Multiple Aggregates and Aggresomes of C-Terminal Truncated Human αA-Crystallins in Mammalian Cells and Protection by αB-Crystallin

Background: Cleavage of 11 (alpha A162), 5 (alpha A168) and 1 (alpha A172) residues from the C-terminus of alpha A-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified alpha A-crystallins is enhanced in diabetes. In the present study, the fate of the truncated alpha A-crystallins expressed in living mammalian cells in the presence and absence of native alpha A- or alpha B-crystallin has been studied by laser scanning confocal microscopy (LSM). Methodology/Principal Findings: YFP tagged alpha Awt, alpha A162, alpha A168 and alpha A172, were individually transfected or co-transfected with CFP tagged alpha Awt or alpha Bwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of alpha-crystallin expression because Western blotting results showed nearly same level of expression of the various alpha-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. alpha A172 interacted with alpha Awt and alpha Bwt better than alpha A168 and alpha A162, interaction of alpha Bwt being two-fold stronger than that of alpha Awt. Furthermore, aggresomes were detected in cells individually expressing alpha A162 and alpha A168 constructs and co-expression with alpha Bwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with alpha Awt co-expression with the truncated constructs, alpha A162 and alpha A168. Double immunocytochemistry technique was used for co-localization of gamma-tubulin with alpha A-crystallin to demonstrate the perinuclear aggregates were aggresomes. Conclusions/Significance: alpha A172 showed the strongest interaction with both alpha Awt and alpha Bwt. Native alpha B-crystallin provided protection to partially unfolded truncated alpha A-crystallins whereas native alpha A-crystallin did not. Aggresomes were detected in cells expressing alpha A162 and alpha A168 and alpha Bwt co-expression with these constructs diminished the aggresome formation. Co-localization of gamma-tubulin in perinuclear aggregates validates for aggresomes.