Sodium 4-phenylbutyrate ameliorates the effects of cataract-causing mutant gammaD-crystallin in cultured cells

Abstract

Purpose: gammaD-Crystallin (CRYGD) is a major structural lens crystallin and its mutations result in congenital cataract formation. In this study, we attempted to correct the altered protein features of G165fsX8 CRYGD protein with small chemical molecules.

Methods: Recombinant FLAG-tagged mutants (R15C, R15S, P24T, R61C, and G165fsX8) of CRYGD were expressed in COS-7 cells and treated with small chemical molecules with reported protein chaperoning properties (sodium 4-phenylbutyrate [4-PBA], trimethylamine N-oxide [TMAO], and glycerol and DMSO [DMSO]). Protein solubility in 0.5% Triton X-100 and subcellular distribution was examined by western blotting and immunofluorescence, respectively. Apoptosis was assayed as the percentage of fragmented nuclei in transfected cells. Expression of heat-shock proteins (Hsp70 and Hsp90) was examined by reverse transcription-polymerase chain reaction analysis.

Results: Unlike WT and most mutants (R15C, R15S, P24T, and R61C) of CRYGD, G165fsX8 CRYGD was significantly insoluble in 0.5% Triton X-100. This insolubility was alleviated by dose-dependent 4-PBA treatment. The treatment relieved the mislocalization of G165fsX8 CRYGD from the nuclear envelope. Also, 4-PBA treatment reduced cell apoptosis and caused an upregulation of Hsp70.

Conclusions: 4-PBA treatment reduced the defective phenotype of mutant G165fsX8 CRYGD and rescued the affected cells from apoptosis. This could be a potential treatment for lens structural protein and prevent lens opacity in cataract formation.

Figure 1

Chemical chaperone 4-PBA improved Tx solubility of G165fsX8 CRYGD expressing in COS-7 cells. Cells were treated with 4-PBA (0 to 3 mM) for two days followed by western blotting for FLAG-tagged CRYGD and band densitometry (represented by histogram). A: Tx insoluble fractions. B: Tx soluble fractions. β-Actin and GAPDH were the housekeeping proteins of Tx-insoluble and soluble fractions, respectively. Asterisks indicate a p<0.05 by independent Student’s t-test. C: Treatment of cells with TMAO (0 to 300 mM) for two days followed by Tx solubility test for FLAG-tagged CRYGD expression. D: Band densitometry analysis showed that TMAO did not affect the mutant protein solubility.

Figure 2

4-PBA improved the solubility of various cataract-causing CRYGD mutants expressing in COS-7 cells. A: western blotting of FLAG and housekeeping proteins in Tx soluble and insoluble fractions from cells expressing WT, R15C, R15S, P24T, R61C, or G165fsX8 CRYGD treated (right) or not (left) with 1 mM 4-PBA for two days. B: Band densitometry of FLAG-tagged CRYGD normalized with housekeeping proteins in cells from A. Asterisk indicated a p<0.05 by independent Student’s t-test.

Figure 3

The corrective effect of 4-PBA on G165fsX8-expressing cells. A-D: 4-PBA corrected the mislocalization of G165fs CRYGD as shown by confocal immunofluorescence. A: Untreated WT cells and B: WT cells treated with 1 mM PBA for two days. C: Untreated G165fs CRYGD cells and D: G165fs CRYGD cells treated with 1 mM PBA for two days. E: 4-PBA reduced apoptosis of mutant cells with nuclear fragmentation. The asterisk indicates a p<0.05 by independent Student’s t-test. F: Semi-quantitative RT–PCR analysis showed specific upregulation of Hsp70 in G165fs CRYGD cells treated with 4-PBA (p<0.05, independent Student’s t-test). No significant change was found for Hsp90.