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

The proteasome is a large protease complex that is thought to be responsibl e for proteolytic removal of damaged proteins. We have previously shown tha t the level of proteolytic activity due to the proteasome is lower in lens epithelium from human cataractous lenses compared to the activity in epithe lium from clear donor lenses. This study aimed to characterize the three ma in peptidase activities of the proteasome in human lens epithelium with res pect to kinetic properties and sensitivity to heat and oxidation. Human len s epithelia were obtained from cataract surgery and analysis performed on p ools of epithelial cell cytoplasm. Using the fluorogenic peptide substrates Sue-Leu-Leu Val-Tyr-AMC (LLVY), Boc-Val-Gly-Arg-AMC (VGR) and Z-Leu-Leu-Gl u-beta NA (LLE), K-m-values of 56, 678 and 108 mu M were obtained. All pept idase activities were inhibited by lactacystin, a specific proteasome inhib itor, but at very different rates; with LLVY-hydrolysing activity being the most sensitive (K-150% = 0.15 mu M). Thermostability was investigated by p erforming 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 a fter a few hours at 37 degrees C. Oxidative inhibition was induced by incub ation of the samples in 0.5 mM H2O2 for 1 or 24 hr. One hour exposure to H2 O2 caused moderate inhibition of all peptidase activities. The activity cou ld be partially restored by adding 1 mM dithiotreitol, indicating the depen dency on intact SH-groups. After 24 hr, peptidase activities were decreased to 25% (LLVY), 73% (VGR) and 44% (LLE) of corresponding control. This inhi bition was irreversible for VGR and LLE, but could be partly prevented by t he presence of heat shock protein 90 (LLVY and VGR) or cc-crystallin (LLVY) . These data show that the peptidase activities of the human lens proteasom e can be modulated by metabolites, such as reactive oxygen species, and by endogenous proteins such as a-crystallin and heat shock protein 90. (C) 199 9 Academic Press.