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

Background: alpha A-crystallin plays an important role in eye lens development. Its N-terminal domain is implicated in several important biological functions. Mutations in certain conserved arginine residues in the N-terminal region of alpha A-crystallin lead to cataract with characteristic cytoplasmic/nuclear aggregation of the mutant protein. In this study, we attempt to gain mechanistic insights into the congenital cataract caused by the R54C mutation in human alpha A-crystallin. Methods: We used several spectroscopic techniques to investigate the structure and function of the wild-type and R54C alpha A-crystallin. Immunoprecipitation, chromatin-enrichment followed by western blotting, immunofluorescence and cell-viability assay were performed to study the interaction partners, chromatin-association, stress-like response and cell-death caused by the mutant. Results: Although R54C alpha A-crystallin exhibited slight changes in quaternary structure, its chaperone-like activity was comparable to that of wild-type. When expressed in lens epithelial cells, R54C alpha A-crystallin exhibited a speckled appearance in the nucleus rather than cytoplasmic localization. R54C alpha A-crystallin triggered a stress-like response, resulting in nuclear translocation of alpha B-crystallin, disassembly of cytoskeletal elements and activation of caspase 3, leading to apoptosis. Analysis of the interactome revealed an increase in interaction of the mutant protein with nucleosomal histones, and its association with chromatin. Conclusions: The study shows that alteration of interactome and nucleosomal association, rather than loss of chaperone-like activity, is the molecular basis of cataract caused by the R54C mutation in alpha A-crystallin. General significance: The study provides a novel mechanism of cataract caused by a mutant of alpha A-crystallin, and sheds light on the possible mechanism of stress and cell death caused by such nuclear inclusions.

Automated summary

This study reveals that alterations in the interactome and nucleosomal association, rather than loss of chaperone-like activity, are the molecular basis of cataract caused by the R54C mutation in alpha A-crystallin. Furthermore, it provides a novel mechanism of cataract caused by this mutant and sheds light on the possible mechanisms of stress and cell death induced by nuclear inclusions.