Nucleosomal association and altered interactome underlie the mechanism of cataract caused by the R54C mutation of αA-crystallin

Abstract

The small heat shock protein (sHSP), αA-crystallin, plays an important role in eye lens development. It has three distinct domains viz . the N-terminal domain, α-crystallin domain and the C-terminal extension. While the α-crystallin domain is conserved across the sHSP family, the N-terminal domain and the C-terminal extension are comparatively less conserved. Nevertheless, certain arginine residues in the N-terminal region of αA-crystallin are conserved across the sHSP family. Interestingly, most of the cataractcausing mutations in αA-crystallin occur in the conserved arginine residues. In order to understand the molecular basis of cataract caused by the R54C mutation in human αA-crystallin, we have compared the structure, chaperone activity, intracellular localization, effect on cell viability and “interactome” of wild-type and mutant αA-crystallin. Although R54CαA-crystallin exhibited slight changes in quaternary structure, its chaperone activity was comparable to that of the wild-type. When expressed in lens epithelial cells, R54CαA-crystallin triggered a stress-like response, resulting in nuclear translocation of αB-crystallin, disassembly of cytoskeletal elements and activation of Caspase 3, leading to apoptosis. Comparison of the “interactome” of the wild-type and mutant proteins revealed a striking increase in the interaction of the mutant protein with nucleosomal histones (H2A, H2B, H3 and H4). Using purified chromatin fractions, we show an increased association of R54CαA-crystallin with these nucleosomal histones, suggesting the potential role of the mutant in transcriptional modulation. Thus, the present study shows that alteration of “interactome” and its nucleosomal association, rather than loss of chaperone activity, is the molecular basis of cataract caused by the R54C mutation in αA-crystallin.