Inhibition of apolipoprotein E degradation in a post-Golgi compartment by a cysteine protease inhibitor

• Published in: The Journal of biological chemistry 1993-04, Vol.268 (12), p.8497-8502
• Main Authors: Ye, S Q, Reardon, C A, Getz, G S
• Format: Article
• Language: English
• Subjects: Albumins - metabolism; Ammonium Chloride - pharmacology; Animals; apolipoprotein E; Apolipoproteins E - metabolism; Brefeldin A; calcium; Calcium - metabolism; Cathepsin B - antagonists & inhibitors; Cathepsin L; Cathepsins - antagonists & inhibitors; Chloroquine - pharmacology; CHO Cells; Cricetinae; Cyclopentanes - pharmacology; Cysteine Endopeptidases - metabolism; cysteine proteinase inhibitor; Cysteine Proteinase Inhibitors - pharmacology; degradation; dependent; Endopeptidases; Golgi apparatus; Golgi Apparatus - drug effects; Golgi Apparatus - metabolism; HepG2 cells; Humans; inhibition; Kinetics; Leupeptins - antagonists & inhibitors; Leupeptins - pharmacology; Molecular Sequence Data; Transfection; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52902-1

In our prior studies on lipoprotein stimulation of apolipoprotein E (apoE) secretion in HepG2 cells, it became clear that a proportion of the newly synthesized apoE was degraded intracellularly (Ye, S. Q., Olson, L. M., Reardon, C. A., and Getz, G. S. (1992) J. Biol. Chem. 267, 21961-21966). The present study was designed to determine the nature of the proteases and the intracellular sites involved in newly synthesized apoE degradation. The effect of seven protease inhibitors on total apoE levels was examined. Only N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cysteine protease inhibitor, significantly blocked apoE degradation in HepG2 cells. The amount of total apoE from cells chased with ALLN for 4 h was increased by 1.58 +/- 0.05-fold relative to the controls (n = 11, p < 0.01). ALLN extended the half-life of apoE from 2.61 h to 4.38 h (p < 0.01). This effect occurs in a post-Golgi compartment since in the presence of brefeldin A, ALLN had no effect on intracellular apoE levels. Chloroquine and NH4Cl significantly reduced apoE degradation; however, ALLN plus either of these reagents appear to have an additive effect. The amount of apoE in cells chased in Ca(2+)-free medium was significantly higher than that in cells chased in Ca(2+)-containing medium (1.70 +/- 0.07-fold, n = 6, p < 0.01). ALLN plus Ca(2+)-free medium had no additive effect. ALLN had no significant influence on the degradation of albumin but had a similar effect on transfected apoE in Chinese hamster ovary cells. Overall, these data suggest that apoE may be degraded in a post-Golgi compartment of HepG2 and Chinese hamster ovary cells by lysosomal enzymes and cytosolic Ca(2+)-dependent cysteine proteases. ALLN inhibits the latter.