens promote the formation of light-scattering aggregates that disrupt vision, leading to cataract: a leading cause of blindness worldwide. To counteract these effects, ∼40% of the lens cytosol is composed of α-crystallins (αAc and αBc isoforms): ATP-independent chaperone “holdases” that work to prevent formation of protein aggregates capable of scattering light. Like other members of the small heat shock protein (sHSP) family of chaperones, the α-crystallins form large and polydisperse oligomeric assemblies that recognize and sequester destabilized proteins, through the formation of soluble chaperone/client complexes. Over time, saturating conditions of unfolding clientele overwhelm lens chaperone capacity, leading to light-scattering co-aggregates. A mechanistic basis for the chaperone/client co-aggregation pathway is not well understood. Here, we applied single-particle electron microscopy and other biophysical techniques to define the morphological transitions associated with chaperone/client co-aggregation, using the model client lysozyme. We observe a mechanism where αAc and αBc chaperone/client sequestration progresses through an “initiation complex” (∼15–20 nm diameter, akin to apo-state α-crystallins), and proceeds through an intermediate elongation/expansion stage where co-aggregates reach dimensions of 50–200 nm. Ultimately, under saturating client conditions, the elongation/expansion complexes appear to cluster (or collapse) to form large light-scattering aggregates (microns in diameter). Ensemble and single-particle analysis techniques show αAc and αBc adopt a similar overall mechanism of expansion/elongation, while some unique isoform-specific features may be attributed to characteristic differences in activity toward lysozyme client. Overall, this work provides a mechanistic basis for understanding how α-crystallins (and perhaps other sHSP's) accommodate destabilized clients and depicts a potential client-induced co-aggregation pathway leading to lens opacification and age-related vision loss.


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