Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R alphaA-crystallin

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.

Figure 1

G98R αA-crystallin was TritonX-100-insoluble and formed aggregates inside cells. A: Triton X-100 (Tx) solubility assay of wild-type (WT) and cataract-causing mutant CRYAA in B3 cells by western blotting of myc (detecting CRYAA), housekeeping GAPDH, and β-actin. The band densitometry analysis showed the drastic reduction of Tx solubility of G98R CRYAA when compared to WT or other mutants. B: Direct sequencing of pHis/myc-CRYAA^G98R to indicate the base change at c.292G>A. C–L: Confocal double immunofluorescence of WT and G98R CRYAA in B3 cells. C and D: A lower magnification to show the expression of WT (C) and G98R CRYAA (D) in cells. E–H: G98R CRYAA (myc staining in F) formed intracellular aggregates and was intensely co-distributed with PDI (G) in the overlay image (H). I–L: WT CRYAA (J, myc staining) was diffusely distributed in cytoplasm and had only mild co-distribution with PDI (K) in overlay image (L). Nuclei were stained with DAPI (blue, E and I). Scale bars: 10 μm (C–L). PDI: protein disulphide isomerase; DAPI: 4'-6-diamidino-2-phenylindole.

Figure 2

Chemical chaperones reduced the cellular aggregation of G98R αA-crystallin. A-C: Distribution of G98R αA-crystallin (CRYAA) in transfected B3 cells. Untreated B3 cells showed aggregated G98R CRYAA (A), which was reduced after 4-PBA (1 mM; B) and TMAO (100 mM; C) treatments. D: Semiquantitative scoring of intracellular CRYAA aggregates in cells. E: Staining index representing phenotype severity showed dramatic reduction after 4-PBA or TMAO treatment. 4-PBA: 4-phenylbutyric acid; TMAO: trimethylamine N-oxide

Figure 3

Trimethylamine N-oxide reduced the detergent insolubility of C98R αA-crystallin (CRYAA). A: Western blot analysis of myc (CRYAA) showed that the WT was mainly Tx-soluble and not affected by 4-PBA or TMAO treatment. Untreated G98R CRYAA was predominantly Tx insoluble, which was significantly reduced after 4-PBA (1 mM) or TMAO (100 mM) treatment for 2 days. The asterisk indicates a p<0.05 by independent Student t test. DMSO (1%) did not affect mutant insolubility. B: TMAO reduced Tx-insoluble G98R CRYAA dose dependantly. Treatment with TMAO (50 mM or higher) for 2 days significantly decreased Tx-insoluble G98R CRYAA. The asterisk indicates a p<0.05 by independent Student t test. C: TMAO (100 mM) reduced Tx-insoluble G98R CRYAA in a time-dependant manner. WT: wild-type; Tx: Triton X-100; 4-PBA: 4-phenylbutyric acid; TMAO: trimethylamine N-oxide; DMSO: dimethylsulfoxide.

Figure 4

Trimethylamine N-oxide treatment degraded reduced G98R αA-crystallin (CRYAA) via the ubiquitin-proteasome pathway. A: Reduced level of Tx-insoluble G98R CRYAA after TMAO (100 mM, 2 days) was reversed by MG132 (10 μM, 8 h). MG132 alone further increased Tx-insoluble mutant protein. B–E: Cytoplasmic G98R CRYAA aggregates were reduced after TMAO treatment (C). Intense aggregation was observed in cells treated with TMAO and MG132 (E), MG132 only (D), and untreated cells (B). F: Percentages of cells with CRYAA aggregates after treatments. G: Null changes of Tx-insoluble G98R CRYAA after treatment with 3-MA, unlike that of MG132 treatment. The asterisk indicates a p<0.05 by independent Student t test. Tx: Triton X-100; TMAO: trimethylamine N-oxide; UPR: unfolded protein response; PERK: phosphorylated protein kinase-like ER-kinase; BiP: binding immunoglobulin protein; CHOP/GADD153: C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153; WT: wild-type.

Figure 5

Trimethylamine N-oxide treatment alleviated the endoplasmic reticulum (ER) stress and apoptosis of cells expressing G98R αA-crystallin (CRYAA). A: TMAO (100 mM, 2 days) reduced expression of ER stress and UPR markers (PERK, BiP, and CHOP/GADD153) that were upregulated in G98R CRYAA cells. No specific change was observed for WT cells. B: Reduced caspase-3 expression in G98R CRYAA cells treated with TMAO. The asterisk indicates a p<0.05 by independent Student t test. C: Fewer G98R CRYAA cells exhibited fragmented nuclei after TMAO treatment.

Figure 6

Heat-shock response and cell stress signaling. Semiquantitative reverse transcription-polymerase chain reaction analysis showed specific upregulation of Hsp70 in G98R CRYAA cells treated with TMAO, whereas other stress-inducible genes remained unchanged. No changes were found for TMAO treatment on nontransfected cells. Hsp70: heat-shock protein 70; CRYAA: αA crystallin; TMAO: trimethylamine N-oxide