Thiol regulation in the lens

The high content of glutathione (GSH) in the lens is believed to protect th e thiols in structural proteins and enzymes for proper biological functions . The lens has both biosynthetic and regenerating systems for GSH to mainta in its large pool size (4-6 mM). However, we have observed that, in aging l enses or lenses under oxidative stress, the size of GSH pool is diminished; and some protein thiols are being S-thiolated by oxidized nonprotein thiol s to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC), We have shown in an H2O2-induced ca taract model that PSSG formation precedes a cascade of events starting with protein disulfide crosslinks, protein solubility loss, and eventual lens o pacification. Recently, we discovered that this early oxidative damage in p rotein thiols could be spontaneously reversed in H2O2 pretreated lenses if the oxidant was removed in time. This dethiolation process is likely mediat ed through a redox regulating enzyme, thioltransferase (TTase), which has b een discovered recently in the lens. To understand if the role of oxidative defense and repair is the physiological function of TTase in the lens, we cloned the TTase gene and purified the recombinant human lens TTase. Althou gh TTase required GSH for its activity, TTase was far more efficient in det hiolating lens proteins than GSH alone. It favored PSSG over PSSC and dethi olated gamma-crystallin-S-S-G better than the a-crystallin counterparts. Fu rthermore, TTase showed a remarkable resistance to oxidation (H2O2) in cult ured rabbit lens epithelial cells when GSH peroxidase, GSH reductase, and g lyceraldehyde-3-phosphate dehydrogenase were severely inactivated. We furth er showed that activity loss in those SH sensitive enzymes could be attribu ted to S-thiolation, but reactivation via dethiolation could be attributed to TTase. We conclude that TTase can regulate and repair the thiols in lens proteins and enzymes through its dethiolase activity, thus contributing to the maintenance of the function of the lens.