Abstract
BackgroundMutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-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 αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy.Methodology/Principal FindingsYFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-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/SignificanceMutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-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.
Highlights
Cataract of the eye lens is the leading cause of blindness worldwide [1]
Since mutants of aA-crystallin contribute to the development of congenital cataract through the formation of aggregated proteins precipitated in the cells of eye lens, we evaluated the expression of mutants of aA-crystallin in mammalian cells (HeLa cells) in terms of identifying the cells having aggregates and aggresomes as the general cellular response to having over expressed mutant proteins [12]
In view of the propensity of aAcrystallin mutants to aggregate in cells, we explored whether there is any involvement of ubiquitin-proteasomal pathway (UPP) contributing to the degradation of unfolded proteins of mutants of aA-crystallin
Summary
Cataract of the eye lens is the leading cause of blindness worldwide [1]. Pediatric cataract of the congenital type is the most common form of childhood blindness and it is clinically and genetically heterogeneous. At least 34 loci in the human genome have been reported to be associated with various forms of pediatric cataracts. Autosomal dominant and recessive forms of cataracts have been caused by mutations in 22 different genes [2]. Mutations of human aA-crystallin cause congenital cataract by protein aggregation. 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 aA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy
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