Differential inhibition of three peptidase activities of the proteasome in human lens epithelium by heat and oxidation

The proteasome is a large protease complex that is thought to be responsible for proteolytic removal of damaged proteins. We have previously shown that the level of proteolytic activity due to the proteasome is lower in lens epithelium from human cataractous lenses compared to the activity in epithelium from clear donor lenses. This study aimed to characterize the three main peptidase activities of the proteasome in human lens epithelium with respect to kinetic properties and sensitivity to heat and oxidation. Human lens epithelia were obtained from cataract surgery and analysis performed on pools of epithelial cell cytoplasm. Using the fluorogenic peptide substrates Suc-Leu-Leu-Val-Tyr-AMC (LLVY), Boc-Val-Gly-Arg-AMC (VGR) and Z-Leu-Leu-Glu-betaNA (LLE), Km-values of 56, 678 and 108 micrometers were obtained. All peptidase activities were inhibited by lactacystin, a specific proteasome inhibitor, but at very different rates; with LLVY-hydrolysing activity being the most sensitive (Ki50%=0.15 micrometers). Thermostability was investigated by performing the proteolytic assay at 20 degrees, 37 degrees and 53 degrees C. The trypsin-like activity, as measured by VGR, was completely stable at 53 degrees C for at least 24 hr whereas hydrolysis of LLVY and LLE declined after a few hours at 37 degrees C. Oxidative inhibition was induced by incubation of the samples in 0.5 m m H2O2for 1 or 24 hr. One hour exposure to H2O2caused moderate inhibition of all peptidase activities. The activity could be partially restored by adding 1 m m dithiotreitol, indicating the dependency on intact SH-groups. After 24 hr, peptidase activities were decreased to 25% (LLVY), 73% (VGR) and 44% (LLE) of corresponding control. This inhibition was irreversible for VGR and LLE, but could be partly prevented by the presence of heat shock protein 90 (LLVY and VGR) or alpha-crystallin (LLVY). These data show that the peptidase activities of the human lens proteasome can be modulated by metabolites, such as reactive oxygen species, and by endogenous proteins such as alpha-crystallin and heat shock protein 90.