XXXX causing mutants of aA-crystallin/sHSP form aggregates and aggresomes degraded through ubiquitin-proteasome pathway.

@entity1 @entity11311 @entity28824 @entity231 @entity1880 @entity28821 @entity28822 @entity28819 @entity28820 @entity28823 @entity6960
BACKGROUND: Mutations of @entity1 aA-crystallin cause congenital @entity231 by @entity6960 . How mutations of aA-crystallin cause disease pathogenesis through @entity6960 is not well understood. To better understand the cellular events leading to @entity6960 , we transfected @entity231 causing mutants, @entity11311 , @entity28819 , @entity28820 , @entity28821 , @entity28822 , @entity28823 and @entity28824 , of @entity1 aA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy. METHODOLOGY/PRINCIPAL FINDINGS: YFP-tagged @entity1 aA-wild-type (aA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The aA-wt and the mutants were individually transfected or co-transfected with CFP-tagged aA-wt or aB-wild-type (aB-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 aA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with aA-wt. Aggresomes were validated by double immunofluorescence by co-localization of y-tubulin, a centrosome marker protein with aA-crystallin. Furthermore, increased ubiquitination was detected in @entity28820 , @entity28823 and @entity28824 as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in @entity28823 transfected cells. Pulse chase assay, after @entity1880 treatment, suggested that @entity28823 degraded faster than the wild-type control. CONCLUSIONS/SIGNIFICANCE: Mutants of aA-crystallin form aggregates and aggresomes. Co-expression of aA-wt with the mutants increased aggregates and co-expression of aB-wt with the mutants significantly decreased the aggregates. The mutant, @entity28823 protein degraded faster than wild-type control and increased ubiquitination was evident in @entity28823 expressing cells.
