Abstract
Purpose: :
Misfolded protein aggregates in the endoplasmic reticulum (ER) generate ER stress. Many cataractogenic stressors also induce ER stress, and prolonged ER stress induces the unfolded protein response (UPR) in lens epithelial cells (LECs) prior to cataract formation. Lens crystallins are protected by molecular chaperones such as α-crystallins. The low molecular weight chemical chaperones 4-phenyl butyric acid (PBA) and trimethylamine N-oxide dihydrate (TMAO), as well as bile salts ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) are reported to stabilize protein conformations. Previously, we have shown that these chaperones and TUDCA suppressed LEC death and alleviated cataract formation in galactosemic rat lenses. The purpose of this study was to investigate whether chemical chaperones and bile acids also suppress the crystallin aggregation induced by in vitro thermal denaturation.
Methods: :
Total human lens water-soluble proteins were prepared by centrifugation of a lens homogenate. Recombinant human crystallins (αA- and αB-subunits, βB2- and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. Crystallin samples (0.1mg/ml) were heated to either 60oC or 80oC; protein aggregation was measured by increased light scattering in the presence and absence of each small compound using a fluorometer set at Ex/Em =400/400 nm.
Results: :
High levels of PBA, TMAO, UDCA, and TUDCA significantly suppressed thermal aggregation of total water soluble lens crystallins. Subsequent spectroscopic analysis with each individual recombinant human crystallin indicated that these compounds significantly protected thermally induced aggregation of αA- and αB-subunits and moderately protected γC-crystallins but did not or so little protected βB2-crystallin.
Conclusions: :
In vitro, naturally occurring low molecular weight chemical chaperones and bile acids can suppress aggregation of eye lens αA- and αB-subunits preventing an aggregation process that can cause light scattering in cataract disease.
Keywords: cataract • stress response • cell survival