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.
Highlights
We have recently described a mechanism for post-translational stabilization of ethanol-inducible CYP2E1’(3-5), an vealed the presence of a Mg’+-ATP-activated proteo- enzyme mainly regulated at the post-transcriptional level
Subfractionation of microsomes revealed that the Mg’+-ATP-dependent proteolytic system activeon CYP2El was present inboth rough and smooth endoplasmic reticulum.It is suggested that hepatic cytochromes P450 are degraded both in a bulk process,accordingtothe autophagosomal/lysosomal pathway and more rapidly, in ahormone- and subrise of CYP2E1 in uiuo following, e.g. acetone treatment [6, 7], in comparison to the slower acetone-dependent increase of CYP2B1 ( 7 ),can be explained by such post-translational enzyme stabilization
By contrast,CYP2B1degradationin the hepatocytes was unaffected by the hormones, and this isozyme has previously been shown to be degraded according to theautophagosomal/lysosomal pathway ( 7, 8 ) .In the present investigation, we have used primary hepatocyte cultures and subcellularliver fractions in ordertoinvestigatethe mechanisms behind the difference in CYPBEl and CYP2B1 strate-regulated fashion, bya specific proteolytic sys- regulation
Summary
Materials-Collagenase (type IV, lot 58F-6832), CAMP-dependent proteinkinasecatalyticsubunit(bovineheart), glucagon, type I collagen (rat tail, type VII), 3-methyladenine, leupepticnh,loroquine, hathophenantrolin, Na,ATP, NADPH, and cycloheximide were obtained fromSigma. Theabbreviations usedare: CYP2E1,ethanol-induciblecytochromeP450 2E1; ER,endoplasmic reticulum;P450,cytochrome P450; CYP2B1, the major phenobarbital-inducible cytochrome P450 2B1; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Hepes,4-(2-hydroxyethyl)-l-piperazineethanesulfonaiccid. Hepatocyteswere challenged with inhibitors o f lysosomal proteolysis for 4h with subsequent determination of CYP2E1/2Bl levels by SDS-PAGE/Western blot on thecell homogenate 2500 X g X 5 min supernatant. I n Vitro Proteolysis-Liver microsomes were isolated as described [9] including washing of microsomal pellets and repeated centrifupation before final homogenization in 50 mM phosphate buffer, p H. Other livers were homogenized in 0.25 M sucrose followed by the centrifugation steps [9] and washing of microsomes in 50 mM phosphate buffer beforefinalhomogenization. SDS-PAGE/Western blot with subsequent densitometric determination of CYP2E1/2B1 levels was performed as described [3], including controls of linear relationship between color intensity and amount of protein. :)Iter reincuhation a t 37 "C, in the presence orabsence of M$+-ATP, fi)llowed by SDS-PAGE/Westernblotquantification of CYP2E1/
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