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Ekta Tiwary, Shylaja Hegde, Om P Srivastava; βA3-Crystallin Reduces the Aggresome Formation of αA- and αB-Crystallins and their Deamidated Mutants in HeLa Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5590.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of the study was to determine the aggresome formation of αA- and αB- crystallins and their deamidated mutants (αA N101D, αA N123D, αB N78D and αB N146D) in the presence of βA3-crystallin in HeLa cells.
WT αA- and αB- crystallins and their deamidated mutants were cloned in pZs Yellow N1 vector and βA3-crystallin was cloned in pAm Cyan N1 vector. The recombinant plasmids were transfected in HeLa cells using lipofectamine, and the expression of CFP-tagged βA3-crystallin and YFP-tagged αA- and αB-crystallins were determined after 24 h. Further, cells were fixed with 4% paraformaldehyde and aggresome staining was performed using a Proteostat staining kit (Enzo Life Sciences). Expression of crystallins and their aggresome formation were analysed under a fluorescence microscope using CFP-, YFP- and red filters.
βA3-crystallin did not show aggresome formation while αA- and αB-crystallins and their deamidated mutants formed aggresomes. A greater than 90% HeLa cells were positive for aggresome staining when they expressed WT αA- or WT αB-crystallins. Similarly, >90% HeLa cells, transfected with deamidated mutants of αA- and αB-crystallins, showed aggresome formation. A significant decrease (about 70-80% reduction) in aggresome formation was observed when either WT αA- or WT αB-crystallin was co-expressed with βA3-crystallin. Similar results were observed on co-expression of deamidated mutants of αA-or αB-crystallins with βA3-crystallin and aggresome formation was reduced to 40-60%.
WT αA- and WT αB-crystallins and their deamidated mutants formed aggresomes on their expression in HeLa cells. The co-expression of βA3-crystallin with them significantly reduces the aggresome formation. The results are consistent with the earlier reports, suggesting that βA3-crystallin plays a role in the clearance of cellular debris. A study of βA3-crystallin knockout mouse model in our laboratory also showed impaired autophagy, which resulted in accumulation of aggregates during cataract development.
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