Congenital Cataract Causing Mutants of αA-Crystallin/sHSP Form Aggregates and Aggresomes Degraded through Ubiquitin-Proteasome Pathway

Background: Mutations of human alpha A-crystallin cause congenital cataract by protein aggregation. How mutations of alpha A-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human alpha A-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy. Methodology/Principal Findings: YFP-tagged human alpha A-wild-type (alpha A-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The alpha A-wt and the mutants were individually transfected or co-transfected with CFP-tagged alpha A-wt or alpha B-wild-type (alpha B-wt) in HeLa cells(Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes(Co-expression of aB-wt with these mutants significantly inhibited protein aggregates where as co-expression with alpha A-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with alpha A-wt. Aggresomes were validated by double immunofluorescence by co-localization of gamma-tubulin, a centrosome marker protein with alpha A-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells(Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control. Conclusions/Significance: Mutants of alpha A-crystallin form aggregates and aggresomes. Co-expression of alpha A-wt with the mutants increased aggregates and co-expression of alpha B-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells.