NADPH-Dependent Microsomal Electron Transfer Increases Degradation of CYP2E1 by the Proteasome Complex: Role of Reactive Oxygen Species

Abstract

Increased levels of cytochrome P450 2E1 (CYP2E1) produced by low-molecular-weight compounds is mostly due to stabilization of the enzyme against proteolytic degradation. CYP2E1, in the absence of substrate or ligand, normally has a short half-life, but the factors which regulate CYP2E1 turnover or trigger its rapid degradation are not known. Since CYP2E1 is active in producing reactive oxygen species, experiments were carried out to evaluate whether reactive oxygen species modulated the degradation of CYP2E1. CYP2E1 present in human liver microsomes was very stable. Addition of the cytosol fraction produced degradation of CYP2E1, and this was enhanced when NADPH was present in the reaction system. Antioxidants or iron chelators which prevent lipid peroxidation, prevented the degradation of CYP2E1 by the cytosolic fraction. Similarly, diphenyleneiodonium chloride, which inhibits NADPH-dependent electron transfer, prevented the degradation of CYP2E1, as did 4-methylpyrazole, a ligand which increases the level of CYP2E1. If microsomes were first incubated with NADPH for 30 min, followed by the addition of these agents, there was no protection against CYP2E1 degradation. Lactacystin, an inhibitor of the proteasome, decreased the degradation of CYP2E1. In intact HepG2 cells transduced to express CYP2E1, proteasome inhibitors elevated steady-state levels of CYP2E1. Steady-state levels of CYP2E1 were increased by about 50% when the cells were incubated with trolox. Trolox decreased the rate of loss of CYP2E1 protein when the cells were treated with cycloheximide. These results suggest that NADPH-dependent production of reactive oxygen species may result in oxidative modification of CYP2E1, followed by rapid degradation of the labilized CYP2E1 by the proteasome complex. It is interesting to speculate that one consequence of the high rates of production of reactive oxygen species by CYP2E1 is its own labilization and subsequent rapid degradation, and this may be a regulatory mechanism to prevent high levels of the enzyme from accumulating within the cell.