ABSTRACT: PURPOSE: Crystallins are major functional and structural proteins in mammalian lens. Their expression, distribution, and protein-protein interaction affect lens development and fiber cell differentiation. Mutated crystallins lead to structural and functional changes of lens structure and could lead to opacity formation and cataract development. The purpose of this study was to investigate the biological effects of the cataract-causing G98R mutation on the alphaA-crystallin (CRYAA) protein and to test the capability of chemical chaperone trimethylamine N-oxide (TMAO) to reverse such effects. METHODS: Myc/His-tagged, human, full-length, wild-type (WT) or G98R CRYAA was expressed in human lens epithelial B3 cells and treated or not treated with TMAO. Triton X-100 (Tx) solubility and cellular localization of CRYAA were examined by western blotting and confocal immunofluorescence, respectively. Ubiquitin proteasome-associated degradation was assayed by MG132 treatment. Endoplasmic reticulum (ER) stress, unfolded protein response, and apoptosis were analyzed by the expression of phosphorylated protein kinase-like ER-kinase, binding immunoglobulin protein (BiP), C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/GADD153), and caspase-3 and immunocytochemistry. Changes in heat shock and stress signaling were investigated. RESULTS: When transfected in lens epithelial B3 cells, unlike WT CRYAA located in the cytoplasm, the G98R CRYAA mutant formed aggregates inside the ER and the protein was predominantly Tx-insoluble. ER stress was induced by G98R CRYAA expression, and cells underwent apoptosis, as shown by a more frequent appearance of fragmented nuclei. Treatment with TMAO reduced Tx-insoluble mutant protein in time- and dose-dependent manners. Other chemical chaperones, 4-phenylbutyric acid, dimethysulfoxide, and glycerol, were much less effective than TMAO. ER-associated aggregates were reduced after TMAO treatment, and the protein was degraded through the ubiquitin-proteasome pathway. This alleviated ER stress and resulted in less apoptosis. Moreover, TMAO treatment induced a moderate upregulation of heat shock protein 70, indicating its effect on heat-shock response to modulate protein folding and assembly. No change was found for nontransfected cells after TMAO treatment. CONCLUSION: The natural osmolyte and chemical chaperone TMAO reduced the aggregation of G98R CRYAA. This alleviated ER stress and rescued the affected cells from apoptosis. Our results showed that the chemical chaperone reduces mutant CRYAA aggregates in lens cells. We suggest a potential chemical-based strategy to reduce lens opacity formation.
