Proteolysis in lens epithelium. Basic Studies and Possible Implications for Cataract Formation

Aims: Proteolysis has been implied in cataract formation, but the proteases involved and their precise role has not been fully elucidated. The objectives of this thesis were to study the activity of three main non-lysosomal proteolytical systems; calpains, the proteasome and the caspases, in lens epithelium from clear and cataractous lenses and during potentially cataractogenic conditions.Methods: Human lens epithelium from cataractous lenses was obtained during cataract surgery (Sahlgrenska University Hospital) and epithelium from clear donor lenses was obtained from the Cornea bank in =rhus. Lens epithelium from rabbit lenses kept in organ culture was studied after exposure to near-physiological levels of hydrogen peroxide (50 and 100 µM). Apoptosis, induced by staurosporin or lactacystin, was studied in cultured bovine lens epithelial cells by staining with Annexin V, Hoechst 33342 and Propidium Iodide. The number of apoptotic cells was quantified with the TUNEL assay. Proteolytic activity of m-calpain, the proteasome and of several caspases was measured using fluorogenic substrates and the amount of m-calpain was determined by immunoblotting.Results: Human lens epithelium exhibits m-calpain protein but no net calpain activity, indicating strict regulation by calpastatin. Epithelium from cortical cataracts have higher levels of immunoreactive m-calpain than other cataract types, supporting a role for calcium-dependent proteolysis in cortical cataract. A 55% upregulation of calpain activity in rabbit lens epithelium was seen after moderate oxidative stress of whole lenses.Comparison of the three main peptidase activities of the proteasome in human lens epithelium from cataract lenses revealed major differencies in kinetic properties; Michaelis-Menten constant was 56, 678 and 108 µM for the chymotrypsin-like, the trypsin-like and the peptidyl-glutamyl peptidase activity respectively. The inhibition rates with respect to lactacystin also differed substantially, indicating separate active sites for these peptidase activities in the proteasome. The trypsin-like activity was the only thermostable peptidase activity and all activities were inhibited by hydrogen peroxide. After 24 hours, remaining proteolytic activity compared to control was 25%, 73% and 44% for the chymotrypsin-like, the trypsin-like and the peptidyl-glutamyl peptidase activity. Oxidative inhibition of the chymotrypsin-like activity could be partially prevented by alpha-crystallin or heat shock protein 90. The chymotrypsin-like activity of the proteasome was lower in lens epithelium from human cataractous lenses as compared to clear donor lenses, suggesting impaired proteolytical removal of aberrant proteins as a cataractogenic cause.During staurosporin-induced apoptosis in bovine lens epithelial cells, caspase-2, -3, -4, -8 and -9 were activated, preceding DNA fragmentation. Caspase inhibition caused a 60% reduction in the number of apoptotic cells. Inhibition of the proteasome by lactacystin increased the number of TUNEL-positive cells and also lead to caspase-3 activation, but apoptosis was not prevented by caspase inhibiton. These data thus demonstrate that both staurosporin- and lactacystin-induced apoptosis can be caspase-independent