Mechanisms and functional effects of fibrillar aggregation in the lens alpha-crystallins

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Project Summary Lens transparency is maintained by the abundance of a pair of small heat-shock proteins, αA- and αB-crystallin. Over time, lens proteins (including the α-crystallins) accumulate chemical damage that lead to destabilized states and the nucleation of light-scattering aggregates, which take on a pathogenic state known as cataracts. Understanding how this age-related process of protein aggregation in the lens occurs, or how to prevent/reverse it by therapeutics, has been hindered by our poor understanding of the pathways of α-crystallin aggregation, and the molecular nature (or structures) that define light-scattering opacities in the eye lens. The most common state of protein aggregation is presumably amorphous in nature (i.e., proteinaceous precipitates). However, recent advancement in the detection of amyloids has also supported the hypothesis that one of the other prominent aggregate pathways in the lens may be amyloidogenesis (or the formation of amyloids). In fact, over 5% of the insoluble extracts of cataract lenses have been detected to be amyloid in nature. Furthermore, I have discovered that subtle chemical changes to α-crystallin structure can both promote amyloid formation in the α-crystallins, as well as lead to the formation of a completely novel type of fibrillar aggregation state of αB-crystallin inside living cells, that potentiate formation of light-scattering aggregation in response to other destabilized proteins. This proposal seeks to: 1) characterize a novel and reversible form of fibrillar aggregation in αB-crystallin; and 2) determine how disruption of a conserved motif present in both αA- and αB-crystallin leads to the enhancement of amyloid formation. To accomplish these aims, I will employ a collaborative and multi-disciplinary approach that leverages the enabling technology of single particle CryoEM, together with various biochemical and biophysical methods to characterize these aggregation states of the α-crystallins. Success in these aims are expected to contribute to our understanding of protein misfolding and aggregation in the eye lens – the hallmark of the world's leading cause of vision loss.