title: Hormone- and substrate-regulated intracellular degradation of cytochrome P450 (2E1) involving MgATP-activated rapid proteolysis in the endoplasmic reticulum membranes.
abstract: We have examined differences in post-translational regulation between rat liver ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible CYP2B1 using hepatocyte cultures and subcellular fractions, prepared from starved and acetone-treated rats. The intracellular degradation of CYP2E1 was rapid (approximate t1/2 = 9 h) and increased by glucagon treatment of the cells in an isozyme-specific manner, whereas CYP2B1 degradation in the same cells, was slower (t1/2 = 21 h). The glucagon effect on CYP2E1 degradation was abolished by either cycloheximide treatment of cells, indicating the involvement of protein components with rapid turnover, or by lowering of the culture temperature to 23 degrees C. The rapid phase of CYP2E1 degradation was not influenced by inhibitors of the autophagosomal/lysosomal pathway. In vitro experiments with isolated liver microsomes revealed the presence of a Mg(2+)-ATP-activated proteolytic system active on CYP2E1, previously modified by phosphorylation on Ser-129 or denatured by reactive metabolites formed from carbon tetrachloride. Imidazole, a CYP2E1 substrate, specifically inhibited the rapid intracellular degradation of CYP2E1 and also prevented phosphorylation and subsequent proteolysis in isolated microsomes. In contrast, no proteolysis of CYP2B1 occurred under the conditions used. The microsomal Mg(2+)-ATP-dependent CYP2E1 proteolysis could not be solubilized with high salt and 0.05% sodium cholate, indicating the action of membrane-integrated protease(s). Subfractionation of microsomes revealed that the Mg(2+)-ATP-dependent proteolytic system active on CYP2E1 was present in both rough and smooth endoplasmic reticulum. It is suggested that hepatic cytochromes P450 are degraded both in a bulk process, according to the autophagosomal/lysosomal pathway and more rapidly, in a hormone- and substrate-regulated fashion, by a specific proteolytic system in the endoplasmic reticulum, active on physiologically or exogenously modified molecules.
authors.affiliation: Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Eliasson
authors.forename: S
authors.initials: S
authors.lastname: Mkrtchian
authors.forename: M
authors.initials: M
authors.lastname: Ingelman-Sundberg
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002458:Cell Fractionation; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D019362:Cytochrome P-450 CYP2B1; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005934:Glucagon / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D005934:Glucagon; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D010088:Oxidoreductases; D019392:Cytochrome P-450 CYP2E1; D019362:Cytochrome P-450 CYP2B1; D010089:Oxidoreductases, N-Demethylating