Metastable Crystallins: Structure and Stabilization

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Abstract α-Crystallin is a complex macromolecule that accounts for nearly 40% of the adult lens proteins. The chaperone-like activity of α-crystallin, which was discovered nearly three decades ago, is implicated as a key component in the maintenance of lens transparency by suppression of crystallin aggregation. It was found that the deletion of 21-28 and 54-61 regions of αB-crystallin leads to increased chaperone-like activity (activation, gain of function). Understanding the molecular organization and properties of crystallin subunits in activated chaperones would help answer questions on how α-crystallin chaperone-like activity might be harnessed and manipulated for the development of protein-based therapeutics. It is hypothesized that the increased αB- crystallin chaperone-like activity in deletion mutants stems from new type of oligomers where subunit–subunit interactions lead to the exposure of “cryptic” chaperone sites in the native oligomers. Studies show a recombinant αB-crystallin expressed after deleting either 54-61 or 21-28 and 54-61 sequences (resulting in a protein designated as αBΔ54-61 and αBΔ21-28,Δ54-61) was found to form smaller oligomers than the wild- type protein but to show up to ~25-fold increase in chaperone-like activity. The experiments proposed in this proposal will uncover the molecular changes that drive the increased chaperone-like activity in αBΔ21-28,Δ54- 61 and αBΔ54-61. The aims of the application are 1) Uncover the molecular changes in the activated αB- crystallins, (αBΔ54-61 and αBΔ21-28,Δ54-61), 2) determine the biological implications of enhanced chaperone-like activity of engineered proteins in the cell culture and whole lens culture system. Novel crosslinker(s) will be used to gain fresh insights into the “cryptic” chaperone sites getting exposed in the activated crystallin. The studies will also make use of site-directed mutagenesis and mass spectrometric analysis to uncover the molecular changes at subunit interaction level in activated oligomers. To see whether the activated αB-crystallin can be exploited to protect cells from oxidative injury, the effects of stress-inducing agents such as H2O2 and sodium iodate will be investigated in HEK293 and ARPE-19 cells in presence of activated crystallins. Further, the ability of activated chaperones to suppress aggregation and toxicity of fibril- forming β-amyloid will be investigated both in vitro and ex-vivo. The long-term goals of the studies are to understand the structure–function relationship of activated αB-crystallins and develop crystallin proteins that have therapeutic value in protein conformational diseases and oxidative stress conditions.